MASSMATTERS
BMSS: 50 years
British Mass Spectrometry Society
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Editor’s letter
Welcome to the December edition of Mass Matters.
This is my first issue as editor, replacing Jackie Mosley, who has left me with rather big shoes to fill. I would like to thank Jackie very much for all of her work with Mass Matters over recent years, as well as her invaluable help and support in recent months. Likewise, this issue would not have been possible without all the hard work and dedication of our publicity secretary, Pat Wright.
I am very enthusiastic and extremely honoured to be involved in the production of this publication; I feel strongly about continuing to build a close-knit and open community, sharing knowledge and discussing science in a friendly and respectful environment.
In this edition, as we continue to celebrate 50 years of BMSS, we review the busy conference season (including the BMSS annual meeting, ASMS and IMSC), report on mass spectrometer world records, interview the BMSS medal winner Frank Pullen, highlight recent research articles from our membership, and more. Our community recently heard the sad news of the loss of two great mass spectrometry pioneers, Frank Hillenkamp and Nico Nibbering, and we remember them, their life and their work, in our tributes here.
As winter settles in, we start to look forward to the year ahead and all that is to come (in the 51st year of BMSS!). Check page 5 for details of upcoming conferences, and keep an eye on the BMSS website www.bmss.org.uk for more dates for your diaries and details of the Special Interest Groups.
Wishing you all the best for the coming months and the holiday season,
Chair’s Report
This is my first Chair’s report and I would like to start by thanking Tony Bristow, who completed his term of office as Chair of BMSS in September, for his leadership of the Society over the last two years. Tony has taken up the new post of Immediate Past Chair, which was created as part of the changes to the BMSS constitution approved in April, so we will not be losing his experience and expertise. There have been several other changes to the Executive Committee and I would particularly like to welcome Gavin O’Connor as Vice-Chair and Jackie Mosely as General Secretary of the Society. Dan Weston takes on the role of Education Officer from Sarah Hart, who retires from the Committee and Peter O'Connor the role of Papers Secretary. We also welcome three newly elected General Members of the Executive Committee, Cristian Lapthorn, Logan Mackay, and Bela Paizs and three Co-opted Members Becky Burnley, Neil Oldham and Mark McDowell. Becky Burnley will be assisting the BMSS Publicity Secretary, Pat Wright, with the
editing of Mass Matters. The names and contact details of all members of the Executive Committee are available on the BMSS web site.
The BMSS has appointed Susan Crosland, a former Chair of BMSS, as a member of the Advisory Board. She replaces Mira Doig who we thank for her contribution to the important work of the Board during her term of office. The Advisory Board is responsible for reviewing research support grants and summer studentships applications and recommending awards. This year the Society supported five summer studentships (one jointly with the Royal Society of Chemistry), so look out for reports on the students’ research in future issues of Mass Matters.
The annual scientific meeting of the Society was traditionally held in two years out of three to accommodate the triennial International Mass Spectrometry Conference (IMSC). However, in 2012 the IMSC changed to a biennial meeting cycle and the BMSS Annual Meeting has since
been held in April and in September in alternate years. This has resulted in a very short gap between the September and April meetings and over a year between the April and September meetings (if you follow me!), which was considered to be less satisfactory than regularly spaced meetings. We have therefore decided that from 2015, the annual scientific meeting will be held in September each year, scheduled to avoid a clash with the IMSC in alternate years. Planning for next year’s 36th BMSS Annual Meeting is currently underway, led by Tony Sullivan (Meetings Secretary) and Peter O’Connor (Papers Secretary) with Helen Cooper as the local organiser, and it is never too soon to get the dates in your diary: 15-17 September 2014 at Birmingham University. Amongst other sessions, we are planning to highlight the activities of our Special Interest Groups at the meeting. The meeting will again be preceded by the successful Introduction to Mass Spectrometry short course and a careers workshop for younger members of the Society.
Finally, many colleagues will have heard the sad news that two highly distinguished mass spectrometrists, Professor Franz Hillenkamp and Professor Nico Nibbering, passed away in the summer. Nico was a particular friend of BMSS and an honorary life member. Their achievements are described in obituaries published in this issue of Mass Matters. They will be greatly missed.
Collin Creaser, Chair
2015 BMSS membership subscription
Don’t miss out, renew your BMSS membership subscription for 2015 Membership entitles you to a substantial discount on the annual BMSS meeting in addition to receiving our newsletter, Mass Matters, three times a year. Members may apply for small grants to help with their research and student members are eligible to apply for travel grants to allow attendance of scientific conferences. Wiley offer a discount on chemistry books to BMSS members. Please renew your membership now and sign up to be included in any or all of our Special Interest Groups. There are 7 to
consider (Environmental and Food Analysis, Protein Analytics, Clinical and Forensic, MALDI and Imaging, Quantitative MS, Ambient Ionisation and Ion Mobility) – and they are free to join! You can use the form that appears on the inside front cover of this newsletter or you can renew on-line via the BMSS website, www. bmss.org.uk. Payment can be made by credit card: VISA and MasterCard.
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Happy Holidays!
And best wishes for the New Year.
DIARY DATES
22nd - 25th Jan 2015 Sanibel Conference on Mass Spectrometry, Clearwater Beach, FL, USA “Security and Forensic Applications of Mass www.asms.org Spectrometry”
2nd - 6th Feb 2015
29th ISMAS-2015 International Mass
Vivanta by Taj Harimahal, Spectrometry Symposium Jodhpur, Rajasthan, India www.ismas.org
8th -12th Mar 2015 Pittcon
New Orleans, LA, USA www.pittcon.org
26th Mar 2015 Emerging Separations Technologies RSC Burlington House London, UK www.chromsoc.com
12th - 13th May 2015 Biopharmaceuticals and Spring Symposium MedImmune, Cambridge, UK
31st May - 4th June 2015 63rd ASMS Conference
St Louis, MO, USA www.asms.org
21st - 25th June 2015 HPLC 2015 Geneva, Switzerland www.hplc2015@symporg.ch
7th - 10th Sept 2015 21st Reid Bioanalytical Forum
15th - 17th Sept 2015
University Surrey, UK www.chromsoc.com
Annual Meeting Birmingham University, UK www.bmss.org.uk
14th Oct 2015 Advances in GC IV
The Heath Business Park, Runcorn, UK www.rsc.org
10th - 11th Nov 2015 (tbc) Analytical Challenges and Solutions for GSK, Ware, UK Difficult Pharmaceutical Molecules
OBITUARIES
Franz Hillenkamp, 1936-2014
Franz Hillenkamp, the co-inventor of matrix-assisted laser desorption/ ionisation (MALDI) and a pioneer in biological mass spectrometry, died on August 22 of pneumonia at the age of 78. Up to his sudden death, he was an energetic and highly active Professor Emeritus and a member of the Scientific Advisory Board at the University of Münster. It was also at the University of Münster where in the 1980s, together with Prof. Michael Karas, he investigated the laser-induced co-desorption of larger biomolecules using small organic compounds as the laser energy-absorbing matrix. This work ultimately led to the highly sensitive and soft desorption/ ionisation technique that is better known as MALDI.
Starting as an engineer in the 1960s (M.S.E.E. at Purdue University; Diploma and PhD at the TUM in Munich) he moved into the world of (laser) physics and further to biophysics, taking up the position of Professor of Biophysics at the
J. W. Goethe University, Frankfurt, Germany in 1976, and in 1986, Professor of Biophysics and Medical Physics and Director of the Institute of Biophysics and Medical Physics at the University of Münster. He was a Visiting Professor at the Universita di Napoli, Texas A&M University, University of Innsbruck, and for more than two decades at the Harvard Medical School, Boston, USA.
Franz Hillenkamp was not only the inventor of MALDI, the true success story in soft laser desorption that led to the 2002 Nobel Prize in Chemistry, he was also a named inventor on more than 30 patents, mostly in the field of mass spectrometry, and consultant for many companies, including Chief Consultant for Mass Spectrometry at Sequenom Inc., San Diego, USA. He has published more than 200 articles and his publications have been cited tens of thousands of times. More importantly, his work in mass spectrometry is regarded as
fundamental to the understanding of many laser-induced desorption techniques, and mass spectrometry and its instrumentation at large. However, mass spectrometry was not the only scientific field he worked in. Franz Hillenkamp has also been recognised for his significant contributions to research in laser medicine and applied optics. His many scientific achievements are also reflected in the numerous prizes he obtained, including the Thompson Medal of the International Mass Spectrometry Society, the Distinguished Contribution in Mass Spectrometry award from the American Society for Mass Spectrometry, the Karl-HeinzBeckurts Prize (Germany’s most important prize for translational research), the Fresenius Medal of the German Chemical Society, and the Torbern Bergman Medal of the Swedish Chemical Society. Furthermore, he was on the editorial board of many well-known journals
Nico Nibbering, 1938-2014
Friends and colleagues will have heard with great sadness that Nicolaas (Nico) M.M. Nibbering died on 25th August, 2014. His career in mass spectrometry and gas-phase ion chemistry spanned almost exactly fifty years during which he established one of the world’s leading research groups in these subjects.
He was born in Zaandam in the Netherlands in 1938 and studied chemistry at the University of Amsterdam, obtaining his Ph.D. cum laude under the supervision of Professor Th. De Boer in 1968. Unusually, because of his exceptional ability, he had been appointed to the permanent staff of the University in the previous year and he became a full professor in 1980. He published over 400 articles on a wide variety of topics including the structures of both positive and negative organic ions and their fragmentation mechanisms, field ionisation kinetics, FTICR MS and a number of instrumental developments. In addition to his many research
contributions, he was very active in promoting mass spectrometry in a variety of other ways. He was a member of the editorial boards of many mass spectrometry journals and was editor of the Journal of Mass Spectrometry and of Mass Spectrometry Reviews for a number of years in addition to editing Volume 4, “Organic Applications” of the Encyclopaedia of Mass Spectrometry. Together with Dom Desiderio, he co-edited the Wiley series of books on mass spectrometry from 1996 until his death.
Nico as also frequently on the organising committee of major international meetings, serving as chairman of the 12th International Congress on Mass Spectrometry in Amsterdam in 1991 and on the organising committees of a series of NATO schools on Gas-Phase Ion Chemistry between 1974 and 1995 . He was instrumental in the formation of the Dutch Mass Spectrometry Society and served as chairman during its early years. More recently, he was appointed Chairman and President of the
in his field.
