MASSMATTERS
The official publication of the British Mass Spectrometry Society Edition 98 – Autumn 2023
Aston Medal 2023: Alexander Makarov
Reflections:
Reflections: Laura Cole... p22
Rob Bradshaw... p22
Editor’s letter
Welcome to the latest edition of Mass Matters!
Another year has just flown by and we have only just sat down after the yet another annual meeting – thanks to everyone who contributed and made it the success it was! In this issue, we look back at some of the highlights from Manchester, as well as forward to some of our new SIG Group meetings still to come.
We have to also reflect on the huge achievements of Prof. Alexander Makarov – and his receipt of the BMSS Aston Medal. Be sure to read about out his thoughts later in this issue!
Krisztina Radi also gets her ‘second welcome’ onto the committee, but this time following her successful election as a full general member. Congrats!
We hope you enjoy the issue... happy reading!
Best, Jon.
Jonathan Jones, BMSS Publicity Secretary
Chair’s Report
Welcome to the latest edition of MassMatters. As ever, a big thanks goes to Jon Jones and Krisztina Radi for all their hard work in bringing it together. Thanks too to all those who have contributed with news and articles: it’s great to see such a wealth of activity in the British mass spectrometry community. Please do check out the upcoming events highlighted in the Diary Dates section. For those whose budgets can stretch to it, IMSC 2024 will be taking place in Melbourne, Australia. Never fear, though, as BMSS44 will also be happening next year. It will take the form of a ‘Super-Meeting’ together with our colleagues and friends from the British Society for Proteome Research (BSPR). BMSS and BSPR have been working closely for some time now on areas of mutual interest, including holding sessions at each other’s annual meetings. I do want to put people’s minds at rest who don’t work on protein mass spectrometry and proteomics that this is not a focussed meeting and BMSS will be offering the usual mix of sessions across the entire field of MS science, and we are looking forward to showcasing the full breadth of our members’ research. The Meetings Secretary and Meetings Sub-Committee are just finalising the contract with the venue, and we are hoping to make an announcement in the very near future.
On the subject of the annual meeting, I’d like to reflect on the great success of BMSS43. Thanks to all of you who were able to attend for making this one of the largest meetings we have ever had. I think the final head count was 445 delegates and 38 exhibitors. There was an incredible buzz in the RNCM over all 3 days, and the decision to a set of parallel sessions on the first day meant that there was additional time for inclusion of more great MS science. Thanks again to the
Meetings and Papers teams for all their hard work in making this meeting such a success. It was a great honour to be able to present, on behalf of BMSS, the Aston Medal to Alexander Makarov for his outstanding contribution to the science of mass spectrometry. I’m sure you’ll agree that he is a worthy winner. It was also a pleasure to present prizes to our delegates for the best posters, oral presentation, and flash oral (please see page 12 for more details). If you’d like to know more about the awards and prizes that BMSS presents, including how to nominate someone, please see the society’s website for details. On sadder news, I was sorry to hear of the passing, in September, of Richard Sleeman. Richard was a pioneer in the application of MS in forensic science and well known to many BMSS members. He will be sadly missed.
I would like to welcome Krisztina Radi to her new role as a general member of the BMSS Executive Committee. Krisztina was previously a co-opted committee member and has been assisting the Publicity Secretary in the important job of producing MassMatters. Thanks too to Patrick Sears, Oliver Hale and Angela Taylor for serving as casual member and co-opted members, respectively, whose terms finished with September’s AGM. I’d also like to thank all current Executive Committee members for their continued hard work on behalf of the Society.
As you read this issue of MassMatters, you will notice the reminders to renew your membership to BMSS. I’m delighted to say that membership figures have bounced back after the drop we saw during COVID, but it is essential for the future of the Society that we keep the numbers up. Membership is
very competitively priced and has many benefits, including access to community news, discounted meeting registration fees, as well as the ability to apply for travel and research funds.