At a personal level he was an inspiration in his drive and knowledge, with only a few being able to match the depth of his scientific understanding, expertise and thinking. His scientific judgement was rarely wrong and he was a great collaborator due to his breadth of experience and meticulous application of his knowledge. Personally, I feel honoured to have learned mass spectrometry through his supervision and work in Münster, and later through his contributions as a collaborator. He was a collaborator on one of our current research grants and authored his last research article with us, and I can truly say that there was still so much more he would have been able to contribute to the field of mass spectrometry. He will be much missed both personally and professionally.
Rainer Cramer
European Mass Spectrometry Society and the International Mass Spectrometry Society. Nico was honoured in a number of ways for his many and varied contributions to mass spectrometry, receiving the Thomson Medal of the International Mass Spectrometry Society in 1991 and the Johannes Marcus Marci Award of the Czech Spectroscopic Society in 1992. He was elected to membership of the Dutch Academy of Sciences and a number of societies appointed him to Honorary Life Membership including the British Mass Spectrometry Society and those of the Netherlands, India and Japan. Although Nico will be admired for his many achievements, those who were fortunate to work with him, either as a student or as a colleague, will remember him for his warm and generous personality, his great enthusiasm for his subject and for his infectious sense of humour. Over the years, many young people have learnt their mass spectroscopy in his laboratory and have gone on to successful careers and have
continued to receive his support long after his retirement. Nico was always ready to give freely of his time to encourage young mass spectroscopists throughout the world and many will have had the benefit of his valuable advice. His enthusiasm for his research work may be judged from his article “Four decades of joy in mass spectrometry” (Mass Spectrom. Rev. 25, 962-1017, 2006) in which he reminisces about his work over the previous forty years. Even as his health was failing, he found the time and energy to write an article “Highlights of 50 Years of ionic reaction mechanistic studies” which will appear shortly in the International Journal of Mass Spectrometry. He was a staunch supporter of BMSS and he will be missed at our meetings.
Keith Jennings
Here is a selection of the latest papers by BMSS members:-
A Divide-and-Conquer Approach to Compute Collision Cross Sections in the Projection Approximation Method
B. Paizs, IJMS, DOI: 10.1016/j.ijms.2014.10.005 (preview on-line).
Zundel-type H-bonding in biomolecular ions. O. Hernandez, P. Pulay, P. Maître, B. Paizs, J. Am. Soc. Mass Spectrom. 25, 1511-1514, 2014
Annual variation of dietary deoxynivalenol exposure during years of different Fusarium prevalence: a pilot biomonitoring study
S. W. Gratz, A. J. Richardson, G. Duncan, G. Holtrop Food Addit. Contam. Part A. 31 (9), 2014 DOI: 10.1080/19440049.2014.93777
Novel formation of [2M–H]+ species in positive electrospray mass spectra of indoles
A. Saidykhan, S. T. Ayrton, R. T. Gallagher, W. H. C. Martin, R. D. Bowen
Rapid Comm. Mass Spectrom. 28 (17), 1948–1952, 2014
DOI: 10.1002/rcm.6976
On-line Monitoring of Continuous Flow Chemical Synthesis Using a Portable, Small Footprint Mass Spectrometer
T. W. Bristow, A. D. Ray, A. O’Kearney-McMullan, L. Lim, B. McCullough, A. Zammataro
J. Am. Soc. Mass Spectrom. 25 (10), 1794-1802, 2014
DOI: 10.1007/s13361-014-0957-1
Immune Response and Suppression of Fish Immunity by the Oomycete Saprolegnia parasitica: The Role of Pathogen-derived Cell
Wall Carbohydrates and PGE2
R. Belmonte, T. Wang, G. Duncan, I. Skaar, H. Melida, V. Bulone, C. Secombes
Infect. Immun. 82 (11), 4518-29, 2014 DOI: 10.1128/IAI.02196-14
Atomic spectrometry update. Review of advances in the analysis of metals, chemicals and functional materials
B. Gibson, S. Carter, A. S. Fisher, S. Lancaster, J. Marshall, I. Whiteside
J. Anal. At. Spectrom. 2014, Advance Article DOI: 10.1039/C4JA90045F
Monitoring of an esterification reaction by on-line direct liquid sampling mass spectrometry and in-line mid infrared spectrometry with an attenuated total reflectance probe
A. W. Owen, E. A. J. McAulay, A. Nordon, D. Littlejohn, T. P. Lynch, J. Steven Lancaster, Robert G. Wright, Anal. Chim. Acta. 849, 12-8 2014
DOI: 10.1016/j.aca.2014.08.009
On-line detection and quantification of trace impurities in vaporisable samples by direct liquid introduction process mass spectrometry
A. W. Owen, A. Nordon, D. Littlejohn, T. P. Lynch, J. S. Lancaster, R. G. Wright
Anal. Methods. 6, 8148-8153, 2014
DOI: 10.1039/C4AY01064G
Parameters affecting ion intensities in transmission-mode direct analysis in real-time
mass spectrometry
L. P. Harding, G. M. Parkes, J. D. Townend
Analyst 139, 4176, 2014
doi: 10.1039/c4an00859f
Optimized DLP linear ion trap for a portable non-scanning mass spectrometer
B. Brkić, S. Giannoukos, N. France, R. Murcott, F. Siviero, S. Taylor
Int. J. Mass Spectrom. 369, 30-35, 2014
DOI: 10.1016/j.ijms.2014.06.004
Experimental Binding Energies for the Metal Complexes [Mg(NH3)n]2+, [Ca(NH3)n]2+, and [Sr(NH3)n]2+ for n = 4–20 Determined from Kinetic Energy Release Measurements
E. Bruzzi , G. Raggi , R. Parajuli, A. J. Stace
J. Phys. Chem. A, 118 (37), 8525–8532, 2014
DOI: 10.1021/jp5022642
Experimental Binding Energies for the Metal Complexes [Mg(CH3OH)n]2+, [Ca(CH3OH)n]2+, and [Sr(CH3OH)n]2+ for n in the Range 4–20
E. Bruzzi and A. J. Stace
J. Phys. Chem. A, 118 (40), 9357-63, 2014
DOI: 10.1021/jp508131h
Hands-on Electrospray Ionization-Mass Spectrometry for Upper-level Undergraduate and Graduate Students
N. L. Stock, R. E. March
J. Chem. Educ. 91 (8) 1244–1247, 2014
DOI: 10.1021/ed500062w
An Interview with the 2014 BMSS Medal Winner -
How did you first get started in mass spectrometry?
In 1971, I went for a job interview at Wellcome Research Labs in Beckenham. The position was for a lab tech to operate a mass spectrometer. During the interview I was shown this enormous piece of hardware called an AEI MS902, and it was ‘love at first site’. It stood there in all its ‘stainless steel glory’, with an instrument console which looked like something out of Dr Who, with loads of knobs and dials; well, who would not want to drive that, I thought, and fortunately I was offered the job. Training was really a thing of trial and error; every knob influenced the
way the ion beam moved through the analyser, and you learnt what to twiddle to get the best results. I learnt how to run chemists’ samples using the wonders of high and low resolution, and how to understand and interpret EI spectral data. I learnt how to take this monster of a machine apart and fix it; learnt about how to clean ion sources, the joys of UV chart paper, the wonders of electronic valves; the joys of cleaning rotary and oil diffusion pumps etc. The job was never boring and always fun.
What are you most proud of in your career?
Wow that’s a difficult one, because, as I look back over the last
40 years, there are so many things that I feel proud of!! But I suppose, if I was pushed to single out one thing, that I am most proud of, it would be the fact that I have had the opportunity to work with some great people and be part of some amazing teams throughout my career. It is people and teams that have inspired me, and helped me create opportunities, and be part of some great science. People working together, sharing ideas, pushing boundaries and having fun is what science is all about, and so I would have to say that it is the people that I have learnt from, worked with, had fun with and helped develop, that give me a personal sense of pride and satisfaction.
What
makes you want to come to work in the mornings?
The opportunity to engage in good science. Mass spectrometry has been part my life for over 40 years, and it still excites me, and I still get a buzz when things do not go to plan and produce different results from expected!! Mass spec is not just another boring detector, it is a tool that allows us to look at ions in a unique way; how cool is that!!!
Who are your scientific heroes?
Well Heisenberg has always been one of my heroes; anyone who can develop an ‘uncertainty’ principle is to be admired!! In history then Thomson and Aston have to be up there in terms of my MS heroes, but also more recent people whose mass spectrometry books I read when I was starting out, people like Fred McLafferty, John Beynon, Dudley Williams and John Fenn etc, who were all down-to-earth people and were always happy to chat to you and encourage you.
Those people inspired me and made mass spectrometry so exciting and rewarding.
What advice would you give a scientist starting out in mass spectrometry?
Don’t be afraid to try things, there are still things which we don’t know, and the only way to find out is by doing the experiment. Also, don’t be afraid to ask questions; there is no such thing as a ‘stupid question’ and if you have ever been told that, then it is usually because the person who said it does not know the answer!! At mass spectrometry meetings, have the courage to go up and talk to the ‘great and good’. When I was starting out, I always set myself a challenge to go and talk to at least one famous mass spectrometrist at every meeting I attended. Those ‘old people’ have a lot of knowledge, and they are always willing to share their experiences, if asked; you can learn so much by talking with those people. Finally, enjoy yourself; after all mass spectrometry could become a big part of your life, as it has been in mine, and I believe that you can only excel at something if it becomes your ‘passion’.
What does the BMSS mean to you?
Firstly I was ‘blown away’ at being awarded the BMSS medal, I never expected it, and I am still amazed that the society considered me worthy of such an honour. BMSS (in its many guises) has been the cornerstone of mass spectrometry within the UK since before I started out in the field. I can still remember the first meeting that I attended. I found that the MS community were an open and friendly bunch of enthusiasts, who were happy to share ideas and
knowledge freely and also have a good time in the process. As I grew in the field I wanted to ‘do my bit’ to help the society, and in time I took over running the London Area Mass Spectrometry group, and then was successful in getting elected to the BMSS committee. I helped out in a number of roles from membership, through publicity secretary and eventually becoming chairman. During that time I was lucky to be part of the team that helped BMSS put together the successful IMSC Edinburgh bid and managed to pull off what I thought was a really great meeting. BMSS has been a big part of my life, and I have made some good friends through being a part of that great family of mass spectrometrists and, hopefully, I will continue to be an active member of the society for many years to come. The success of BMSS is, firstly, down to the committee who work very hard to ensure that the needs of the society members are catered for (not an easy task), but secondly and probably the most important factor, is it is down to the BMSS membership. It is the membership that has ensured the success of the society to date, and it will be the current and future membership that will ensure that the society is successful and continues to grow within the UK. I hope that the fun and great science that I have enjoyed over the years will continue to be experienced in future generations of mass spectrometrists within BMSS.
What has been your contribution to the development of the next generation of mass spectrometrists?
This is another tough question! I could ramble on about the things that I have been involved in and the things that I am still working on, and how they have helped progress MS, but that would be boring and not what I think this is all about. Hopefully my contribution to the development of the next generation of mass spectrometrists has been through introducing young people to mass spectrometry, and sharing my enthusiasm and enjoyment of the technique. It is a ‘hands on’ subject, and I have always encouraged people to ‘have a go’. You can only read so much in books and scientific publications
about mass spectrometry and how ions move under vacuum, but it is only when you have sat at that console and experienced how you can change a microscopic amount of sample into ions in the gas phase, under vacuum, (which in my mind is something of beauty), and eventually produce a mass spectrum, that you understand what an incredible technique this really is. There is no other analytical technique, in my humble opinion, which gets anywhere near to excitement of mass spectrometry.
What do you think the future holds for mass spectrometry?
I think that we have seen some great advances in the field of mass spectrometry over the last 40 years and I cannot see that changing. At meetings I am always amazed at the variety of MS research that is going on, and the enthusiasm of the people presenting their work; as long as we have scientists and engineers with new ideas and a desire to push back the frontiers, then this powerful technique, that I have had the honour to work with, will continue to develop and grow. I think the future looks good, and I look forward to seeing our next generation of mass spectrometrists continue to come up with new and exciting ways to solve problems and develop new instruments and technology. The bottom line is that BMSS is a vehicle that enables the MS community to share ideas, celebrate successes, and push the boundaries. It has a great history, and hopefully a great future, and I am proud to be part of it and wish it continued success in the future.
Some
The names caused a problem and to this day we are known as "Big Frank" and "Little Frank" - I'll leave you to guess. Frank Cottee, Wellcome Research (retired).
thoughts from former colleagues...
I first met Frank when he was at Wellcome - longer ago than we would both wish to remember. I always found Frank enthusiastic about analytical chemistry and always encouraging to those less expert than himself. A really nice guy. Pete Ryan, Director, KR Analytical. Frank, or more correctly Francis, joined the Wellcome labs at Beckenham as a very young man and as the son of a policeman I can remember him being dressed
During the 18 years I reported to Frank at Pfizer, I found him to have the confidence and enquiring mind needed to try new ideas. He was not afraid of the occasional failure in pursuing novel objectives. I really enjoyed scientific discussions with Frank and it was typical of him that some of our off-the-wall discussions led on to major international projects. One of his greatest strengths is inspiring those around him to strive to innovate. Adrian Wright, Head of Analytical Science, Cyclofluidic Ltd.
A Short History of the BMSG
That’s right! The BMSG (Biomedical Mass Spectrometry Group) was the junior sister of the BMSS and merged with it in the early 1980s. So who or what was the BMSG?
The group started in 1973 as the first incarnation of the London Area Mass Spectrometry Group and its first chairman was Brian Millard at The School of Pharmacy.
The mid-1970s was a period of
as a self-help group to spread the experience and practicalities of new technologies, and enable more groups to get up to speed faster with these new approaches.
So what were the changes taking place and how did they affect the use of mass spectrometers at this time? Well firstly, gas chromatography was a problem as it was carried out using packed
higher gas flow than most source pumping systems on the mass spectrometer could cope with and the problem was overcome by using gas separators between the chromatograph and the mass spectrometer, such as the Ryhage jet separator, and by using quadrupole mass analysers which could tolerate higher working pressures than the sector machines prevalent at the time. Also at this time, the first computerised data acquisition devices were becoming available and again it proved easier to develop these with quadrupole systems rather than sectors.
These systems were invaluable to the use of GC-MS for studying drug metabolites in biofluids and also quantitation of target compounds. These studies were helped by the introduction of a new ionisation technique, Chemical Ionisation (CI), which generated intense protonated molecules ideal for selected ion monitoring. Initially using methane as the reactant gas, most drugs and metabolites were amenable to the use of ammonia which gave cleaner protonated molecules. So our little group started swapping experiences of these new systems and the nuances of dealing with dirty sources, the switch from packed column gas chromatography to initially glass capillary columns and then later fused silica columns, and the introduction of computerised mass spectrometers which not only acquired data for processing but also allowed digital control of the spectrometer itself. By now, news of our meetings and discussions had spread beyond the limits of the Home Counties; and the group was renamed the Biomedical Mass Spectrometry Group.
One of the regular meetings of the group was ‘Christmas at Cardiff’. After Dai Games became involved with our group, we set up a meeting at Cardiff which became a byword for discussion on practical issues as well as innovations affecting the application of mass spectrometry techniques to various chemical analyses. And also a very nice place to meet and socialise with like-minded people just before Christmas! Dai
placed great emphasis on the development of applications and some of the early uses of LC-MS were pioneered at Cardiff and thereby introduced to the Group. The emphasis on application also spawned a new journal, Biomedical Mass Spectrometry! But perhaps our greatest contribution to the BMSS following the merger was our regular newsletter which was recognised as a ‘good thing’ and continued to this day (you’re reading it now!).
Nevil Haskins
Members of the London area Mass Spectrometry Group at the 2nd International Symposium on Mass Spectrometry in Biochemistry and Medicine, Milan, Italy, 1974. L to r : Dai Games, Brian Millard, Nev Haskins and Dave Millington.
Finnigan 3200E, 1974. Early quadrupole mass spectrometer for GC/CI/ MS. The large cabinet to the left is the GC! This instrument has been fitted with isolation valves because of the frequent source cleaning needed using chemical ionisation.
Views of a Finnigan 4000 from 1976. Note the disc drive and keyboard for the in-built data system.
BMSS@50: a community that continues to deliver British science.
This year the British Mass Spectrometry Society is celebrating its golden jubilee. It was founded in 1964 to promote knowledge and advancement in the field of mass spectrometry. It has developed over the years into an inclusive society of scientists from many fields and a variety of backgrounds all of whom share an interest in mass spectrometry and its applications.
The society’s roots date back to the 1950s with the establishment of the Mass Spectroscopy Panel by the Institute of Petroleum’s Hydrocarbon Research Group which combined forces with MetropolitanVickers Company (M-V) Users meetings to host a series of meetings to support the growing field of MS-based applications. The first conference was held at M-V in 1950, inviting international participation in 1953. From then on similar conferences were organised every three years; the precursor to IMSC. In the 1960s a number of UK groups joined forces to provide a platform that supported all areas of mass spectrometry such as organic chemistry, quantitative inorganic analysis, physical chemistry, fragmentation and instrument development. This began as the Mass Spectrometry Group (MSG) in 1964 with key like-minded figures such as John Beynon, Alan Maccoll, Alan Quayle and John Waldron realising the importance of forming a mass spectrometry community to promote and develop the technique.
Over the years, much has been written about the origins of the BMSS. There is an excellent book edited by Keith R. Jennings ‘A History of European Mass Spectrometry’ (IM Publications 2012), in which the UK features heavily and the origins of its mass spectrometry community are clearly charted. In order to remember the people in this community and their achievements in more detail, Susan Crosland has documented the heritage of the Society and this
can be viewed at www.bmss.org. uk/historyintro. Two recent posters celebrating 50 years of BMSS can also be viewed at www.bmss.org.uk/ historyintro.
• The History of the British Mass Spectrometry Society.
Jackie Mosely, with help from John Monaghan and Eddie Clayton, and Stan Evans. 35th BMSS annual meeting, Alderley Park, 2014.
• The British Mass Spectrometry Society: the first 50 years.
Alison Ashcroft, Keith Jennings and Susan Crosland together with Michael Grayson. 62nd American Society of Mass Spectrometry conference on mass spectrometry and allied topics, Baltimore, 2014.
In 1990 Neville Haskins wrote the article ‘BMSS Silver Jubilee – 25 years’ service to British Science (Spectroscopy World 1990, 2(1),1622). Still writing for BMSS (although hopefully not on the type writer he originally used to produce the Society’s newsletters on in 1986!) the first Publicity Officer, Neville has recently penned the article which appears in this issue of Mass Matters, ‘A short History of the BMSG [British Mass Spectrometry Group]’.
Starting where Neville Haskins’ 1990 article left off, we will focus on the last 25 years of the BMSS. The past twenty five years has seen a massive change in mass spectrometry technology which has made mass spectrometry accessible to many more scientists. In 1989, mass spectrometry was in the hands of a limited number of experts who would take a sharp intake of breath if asked to obtain a mass spectrum on much less than 1mg of relative clean material. The majority of analyses were performed either by GC/MS or direct insertion of pure compound, as robust methods to combine the liquid eluent from HPLC with high
voltage ionisation sources were not widely available. Both structural elucidation and quantitation by mass spectrometry was technically challenging, time-consuming and considerably less sensitive than it is today. The revolution came with the development of commercially available atmospheric pressure ionisation techniques such as thermospray and then electrospray. These ionisation sources enabled hyphenation between liquid chromatography and mass spectrometry, the early development of which can be followed in the work of Dai Games, Andries Bruines and Jack Henion among others. These were highly sensitive and robust. Think of where we would be today without LC-MS!