Finally, I’d just like to remind everyone that BMSS is your society, and that if you have any thoughts or suggestions on its activities, you are very welcome to pass them on to members of the Committee, and I promise that they will be discussed. I hope you enjoy this issue of MassMatters!
With very Best Wishes,
Prof. Neil Oldham BMSS Chair
Neil Oldham, BMSS Chair
Renew your BMSS membership subscription for 2024!
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Recent publications by BMSS members...
Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target
Vande Voorde, J, et al, Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target. Nat Metab 5, 1303–1318 (2023). https:// doi.org/10.1038/s42255-02300857-0
The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometrybased metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine.
The Edge Effect in HighThroughput Proteomics: A Cautionary Tale.
Colleen B. Maxwell, Jatinderpal K. Sandhu, Thong H. Cao, Gerry P. McCann, Leong L. Ng, and Donald J.L. Jones, Journal of the American Society for Mass Spectrometry 2023 34 (6), 1065-1072. https://pubs.acs.org/ doi/10.1021/jasms.3c00035
We present an MS-proteomics batch effect within multiwell plates termed the “edge effect” arising from temperature gradients across plates, prevalent in cell culture but not yet reported in a clinical proteomics setting. We present methods herein to ameliorate the phenomenon including proper assessment of heating
techniques for multiwell plates and incorporation of surrogate standards, which can normalise for intraplate variation.
Mass Sportrometry: an annual look back at applications of mass spectrometry in sport and exercise science.
Analytical Science Advances 2023;4:60-80
This annual review provides a summary of the applications of MS across studies investigating aspects related to sport or exercise science (SES) across topics such as targeted analyses, metabolomics, lipidomics, proteomics, and isotope ratio/elemental MS. The advantageous analytical opportunities afforded by MS technologies in SES are considered across a selection of relevant articles and discussions are provided on possible future directions for the use of MS in SES.
Applications of ambient ionization mass spectrometry in 2022: an annual review
Rankin-Turner S, Sears P, Heaney LM. Analytical Science Advances 2023;4:133-53.
This annual review provides an overview of ambient ionisation MS techniques and applications throughout 2022, with a specific focus on some of the major fields of research, including forensic science, disease diagnostics, pharmaceuticals and food sciences. New techniques and methods are introduced, demonstrating the unwavering drive of the analytical community to further
advance this exciting field and push the boundaries of what analytical chemistry can achieve.
A Comparison of Advertised versus Actual Cannabidiol (CBD) Content of CBD Oils, Aqueous Tinctures, E-liquids and Drinks Purchased in the UK
Johnson DA, Hogan M, Marriot R, Heaney LM, Bailey SJ, Clifford T, James LJ. Journal of Cannabis Research 2023;5:28.
Mislabelling of cannabidiol (CBD) content is commonplace in the consumer CBD market. However, it's not clear whether divergence between advertised and actual content differs between product types. Therefore, CBD levels were quantitated using HPLC or GCMS in 63 products, purchased online in the UK, including oil-tinctures, aqueous-tinctures, e-liquids and drinks. It was observed that oil-tinctures deviated least from advertised concentrations, followed by e-liquids, aqueous-tinctures, and drinks.
Trimethylamine N-oxide concentrations and blood pressure in young healthy men and women: a replicated crossover study.
Rowland SN, Heaney LM, Da Boit M, Bailey SJ. Metabolites 2023;13:876.
Trimethylamine N-oxide (TMAO) is a gut-derived cardiometabolic risk marker associated with hypertension and is commonly measured in circulation using LC-MS/
MS. Despite observing a sex difference in brachial systolic blood pressure, plasma TMAO concentrations were consistent across the menstrual cycle in eumenorrheic women, unchanged between pill withdrawal and pill consumption days in oral contraceptive pill users, and not different between young and healthy, males and females. These findings suggest that the link between TMAO and blood pressure is limited in healthy young adults.