MALDI also emerged in the mid-80s and biological mass spectrometry really erupted. In 2002 a share of the Nobel Prize in Chemistry
techniques enabled British mass spectrometrists to really push the boundaries of science forwards. Pioneering work by Dame Carol Robinson took mass spectrometry into the world of structural biology. This work would not have been possible without the concurrent development of mass analysers, in particular quadrupole time-offlight with orthogonal acceleration. Micromass (now Waters) set a commercial bench mark with these instruments which were able to offer so many of the benefits of ToF, such as mass range, sensitivity, speed etc to the continual beam of ions from an electrospray source. Almost a decade later, and also in the Manchester area, a company called HD Technologies started work on what would eventually become the Orbitrap.
In addition, the instrumentation software is now sophisticated
spectrometry is now user-friendly and generally robust, putting it into the hands of scientists from many fields to use as a tool in their research. This is reflected in the BMSS membership. We are no longer a society of mass spectrometry purists, seen roaming the corridors with a voltmeter in one hand and a spanner in the other. Our members are now drawn from a variety of backgrounds, but all have the common desire to understand how best to apply mass spectrometry to support their own field.
All these developments, and so many more, can be tracked in the history of BMSS through the Special Interest Groups (SIGs). In 1992 the first SIGs were formed from subsets of the British MS community to support five areas: LC-MS, quality issues, networking & computer systems, new users and MS/MS. The LC-MS SIG ran very successfully for about 16 years before morphing into the quantitative (LC) MS group to meet current needs, and was central to the first BMSS training course.
More recently, in the last 8 years or so, there has been a huge explosion in the area of atmospheric pressure ionisation techniques as the need for direct analysis and
this. That same year two other SIGs were also founded, one to support ion mobility mass spectrometry and one to support MALDI and imaging, Both groups have held very successful meetings since their birth. This brings the current set of active SIGs to 7:
• Environmental and food analysis
• Protein Analytics
• MALDI and imaging
• Quantitative MS
• Clinical and forensic
• Ambient ionisation
• Ion mobility
Part of the BMSS’s role is to celebrate and encourage excellence in the field. The Aston Medal was established by the British Mass Spectrometry Society in 1987 as the Society's prestigious scientific award, to be given to individuals deserving special recognition by reason of their outstanding contributions to knowledge in the biological, chemical, engineering, mathematical, medical, or physical sciences relating directly to mass spectrometry. To date, 12 awards have been made to individuals who have shaped mass spectrometry globally, starting with Allan Maccoll in 1989 and most recently Tony Stace in 2013.
established. This very occasional award was established to recognise sustained contributions by individual members of the British Mass Spectrometry Society: product champions of mass spectrometry, for developing student and individual education, increasing awareness of mass spectrometry and its utility in science through long-term contributions to the BMSS and/or scientific literature and the representation of mass spectrometry within the wider scientific community. To date only four awards have been made:
Professor Edward Houghton 27th Annual Conference, Derby 2004
Professor Tony Mallet 29th Annual Conference, Heriot-Watt 2007
Professor John Monaghan 32nd Annual Conference, Cardiff 2011
Professor Frank Pullen 35th Annual Conference, Alderley Park 2014
In fact, for much of the last 25 years, BMSS has focussed a lot of effort on MS education. There have
travel grants (over £150,000 travel grants awarded since 2003), training courses and more recently, three BMSS lecturers have taken to the touring circuit (Professors Malcolm Clench, Frank Pullen and Mike Morris).
So what is next for BMSS? There will be continued support for education in mass spectrometry and related topics, especially as BMSS develops its links with fellow societies in areas such as separation science, proteomics and other spectroscopies. As the breadth of MS-based applications continues to grow, so the number of users new to this field of science, and its instrumentation, grows. The BMSS will reach out to new MS-users in these disciplines through continued development in communication, from the latest format of the BMSS newsletter, Mass Matters, through public meetings such as Scibar and the BMSS website. The annual meeting and the SIG meetings will continue to pull the community together to disseminate the latest research and developments with meetings formats and training courses tailored to meet the current needs as BMSS continues to help shape the future of mass spectrometry in the UK.
Curt Brunnée Award Winner, Dr Dimitris Papanastasiou
Dr Dimitris Papanastasiou, a BMSS member and recent Curt Brunnée Award winner, started his differential mobility spectrometry (DMS) adventure in 2006 during a postdoc in New Mexico University. The journey continued via Manchester (Shimadzu Research Laboratories) all the way to Athens, Greece (Fasmatech Science and Technology) and involved deep exploration of vacuum gas dynamics, which in its own right unfolded new directions in mass spectrometry instrumentation design and development. New tools and methods were explored and applied for the first time to understand the properties of supersonic and highly diffusive gas flows in intermediate vacuum regions (e.g. particle tracking velocimetry with novel tracer particles and optimisation of computational simulation methods). The gasdynamics informed DMS work has produced a novel differential ion mobility platform operated in
the fore vacuum region of mass spectrometers equipped with atmospheric pressure ionization sources. Results were first reported at the 2013 ASMS conference in Minneapolis. The significance of these new approaches was recognized by the International Mass Spectrometry Foundation (IMSF) and the presentation of the 2014 Curt Brunnée Award to Dr. Dimitris Papanastasiou for his contributions to the development of the theoretical understanding of ion behaviour and implementation of this knowledge in new devices for ion optics and ion mobility. Dr. Papanastasiou is R&D Director of Fasmatech Science & Technology, a high tech start-up where most of the vacuum gas dynamics and DMS work has been carried out. Dr. Papanastasiou co-founded Fasmatech in 2010 together with Dr. Emmanuel Raptakis, also a BMSS member and Warwick-Manchester alumnus.
Emmanuel Raptakis
Dimitris receiving the Curt Brunnée award from Professor Cathy Costello (Boston University) and Dr. Ken Miller (Thermo Scientific) at the 2014 International Mass Spectrometry Conference in Geneva.
Call for Research Support Grants
Members (of at least 12 months standing) may apply for small grants of up to £2,000, with matched funding, to aid their research.
The BMSS Research Support Grant aims to support small research endeavours including, but not exclusive to:
• Generating pump priming data for grant applications or research areas
• Incoming or outgoing visits to initiate new MS-relevant collaborations/ training (standard class travel, budget accommodation)
• MS taster visits for new MS users (who must join the society)
• Instrumentation (updates, repairs, add-ons, new developments of existing kit, etc.)
• Promotion of industry-academic collaborations.
Full details of eligibility, how to apply and conditions for awarding grants are available on the BMSS website www.bmss.org.uk
International Mass Spectrometry Conference
24th -29th August 2014
Rebecca Burnley
The 20th International Mass Spectrometry Conference was held in August in Geneva, Switzerland, at the CICG congress centre. The CICG is a short walk or tram-ride away from the city centre, located near the Palais des Nations, the UN office. Geneva itself is a diverse city with an interesting mix of styles, the bridge across the stunning Lake Geneva separating the old town from the more modern districts. The lake is one of the defining sights of this city, with the enormous Jet d’Eau, one of the largest fountains in the world, shooting water some
Over 1600 delegates attended the conference, representing countries around the world. Following short courses and tutorial lectures, the main event kicked off on the Sunday afternoon, with an opening ceremony hosted by the conference chair, Renato Zenobi, showcasing traditional Swiss music with some very zealous performances. An interesting plenary from Jules Hoffmann about innate immunity followed, before the welcome mixer, providing the opportunity to meet up with old and new colleagues, associates and friends.
A packed agenda saw 5 parallel
afternoon, Monday to Friday, sandwiching mid-day poster sessions and lunch symposia. The oral sessions I attended included Instrumentation, Biomarkers and Diagnostics, Top-Down Proteomics, Non-Covalent Interactions and Ion Mobility MS, but the topics covered a very broad range of interests. The long lunch breaks allowed plenty of time for networking and exploring that day’s selection of posters. The poster hall was a little cosy, but this in no way precluded the usual healthy debate and vigorous discussion, amongst all different delegates, from student to director to professor. The symposia gave the
and discuss new developments in their instrumentation and software products… in exchange for lunch. A variety of workshops followed the afternoon lecture sessions; on Wednesday, BMSS and Tony Bristow presented a workshop on Careers in MS. This included varied and inspiring talks from Perdita Barran and Alexander Makarov, giving great insight into not only the different pathways possible in this field, but also providing food for thought about other factors influencing both our jobs and the progression of science.
Throughout the week, plenary lectures from world experts, both within and outside of the field of mass spectrometry, were extremely well-received. These additional sessions also included the presentation of prestigious awards: Carol Robinson (Oxford) and Renato Zenobi (Zurich) received the Thomson Award; Yury Tsybin (Lausanne) received the SGMS Young Investigator Award; Dimitris Papanastasiou (Athens) received the Curt Brunée Award (see the feature article within this issue); and the Journal of Mass Spectrometry award for postgraduate students was jointly awarded to Kathirvel Alagesan (Berlin), Denis Chernyshev (Moscow), Stamatious Giannoukos (Liverpool), Jozef Lengyel (Prague) and Michael Wleklinski (Lafayette).
A gala dinner was held on the Thursday evening at the impressive Bâtiment des Forces Motrices on the banks of the Rhône, and the meeting drew to a close the following lunchtime with a farewell cocktail session. This busy and rewarding week of sharing science, and accumulating new knowledge and ideas, was over. Next stop,
Breath Analysis 2014
The authors of these reports all wish to express their thanks to the BMSS for the award of a travel grant to allow them to attend these conferences. ,
6-9th July, Torun, Poland
Liam Heaney, Loughborough University
This was the second year that I had been fortunate enough to visit the Breath Analysis Conference. A second visit allowed me to build on professional relationships that had be founded in 2013 and allowed a more confident approach to discussing current works and possible future ideas. The meeting was held in Toru , a small city in the northern region of Poland. The city had many buildings with inspiring architecture and was situated on the banks of the river Wisła. Travel to the city was not the easiest of tasks on this occasion, a flight to Warsaw followed by a slow, cramped and extremely hot train for 3 hours was required. Nevertheless, once we had arrived at our destination, the rest of the time in Poland was extremely pleasant. Food and drink was very reasonably priced, with many visits
was a most rewarding trip, both in an academic and a personal interest sense. My presentation at this conference is outlined below.
Swimming is often recommended to people with respiratory complaints; exercising in a warm, humid environment prevents airway drying, reducing the likelihood of an exacerbation. However, it has been postulated that the inhalation, ingestion and absorption of disinfection by-products (DBP), may damage the respiratory system of regular swimming pool attendees. The answer to whether swimming is good or bad for your lungs is of interest, especially to those swimmers who make up the largest body of recreational exercisers in the UK. With approximately 3 million individuals attending a swimming pool for at least 30 minutes per week, the impact of any aspect detrimental to health will be widespread. The experiment aimed
30 min self-paced swim of moderate to hard intensity. Exhaled breath samples (2 L) were collected prior to, and at 5, 90, 300, 510 and 600 mins post swim. An adaptive distal breath sampling method was used where participants breathed normally through a full face mask supplied with filtered air, while samples were collected onto a dualbed thermal desorption tube and subsequently analysed by thermal desorption (Unity, Markes Int. UK)gas chromatography (3800GC, Varian, USA)-quadrupole ion trap mass spectrometry (4000MS, Varian, USA). Sample tubes were post-loaded with 20 ng toluene-D8 internal standard and analysed by two-stage thermal desorption with cryogenic enrichment using a chromatography gradient temperature program from 40 to 300°C at 5°C min-1. At 5 minutes post-swimming, exhaled chloroform levels were measured to average 14ng L-1, an increase by a factor of
reported. Two washout profiles were observed, with 3 participants displaying peak intensity at 90 mins and all other individuals showing peak intensity at 5 mins. Exhaled bromodichloromethane levels increased by a factor of 17.5 (p<0.05) and remained above preswimming values for up to 300 mins (p<0.05), all participants produced a similar wash out profile of peak intensity seen at 5 mins. Previous experiments involved single breath sampling techniques; however larger volume, multiple breath samples increased sensitivity and reduced breath-to-breath errors. Participants were infrequent recreational swimmers and the DBP exposure was a transient result. Further studies are planned with swimming athletes who are subject to chronic long term exposure to DBP. Although this experiment cannot determine whether the inhaled DBP are causative of respiratory conditions, if further research could
ASMS Conference
Baltimore, 15-19th June 2014
Sophie Thurlow, University of Edinburgh
It was a pleasure to attend the “50 Years of the British Mass Spectrometry Society” workshop to meet many British mass spectrometrists and to hear about ongoing research. My presentation ‘Investigating Redox Regulation in the Apoptotic Pathway using High Resolution Mass Spectrometry’ is detailed below.
Introduction
The redox environment of our cells is maintained in a tightly regulated homeostasis as the chemical reactions involving the aberrant transfer of electrons can have severely detrimental effects on biological molecules. Proteins are sensitive to redox reactions through the valence electrons of sulphur atoms contained in cysteine residues. Although it has been observed for many years that redox potential correlates with a number of cellular processes such as proliferation or death, it has recently been hypothesised that the proteins of the apoptotic pathway are arranged in order of an electrochemical cascade [1]. We have been investigating the redox regulation of a key component of the apoptotic (cell suicide) pathway, caspase-3. Both the intrinsic and extrinsic apoptotic pathways converge on caspase-3, the activation of which is considered a point of no return in this cell death pathway. Once activated, as an executioner caspase, the enzyme cleaves a number of protein substrates in the cell using the active site catalytic cysteine as a nucleophile in the hydrolysis of the peptide amide backbone after aspartic acid residues. Activity of the protein has previously been shown to be pH and oxidation sensitive [2] [3].
Methods
Recombinant human caspase-3 was expressed in Escherichia coli with a C-terminal 6-His tag to facilitate HisTrap purification. To investigate the protein under different redox environments, the protein was incubated in dithiothreitol (DTT) or glutathione
(GSH) and glutathione disulfide (GSSG). Because the glutathione solutions were of known concentration and buffered to pH 7.0 at 25 ˚C, and the glutathione redox couple has a known cell potential at standard biochemical conditions (Eθcell) of -240 mV, the Nernst equation can be used to calculate the redox potential (Ecell) of the glutathione solution. To determine which cysteine residues in the protein were redoxmodified, after equilibration for 2 hours, the protein was isolated from the solution using a TCA precipitation. The protein was resuspended in 6M Gu-HCl, 0.1 M sodium phosphate pH 7.0, 1 mM N-ethylmaleimide and incubated in the dark at 25 ˚C for 30 minutes to allow for alkylation of the reduced cysteine residues. The remaining cysteine residues were reduced by addition of 10 mM DTT for 30 minutes which also quenched the excess N-ethylmaleimide. Subsequent analysis was performed by online liquid chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry using a monolithic PS-DVB reverse phase analytical column and a solariX FTICR equipped with a 12 T magnet (Bruker Daltonics).
Results and Discussion
The recombinant caspase-3 was analysed by LC-MS which confirmed that during expression self-catalysed cleavage of the protein occurred after positions Asp28 and Asp175 resulting in the formation of the p17 and p12 subunits.
Figure 1 shows a typical chromatogram of the recombinant protein illustrating that the two subunits are separated by reverse phase LC. It also shows that two proteoforms of the p17 subunit, differing in mass by approximately 600 Da, are observed. Through analysis of the intact mass of the smaller species (p17trunk) and application of top-down fragmentation techniques, it has been possible to determine that the truncation involves the loss of six amino acids from the C-terminus of the p17 subunit. This is observed as an increase in the number of y and z ions in the fragmentation maps shown in Figure 2. The
“Figure 1: The total ion chromatogram for the reverse phase LC-MS of the recombinant caspase-3, the mass-to-charge spectra for each subunit, the deconvoluted spectra and the expected mass for each subunit, p12 (blue circles) & p17 (red circles).
Figure 2: Maps illustrating the fragments observed in the CID and ECD spectra in the top-down analysis of the full length and truncated proteoforms of the p17 subunit.
Figure 3: Schematic of the recombinant caspase-3 protein showing the sites of self-catalysed cleavage. Cysteine residues are highlighted in red with the active site in dark red.
Figure 4: The deconvoluted mass of the equilibrated p12 subunit under reducing (red) and oxidising (blue) conditions and the expected mass of the fully reduced polypeptide sequence (•). The insets show the mass-to-charge spectra.
TRAVEL GRANT REPORTS
fragmentation site is after Asp169 agreeing with the P4 site specificity for substrates of caspase-3 and thus may be a result of the catalytic activity of the protein.
To investigate the redox sensitivity of the cysteine residues to redox environment, after equilibration in reducing agent (DTT) or under oxidising conditions (-51 mV), caspase-3 was analysed by reverse phase LC-FTICR-MS. The mass-tocharge spectra for the p12 subunit elution peaks for each condition were deconvoluted and the intact masses investigated. For the p12 subunit shown in Figure 4, the intact mass of the polypeptide matched the expected mass of the fully reduced species under both reducing and oxidising conditions and thus, as the polypeptide sequence contains three cysteine residues, it would be expected that after the alkylation step, the addition of three N-ethylmaleimide moieties would be observed (+375 Da).
Figure 5 shows that when the protein is equilibrated in reducing conditions, the p12 subunit does indeed label with three N-ethylmaleimide moieties but when the protein has been equilibrated in oxidising conditions, two differently labelled populations are observed. The first is also labelled with three N-ethylmaleimide moieties suggesting that a proportion of the population was fully reduced initially, however the second population has only been alkylated with two N-ethylmaleimide moieties suggesting only two cysteines were reduced. This implies a single cysteine residue on the polypeptide chain was oxidised after the equilibration step and on closer inspection of the mass-to-charge spectrum shown in Figure 4 it is observed that two p12 subunits are covalently associated by an intermolecular disulphide bond. To localise the cysteine residues involved in the disulfide bond formation, CID and ECD were employed to fragment the p12 subunit labelled with two N-ethylmaleimide moieties i.e. the unlabelled residue was the residue inhibited from alkylation by involvement in the disulfide bond.
Figure 6 shows sample spectra achieved when applying CID and ECD fragmentation techniques to the p12 subunit alkylated with two N-ethylmaleimide moieties. Figure
7 combines the fragments observed in the CID and ECD experiments and illustrates that the cysteine not labelled with N-ethylmaleimide is Cys184 at the N-terminus of the p12 subunit. Although it can be determined from the fragmentation of the alkylated protein and intact mass analysis of the equilibrated protein that the p12 subunit forms an intermolecular disulfide bond, it is has not been possible to determine whether the two p12 subunits come from the same tetramer or from two different tetramers.
Conclusions
Recombinant caspase-3, expressed in E. coli, self-catalyses its cleavage into the active p17-p12 heterodimer. The p12 subunit is sensitive to oxidation through formation of an intermolecular disulfide bond between the Cys184 residues at the N-terminus of two p12 subunits.
References
1 V. Mallikarjun, D. J. Clarke and C. J. Campbell, “Cellular redox potential and the biomolecular electrochemical series: A systems hypothesis,” Free Radical Biology and Medicine, vol. 53, pp. 280-288, 2012.
2 H. Stennicke and G. Salvesen, “Biochemical characteristics of caspases-3, -6, -7, and -8,” The Journal of Biological Chemistry, vol. 272, pp. 25719-25723, 1997.
3 Z. Huang, J. T. Pinto, H. Deng and J. P. Richie Jr, “Inhibition of caspase-3 activity and activation by protein glutathionylation,” Biochemical Pharmacology, vol. 75, pp. 2234-2244, 2008.
Figure 5: The deconvoluted mass of the alkylated and reduced p12 subunit after equilibration in reducing (red) and oxidising (blue) conditions.
Figure 6: Annotated CID & ECD for the fragmentation of the p12 subunit alkylated with two cysteine residues.
Figure 7: Map illustrating the fragments observed in the CID and ECD spectra in the top-down analysis of the p12 subunit alkylated with two N-ethylmaleimide moieties (red).
ASMS Conference
Baltimore, 15-19th June 2014
Elizabeth Randall, University of Birmingham
As my first large-scale conference outside the UK, ASMS was a great opportunity for me to appreciate the scale of the mass spectrometry community. It was fantastic to be able to present my own work in such a setting, and have the opportunity to receive feedback from so many people and such a wide range of expertise. It was very encouraging to hear positive comments from people who have been working in the field for a lot longer than me. One particularly inspiring aspect of the conference was attending presentations about clinical mass spectrometry which has impacted on patient care and ultimately prognosis. In particular Todd Hembrough discussed the clinical applications of mass spectrometry with respect to personalised oncology patient care. Their work has been demonstrated to more accurately predict patient response to treatment and has already impacted positively on prognosis. Within my own field it was interesting to meet other people experiencing the same problems and obstacles and was helpful to see the solutions they had come up with.
The work I presented at this year’s ASMS described a method using liquid extraction surface analysis for the detection and identification of intact bacterial proteins.
The chemical analysis of bacteria by mass spectrometry was first reported nearly 40 years ago; when different bacterial species were identified through pyrolysis mass spectrometry.(1) Since then methods have been detailed for the robust identification and differentiation of bacterial species, generally via MALDI-TOF mass spectrometry. Applications of bacterial analysis have not been limited to identification however, with applications ranging from characterisation of microbial processes to the search for novel natural products.(2, 3) For example, the development of bacterial antibiotic resistance has motivated this field of research, and liquid extraction surface analysis (LESA)
LESA involves using a droplet of an appropriate solvent (which can be optimised for certain analytes) to extract soluble analytes from a predefined region on a sample surface. The droplet is held in contact with the surface; the interface between the solvent and surface is termed the liquid micro-junction and is maintained for optimum extraction of analytes. The solution can then be aspirated and injected into the mass spectrometer via an electrospray ion source. This is a highly sensitive mass spectrometric technique and has the potential for quantitative analysis by incorporation of calibration standards into the extraction solvent. LESA in conjunction with electrospray mass spectrometry is particularly useful for the analysis of proteins; reported applications of this include the extraction of proteins from thin tissue samples and dried blood spots.(5, 6)
to directly sample surfaces at atmospheric pressure is particularly useful in the analysis of bacteria because ‘on-site’ sampling and analysis could be used for clinical diagnostics or environmental monitoring.
The analysis of intact proteins and identification via top-down proteomics presents several advantages over the more established bottom-up methods for protein identification, in which proteins are digested into constituent peptides before entering the mass spectrometer. Top-down methods involve detecting the intact protein ion before selecting it for tandem mass spectrometry fragmentation; this somewhat overcomes issues such as information loss, difficulties matching peptide masses and loss of post-translational modifications, commonly encountered with bottom-up methods. Bottom-up
protein identification involves enzymatic digestion of complex mixtures of proteins before they
The mixture of peptides can then database searching methods such as mascot can be used to identify
The poster we presented at ASMS involved the detection of intact proteins from bacteria and their top-down methods and database
bacterial proteins and could deduce bacterial proteomic response to
Anhalt JP & Fenselau C (1975) Identification of bacteria using mass Analytical Chemistry
Watrous JD & Dorrestein PC (2011) Imaging mass spectrometry
Watrous J, Hendricks N, Meehan M, & Dorrestein PC (2010) Capturing bacterial metabolic exchange using thin film desorption electrospray ionization-imaging mass spectrometry. Analytical Chemistry 82(5):1598-1600.
4. Kai M, González I, Genilloud O, Singh SB, & Svatoš A (2012) Direct mass spectrometric screening of antibiotics from bacterial surfaces using liquid extraction surface analysis. Rapid Communications in Mass Spectrometry 26(20):24772482.
5. Schey KL, Anderson DM, & Rose KL (2013) Spatially-directed protein identification from tissue sections by top-down LC-MS/MS with electron transfer dissociation. Analytical Chemistry 85(14):6767-6774.
6. Edwards R, Griffiths P, Bunch J, & Cooper H (2012) Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants. J. Am. Soc. Mass Spectrom. 23(11):1921-1930.
BMSS Annual Meeting AstraZeneca Conference Facility
Alderley Edge, 1st-2nd April 2014
Ammar Nasif, University of Southampton
This was the first BMSS conference I have attended, and had the chance to meet interesting people in the field of mass spectrometry and gain their insight to where mass spectrometry could be applied. My PhD project is about characterising and quantifying polar molecules in crude oils using hyphenated mass spectrometric techniques.
The content of the conference was very diverse from protein analysis using the new Synapt G2-S high definition MS, where ion mobility spectrometry is introduced to separate proteins of the same m/z but with different shapes, to the use of MS in space “the Rosetta challenge” where isotope ratios are measured tracing the age and origin of compounds. The use of an electrospray source for monitoring chemical reactions accelerated in microdroplets was demonstrated where the chemical reaction occurs within the droplet. Mass spectrometry imaging was one of the key highlights of the conference as well, where the technology has been employed in brain surgery to differentiate between malignant and healthy tissues.
The focus of my report here is ambient ionisation, in particular Graham Cooks work on paper spray and leaf spray. Analysing drugs from a blood spot by paper spray is analogous to analysing polar compounds in crude oil as both matrices, blood and crude oil, are considered to be complex. Thus the application of this technology greatly reduces analysis time. Furthermore, tissue analysis in real-time will be discussed, where the concept Iknife was introduced by Emrys Jones and IEndoscope by Juzheng Huang.
The invention of ambient ionisation methods allows analysis without sample preparation steps, thus analysing the sample in its native state. The ambient ionisation methods include spray-based (S),
plasma-based (P), laser assisted methods (L) and heat-based (T). 50 different ambient ionisation techniques have been invented since 2004 and 5 have been commercialised. Table 1 lists some of them.
The first ionisation technique to be introduced was DESI. All spraybased methods have a common mechanism, which is directing a stream of ion-desorbing fluid onto a sample surface from which analyte ions are withdrawn and transported to the mass analyser via a standard API interface. Surface materials such as waxes, alkaloids, pesticides, explosives and drugs of abuse from luggage or banknotes can be analysed without
Ambient ionisation
sample preparation or sample pretreatment. Desorption atmospheric pressure photoionisation (DAPPI) was developed for low-polarity compounds. DAPPI represents the adaptation of atmospheric pressure photoionisation (APPI) for the ambient analysis of surfaces. A microchip nebuliser produces a heated jet of solvent vapour and nebuliser gas, which desorb the solid analytes from the surface. A krypton lamp produces photons of 10eV, which ionise the analytes after they are thermally desorbed from the surface (see figure 1). However analytes with no UV chromophore can be ionised via ion-molecule reactions with dopant ions such as toluene. The dopant is added to the microchip nebuliser solvent
desorption electrospray ionisation DESI S easy ambient sonic-spray ionisation EASI S desorption ionisation by charge exchange
flow. The ionisation of molecules is through a charge exchange reaction where the dopant ion receives an electron from the analyte molecule producing a radical cation.
GC-MS/MS and LC-MS/MS show excellent analytical performance but both require time-consuming steps for sample preparation prior to the instrumental analysis step. Paper spray (PS-MS) is an ambient ionisation technique where the sample is spotted on a piece of paper, a porous material, where the high voltage is applied (see Figure 2). Therapeutic drugs such as citalopram and verapamil were analysed in 60 seconds and detected at 1 ng/mL. These drugs were quantified from a blood sample with RSD of 10%, thus ambient ionisation technique such as PS can be used for quantitative analysis.2
S transmission mode desorption electrospray ionisation
S
nanospray-desorption/electrospray ionisation nanoDESI S probe electrospray ionisation PESI S liquid micro junction-surface sampling probe LMI-SSP S paper spray
PS S direct analysis in real-time DART P low-temperature plasma probe
flowing atmospheric pressure afterglow
LTP P
P desorption atmospheric pressure chemical ionisation
P
desorption corona beam ionisation DCBI P
dielectric barrier discharge ionisation
electrospray-assisted laser desorption ionisation
P
L, S laser ablation electrospray ionisation
L, S laser desorption electrospray ionisation LDESI L, S laser diode thermal desorption LDTD L, P
laser-induced acoustic desorption/electrospray ionisation
L, S
laser electrospray mass spectrometry LEMS L, S atmospheric pressure thermal desorption ionisation APTDI T
Table 1. Different ambient ionisation techniques.
Leaf spray (LS), a variant of paper spray but the difference is that LS uses the sample itself (e.g. plant tissue) to generate gas phase ions for the mass spectrometer instead of ionising the sample loaded onto a paper substrate. Malaj et al.4 have analysed pesticides such as thiabendazole, diphenylamine and imazalil directly from plant tissue (“leaf spray”). It was demonstrated that the simple and rapid leaf spray ionisation method (analysis time is about 100 seconds) is appropriate for fast discrimination between organic and non-organic fruit and vegetables.
Rapid evaporative ionisation mass spectrometry (REIMS) for real-time identification is used to obtain a biochemical fingerprint by introducing the evaporate of a solid or liquid sample to the atmospheric inlet of a mass spectrometer. Instantaneous sampling is obtained, making it ideal for real-time applications. As there is no separation, the dominant signal from complex mixtures will be the most abundant, typically phospholipids in tissue analysis. The main application of REIMS is in tumour resection and ensures that the entire tumour is removed and minimises the removal of healthy
tissue by informing the surgeon in real-time. Figure 3 explains tissue identification with REIMS in surgical procedures, called Iknife. Mechanisms of REIMS in tissue analysis involve the use of smoke generated by commonly used electrosurgical knives. Patients are fitted with a return electrode. Joule heating occurs due to the
non-zero impedance of the tissue. Macroscopic aerosolation of the tissue leads to disruption of its structural integrity. Evaporated materials are drawn up past the blade into PTFE tubing that is attached to the mass spectrometer. Ionisation occurs within the droplets with charge segregation occurring similar to the sonic spray mechanism
TRAVEL GRANT REPORTS
as the droplets evaporate on the way to the atmospheric pressure inlet. Gaseous ions are introduced to the mass spectrometer for high resolution and high mass accuracy analysis. For the technique to work, a database of all expected tissues types from a large cohort of patients for a given surgical procedure is required for each application.5
Another interesting new concept for REIMS is the IEndoscope, which employs mass spectrometry for the analysis of upper gastro-intestinal tissue during endoscopic procedures. Aerosols come from the endoscopic resection through the sampling line to the T-piece where they can enter the mass spectrometer coupled to an atmospheric with a heated collision surface or to the endoscope stack.
References
1 http://commons.wikimedia.org/ wiki/File:DAPPI.jpg
2 Manicke, N. E.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R. G. Assessment of paper spray ionization for quantitation of pharmaceuticals in blood spots. International Journal of Mass Spectrometry 2011, 300, 123-129.
3 Wang, H.; Manicke, N. E.; Yang, Q.; Zheng, L.; Shi, R.; Cooks, R. G.; Ouyang, Z. Direct analysis of biological tissue by paper spray mass spectrometry. Analytical chemistry 2011, 83, 1197-1201.
4 Malaj, N.; Ouyang, Z.; Sindona, G.; Cooks, R. G. Analysis of pesticide residues by leaf spray mass spectrometry. Analytical Methods 2012, 4, 1913-1919.
5 Balog, J.; Sasi-Szabó, L.; Kinross, J.; Lewis, M. R.; Muirhead, L. J.; Veselkov, K.; Mirnezami, R.; Dezső, B.; Damjanovich, L.; Darzi, A. Intraoperative tissue identification using rapid evaporative ionization mass spectrometry. Science translational medicine 2013, 5, 194ra193-194ra193.
Figure 1.Schematic of DAPPI source. Adapted1
Figure 2. A schematic of paper spray experiment. Adapted3
Figure 3. Schematic of iknife main components. Adapted5
SPEC 2014 ‘Shedding
New Light on Disease’
17-22 August 2014, Krakow, Poland
Joanna Denbigh Manchester Institute of Biotechnology, University of Manchester
Acute myeloid leukaemia (AML) is an aggressive cancer made up of dysfunctional cells from the myeloid lineage, whereby the bone marrow rapidly produces abnormal excess monocytes and granulocytes which accumulate in the bone marrow and hinder the production of healthy blood cells. If untreated, patients can die within weeks of diagnosis and unfortunately the response to treatment and overall survival of patients post-treatment generally remains poor [1]. Current treatment includes high grade chemotherapy, which has the disadvantage of being very toxic and thus poorly tolerated in a major cohort of elderly AML patients. Relapse is very common and in older patients, more than three quarters experience relapses after complete remission. There is thus an urgent clinical need for novel therapeutic approaches for the treatment of both primary AML and for the elimination of residual leukaemia in remission.
Drug redeployment represents an alternative to chemotherapy and involves the use of existing drugs for situations they were not originally designed for. It has recently been shown that the combination of
the cholesterol lowering drug, bezafibrate, and the female contraceptive, medroxyprogesterone acetate (combination denoted BaP) shows anti-leukaemic activity without haematological toxicity in patients [2].
Fatty acids play an important role in many biochemical pathways. Synthesis of fatty acids endogenously is known as de novo lipogenesis and traditionally serves the purpose of converting excess carbohydrates into lipids for storage. This is highly active in foetal lungs to produce surfactant and in lactating adults to produce breast milk, but otherwise de novo lipogenesis should be low in healthy circumstances. Lipid biosynthesis has, however, been the focal point of much research for many years primarily because of the large numbers of reports that link over-expression of several enzymes involved in de novo lipid biosynthesis with a variety of human tumours [3].
The analysis of lipids is thus a powerful tool for understanding AML and the effect of drugs on AML cell lines and is the focus of this multi-dimensional project in which Raman microspectroscopy, Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS) and Liquid Chromatography-Mass
Spectrometry (LC-MS) have been employed to probe AML cellular lipid content. A summary of data obtained from this project to date were presented during a dedicated poster session at SPEC 2014. Lipidomic studies of BaP treatment in HL60 and K562 AML cell lines have revealed an increase in saturation of diacylglycerols, in particular a relative increase of DAG (36:0) with a corresponding decrease of DAG (36:2) which suggests more palmitic acid (C16:0) present in BaP treated cells with less conversion of palmitic acid to stearic and subsequently to oleic acid (C18:1). Figure 1 shows ToFSIMS data, specifically, average normalised peak areas for intact diacylglycerol chains illustrating the aforementioned change in saturation with BaP treatment. UHPLC-MS analysis was carried out on an Accela UHPLC coupled to an electrospray LTQ-Orbitrap XL hybrid mass spectrometry system (ThermoFisher, Bremen, Germany). Analysis was carried out in both positive and negative electrospray ionisation (ESI) modes, with all extracts analysed in a random order to eliminate any bias and data processing was performed using in-house peak deconvolution software containing the XCMS algorithm for peak picking and the TAVERNA work flow for peak identification. Principle component
analysis (PCA) was performed to reduce the dimensionality of the LC-MS data whilst maintaining intersample variance. Figure 2 shows a PCA scores plot for chloroform extracts run in negative (a.) and positive (b.) ESI, separated according to class (control (C) vs BaP (B)). Separation according to BaP drug treatment can be clearly seen along PC2, with clear grouping observed. Raman spectra and images were obtained with a Renishaw inVia Raman microscope, using a 785 nm excitation beam, ~30 mW at the sample, 50 x objective, exposure time of 10 s for point spectra and 10 s for mapping. Mapping was carried out in high confocal mode. Figures 3 and 4 show representative raman spectra and images respectively. Raman images show localisation of regions of interest for extracted lipid bands in control and BaP treated cells, with inter-cellular differences apparent.
References:
1 Peloquin G, Chen YB, Fathi AT, The evolving landscape in the therapy of acute myeloid leukaemia. Protein & Cell, 2013, 4(10): p. 735746.
2 Murray JA, Khanim FL, Hayden RE, Craddock CF, Holyoake TL, Jackson N, Lumley M, Bunce CM, Dryson MT, Combined bezafibrate
Figure
and medroxyprogesterone acetate have efficacy without haematological toxicity in elderly and relapsed acute myeloid leukaemia (AML). British Journal of Haematology, 2010, 149(1): p. 65-69.
3 Abramson HN, The Lipogenesis Pathway as a Cancer Target. Journal of Medicinal Chemistry, 2011, 54(16): p. 5615-5638.
I was very much looking forward to attending my first dedicated spectroscopy conference, having presented my research at a larger analytical science conference and at BMSS in the past. Since my project has a multidisciplinary focus I was keen to present the benefits of using both mass spectrometry techniques and spectroscopy techniques to complement each other and SPEC 2014 was an ideal forum for this. The conference was a full week of learning about new clinical and medical spectroscopy applications, organised networking activities and making new friends. Krakow was a perfect location for this, being a beautiful historical city with everything within walking distance. From the first evening on which the conference organisers greeted us with a warm welcome in the City Hall to the final night’s Gala Dinner at a breath-taking Polish Castle (with traditional Polish dancing to be
enjoyed by all), SPEC was an actionpacked week. Plenary speakers imparted a wealth of information and of particular enjoyment to me were the discussions before lunch each day. These gave a platform for participants to engage in topical conversations and sometimes debates on the more specific problems encountered in this field and how the future would look moving forward in an ideal world and what we could do to shape that. From this I brought back a number of ideas to implement in the final year of my project. I thoroughly enjoyed the busy poster session in which I received lots of useful feedback and during which wheels were set in motion for a potential new international collaboration next year. I would like to thank BMSS for providing me with a travel grant to attend this conference which I very much enjoyed.
Figure 3. Representative processed Raman spectra for control and BaP treated cell and whole HL60 cell extracted images for lipid region of interest. Rainbow colour mapping shows regions of high intensity in red, orange and yellow, decreasing to lower intensity regions displayed in green, blue and violet with box and whisker plots representing intensity of colour shading.
TRAVEL GRANT REPORTS
IMSC 2014
24th-29th August, Geneva
Kirsty Skeene, University of York
The 20th international mass spectrometry conference was held in Geneva this year at the centre international de conférences Genève. I was able to present a poster entitled “Development of a mass spectrometric approach to study disorders of protein O-glycosylation”. The aim of the study is to develop and optimise a method to release and isolate O-glycans from glycoproteins from whole cultured cell lysates, with application to cultured cell models of congenital disorders of glycosylation (CDG).
Protein glycosylation, the most common post translational modification of proteins, involves attachment of one or more carbohydrate moieties to amino acid side chains, in N- or O-glycosylation. In N-glycosylation, a chain of monosaccharides (GlcNAc2Man9Glc3) is biosynthesised on a lipid carrier (dolichol) in the endoplasmic reticulum (ER). This lipid-linked precursor oligosaccharide is transferred to the amide nitrogen in asparagine’s side chain, when in the specific consensus sequence -Asn-X-Ser-, or -Asn-X-Thr- (where X is any amino acid except proline). Following transfer in the ER, the N-glycan undergoes trimming and migrates into the Golgi complex where modifications are made to the glycan by further trimming, or elongation whereby carbohydrate moieties are added to the glycan core by glycosyl transferase enzymes. In contrast, O-glycans are bound through the hydroxyl oxygen atom, typically to serine or threonine; there is no consensus sequence and no precursor oligosaccharide. Instead, monosaccharides are added stepwise to the growing glycan chain. Glycans coat all cell surfaces and are crucial for cellular development, interactions and processes. However, we have little understanding of the glycan biosynthetic code because, unlike protein and nucleic acid biosynthesis, there is no template for glycan biosynthesis. Glycan biosynthesis is managed by a large number of glycosyltransferase
enzymes (located in the ER and the Golgi) and is dependent on their expression levels, location and activity, each of which can change in response to environmental stimuli and cell differentiation, resulting in a range of possible glycan structures.
CDGs, a family of rare genetic disorders that are not well diagnosed, result from aberrant glycosylation. Glycosylation defects may be due to mutations in the enzymes building the glycan, or those enzymes not localising correctly in the ER or the Golgi complex of the cell; such mislocalisation makes it difficult to predict the structural consequences for glycosylation. CDGs can be separated into two main types. The first type typically presents normal N-glycans attached to glycoproteins but the sites of attachment are not fully utilized. This is caused by defects in the assembly of the precursor oligosaccharide and its transfer to the protein. The most common defect of this type (CDG1a) is in phosphomannomutase, the enzyme that catalyses isomerisation of mannose 6-phosphate to mannose 1-phosphate, required to build the lipid-linked precursor in N-glycosylation. A less common form (CDG-1b) results from defects in the phosphomannose isomerase gene, which generates mannose 6-phosphate from fructose 6-phosphate. Freeze et al1 reported a treatment for this defect, by administering high levels of mannose in the diet which was shown to be sufficient to restore normal levels of the dolichol donor, preventing hypoglycosylation. In CDG-2 almost all of the usual glycosylation sites are used but the glycans attached are truncated. This is a result of defects further along the glycosylation pathway during processing of the proteinbound glycans. CDGs, although rare, make up around 70 genetic disorders known to be caused by glycosylation-related mutations, and effects are expressed in most organ systems, particularly the central nervous system. The disorders present a vast array of abnormalities such as developmental delay in infancy and childhood, muscular dystrophy, strokes, seizures, immune
dysfunction and cardiac failure.
We are therefore studying glycan structures expressed on healthy cells in culture and those with a range of glycosylation defects, in order to understand these diseases better. Consequently, methods are needed to extract glycoproteins from cells in culture and to release and isolate their glycans for mass spectrometric analysis and glycan profiling.
Recently in the group a simple protocol, named Filter-Aided N-Glycan Separation, or FANGS2, has been developed to address this challenge, for release and isolation of N-glycans from glycoproteins in whole cell lysates using a centrifugal filter. The simple protocol makes use of the filter-aided sample preparation (FASP)3 approach for SDS removal and involves solubilisation of membrane proteins by boiling in SDS, followed by exchange of SDS for ammonium bicarbonate buffer in a centrifugal filter. Using the filter to remove the SDS is very important as although SDS is the most efficient way to extract soluble and membranebound proteins, it is a huge problem in MS protocols, and so being able to remove all the detergent is desirable. The glycoprotein extract is then incubated with PNGase F to release the N-glycans which are subsequently collected on centrifugation. My poster presented progress in developing a FANGScompatible approach for O-glycans. We have now developed a simple and efficient protocol for release and isolation of O-glycans directly in a filter unit, using our established non-reductive β-elimination method4 which involves treatment with ammonium hydroxide. The method has been demonstrated using standard glycoproteins, fetuin and mucins, down to 2 pmol. We are currently working on applying our O-glycan release method to cultured whole cell lysates, ready for application to models of CDGs.
References:
1. K. Panneerselvam, H. H. Freeze, J. Clin. Invest., 1996, 97, 1478-1487
2. S. A. Rahman, E. Bergström, C. J. Watson, K. M. Wilson, D. A. Ashford, J. R. Thomas, D. Ungar, J. E. Thomas-
Oates, J. Proteome. Res., 2014, 13, 1167-1176
3. J. R. Wisniewski, A. Zougman, N. Nagaraj, M. Mann, Nat. Meth., 2009, 6, 359-363
4. J. G. Rademaker, S. A. Pergantis, L. Blok-Tip, J. I. Langridge, A. Kleen, J. E. Thomas-Oates, Anal. Biochem., 1998, 257, 149-160
BMSS SUMMER STUDENTSHIP
Proteomic analysis of the medicinal plant Echinacea purpurea (Purple Coneflower) – Towards elucidating the biosynthetic pathways of its natural products
Ryan Joshua Williamson, University of Reading
I completed a nine week summer project within the research group of Professor Rainer Cramer in the Department of Chemistry, University of Reading. The aim of the project was to undertake a large-scale proteomic analysis of the medicinal plant Echinacea purpurea, gaining experience in current procedures of protein extraction and digestion, and operation of modern analytical instrumentation.
At the start of the placement, thirty E. purpurea plants had germinated and grown to a height of approximately 7 cm. Each of these plants was transferred to fresh soil, two or three plants per pot, and each pot covered with antifungal ‘gravel’ to prevent disease. The plants were left to grow on the windowsill of the laboratory until processing. The plants were monitored daily to make sure they were growing well and without disease.
Due to time restrictions on the instrument, analysis was not possible until week 6 of the placement. To ensure the quality of the sample, it was decided that preparation should not begin until the week prior to analysis. To compensate for this time, the project was extended from eight to nine weeks, and a short vacation taken in the middle week. This time outside the lab allowed for background reading to gain some further understanding of proteomics and its various techniques, as well as of the current knowledge of E. purpurea and its biosynthetic pathways. An online training course outlining the basic functions of the MASCOT search engine was
completed, provided by Matrix Science, and practice searches were run with previously obtained data. MASCOT is a powerful tool in proteomic analysis, and having plenty of practice was appreciated. The time was also used for practicing the protein extraction process.
Following this first part of the project, four plants, approximately 10 weeks old and with a maximum length of 35 cm root end to leaf tip, were removed from their soil and cleaned. No defined stems had grown, so the roots were separated from the leaves and both parts immediately flash frozen in liquid nitrogen. After crushing to a fine powder, the mass of the root sample was measured and the leaf sample weighed out to the same mass. The samples were washed with a solution of trichloroacetic acid (TCA) and 2-mercaptoethanol (2-ME) in cold acetone, and then washed three times further with a rinsing solution of 2-ME in cold acetone. The proteins in the samples were extracted into a solubilisation solution of urea, thiourea and dithiothreitol (DTT) in water. The concentration of the extracted protein in solution was measured by Bradford Assay.
The proteins were digested to peptides overnight with trypsin, following cysteine reduction and alkylation. Bovine serum albumin (BSA) was added prior to each digestion as an internal standard. After digestion the solvent was removed completely under vacuum, and the dry sample was dissolved in the solubilisation solution for a desalting step using ZipTips. The samples were analysed by nano-UHPLC-ESI MS/MS using a Dionex nano-UHPLC system
hour gradient to increase separation on the column. A blank separated each sample and a BSA standard was run before each sample type and at the end of the analysis to ensure the instrument performed consistently throughout the analysis.
The resulting data for each replicate were searched using the Mascot software against a published E. purpurea predicted protein sequence database which had been modified to include BSA. The optimum search parameters were found by searching the data from a single leaf run and altering the peptide mass tolerance, MS/MS mass tolerance, number of missed cleavages allowed and peptide charge. The optimum parameters found for this database search were 1 missed cleavage, +2,+3 and +4 peptide charge, 0.8 Da MS/MS mass tolerance and 20 ppm peptide mass tolerance. These settings were then used to search all of the data. The resulting list of proteins was filtered to show only proteins with two or more significant peptide sequence matches, and an ion score cut off of 0.05 was applied. The protein lists with emPAI values were exported as
with the individual replicates of each search using Mascot Daemon.
It is now the intention that these results will be further analysed by BLAST searching to identify the peptides and proteins present in the plant, and that these will be compared against the literature. Further, the proteins common to both leaf and root will be determined, and the emPAI values used for quantitative analysis of the data to find their relative concentrations. Unfortunately, due to time constraints it was not possible to complete this part of the work before the end of the placement. However, we hope to return to these data at a later date to achieve these aims.
Overall, I believe that my summer studentship was a huge success. Working in a research environment has allowed me hands on experience with techniques and instrumentation which I would otherwise not have come into contact with in my undergraduate studies. I would like to thank the BMSS and Prof. Rainer Cramer for supporting me during this 9-week opportunity.
Committee Member Spotlight
I was first exposed to mass spectrometry during an A-level graduate summer job for 10 weeks at the pharmaceutical giant Pfizer, Kent. I used a Fisons MD800 to run EI GC-MS on a series of derivatised steroids, I took the machine apart, sandblasted it and saw it work again occasionally. It was there that I met some of the key people who have inspired me, for over 20 years, to always be keen to learn how things work and to enjoy it! I was struck by how passionate and determined they were, how they were unfazed by setbacks. Things made sense, or would make more sense after that vital experiment, and after some stunningly low yielding undergraduate synthesis projects I was determined to put glassware away and stick with analytical chemistry.
I worked in Structural and Separation Sciences and eventually led the Open-Access mass spectrometry, chromatography and NMR team, helping over 100 chemists run 500K samples per year. Far from doing the same thing year in, year out, we strived to implement new, better ways of doing things, pioneering new technologies while they were very fresh, things that nowadays are taken for granted...mixed-mode dual-polarity ionisation, a TOF mass spectrometer to run openaccess accurate mass, 2 open-access mass and/or UV triggered autopurification systems that were trusted by chemists with their samples rather than using silica columns. I am particularly proud of the developments achieved by introducing Direct Analysis in Real-Time (DART) ambient ionisation to increase compound coverage and for real-time reaction monitoring, also creating fast LC-MS methods that finally competed and beat the tried and trusted tool of chemist...the thin-layer chromatography (TLC) plate. These were drastically different approaches to tackling age-old problems and they eventually garnered a lot of support. Later I realised that specialising in ‘pure’ mass spectrometry would probably give me more time to do novel work and some greater recognition and I joined the mass spectrometry
biology with peptide, protein and oligonucleotide approaches, in addition to supporting traditional organic synthesis.
The devastating closure announcement from Pfizer in 2011 gave me an opportunity to rethink where my career was going and what I wanted from life and I became dedicated to finishing the PhD I had started in ion mobility mass spectrometry with Prof. Frank Pullen. My study has introduced me to a wealth of new topics including molecular modelling using ab initio methods, deeper understandings of fragmentation processes and ion mobility. I’ve crossed over to perform high performance computing and presented at the NSCCS at Imperial College. I’m excited to see what develops and grateful that opportunities such as BMSS and ASMS meetings have enabled me to discuss and ‘pick up the ball and run’ with such burgeoning science.
In my spare time I’ve always enjoyed music, from being a music journalist on Student Union and self-published ‘zines to being a sound engineer on Resonance FM 104.4, the world’s first radio art station and ‘the best radio station in London’ according to The Guardian. Highlights have included engineering a record-breaking singing attempt from Wembley stadium, a live session with Penny Rimbaud (from punk band Crass) and playing a sound-art piece at the Vortex Jazz Club, London. I am married to a creative and brilliant fashion designer, Jiyun, we are blessed with a terrific young son, and live in London.
When I was nominated to join the BMSS committee, I was reminded to reflect on my involvement with the BMSS and what it means to me. I owe much to the many members of the mass spectrometry community who told me their concerns and helped me understand how to do things. I have been on BMSS courses and given workshops, regularly attended meetings
Cris Lapthorne
and offered equipment donations, won a prize and co-ordinated a Special Interest Group. Amazingly, this is still a small reflection of the activities of the BMSS and what opportunities there are, and why I am proud to be part of the society, and a Committee member. I believe, particularly through new ionisation methods, ion mobility and miniaturisation, that mass spectrometry will find ever greater applications and be in the hands of more users than before. It is important therefore, I feel, for the BMSS to maintain its welcoming feel and to help steer mass spectrometry on the right course.
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