Cannabidiol (CBD)-Oil Ingested as Sublingual Drops or Within Gelatin Capsules Shows Similar Pharmacokinetic Profiles in Healthy Males.
Johnson DA, Funnell MP, Heaney LM, Cable TG, Wheeler PC, Bailey SJ, Clifford T, James LJ. Cannabis and Cannabinoid Research 2023; doi: 10.1089/ can.2023.0117
Most cannabidiol (CBD) products purchased in the UK are tinctures, designed for the sub-lingual application of CBD oil. However, it is not clear whether CBD can enter circulation via the oral mucosa in an amount that would impact centrally circulating levels.
Therefore, pharmacokinetic responses of circulating CBD were investigated using LCMS/MS for CBD oil applied sublingually or consumed as oral capsules. It was considered that the bioavailability/ circulatory load of both approaches were equal as no differences were observed for circulating CBD levels across trials.
Diary Dates
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UK Technology Specialist Network meeting
Tony Larson
University of York 18-19th April 2023, Edinburgh
Over the 18th and 19th April 2023, I attended the inaugural meeting of the UK Technology Specialist Network (TSN), held at Pollock Halls in Edinburgh with local organisation expertly provided via Lee Murphy and Natalie Homer (MS core manager at the Edinburgh Clinical Research facility). Overall organisation was provided by the committee representing the founding TSN institutions from the Universities of Cambridge, Edinburgh, Manchester, Nottingham, Southampton Warwick, and York. Formed as one output from the UKRI commissioned TALENT commission report (published early 2022), the TSN aims to bring together Technical Specialists from across the UK to share our roles, skills, and work environment experiences, and to discuss career progression, training, and funding opportunities. This is all to help build a sustainable future for core facilities and the people who run them. I run a bioscience-focussed MS core lab as part of the Technology Facility at the University of York, and travelled with four of my colleagues to Edinburgh to understand what the TSN could offer.
The first day focussed on finance and funding models for core facilities, both from the perspective of the facilities themselves and the UKRI funding bodies. Danielle Hoyle from the Babraham Institute
gave an excellent overview of the challenges in achieving cost recovery and forecasting demand, with a following panel discussion including Joanne Marrison (York TF), Ian Brewis (Cardiff Core Facilities), Tony Chapman (EPSRC) and Nik Ogryzko (UKRI TALENT manager). This was followed by further in-depth talks from Tony Chapman (EPSRC) and Nik Ogryzko giving UKRI perspectives, and then a range of quick-fire facility talks from across the country where managers and RTPs introduced themselves. This was followed by a fantastic networking dinner in the wonderful and historic Playfair library on Edinburgh’s South Bridge.
Day two provided discussion on career pathways, with an inspiring keynote from Kelly Vere MBE (Nottingham), highlighting buy-in to the Technician Commitment across the UK HEI sector and the imminent launch of the UK Institute for Technical Skills and Strategy (ITSS), scheduled for August this year. There was also fantastic talks from David Dickson on programmes for Scottish Schools providing training pathways into industry, and Gareth Savage (Southampton University) on their efforts to make better use of the Apprenticeship Levy to support technical career paths in Universities. Anneke Lubben (Bath) gave a personal experience talk on her career trajectory of moving from Oceanography to Mass Spectrometry, then onto overall facility management and advising on capital
infrastructure grant applications. Anneke highlighted the BMSS community efforts on the C-MASS bid. I had the privilege of speaking in the following panel session organised by Julie Herniman (Southampton) on my experience in winning successful ALERT bids for MS equipment, sharing my experiences together with those from colleagues from Warwick, Newcastle, and Edinburgh.
Overall, it was fantastic to see Technical Specialists can and do lead and win equipment bids, and this is something being actively encouraged by the research councils. It was great to make new connections and be at the first of what will hopefully be more meetings to come – see https://www. mitalent.ac.uk/TSN.
Biomacromolecular Structure SIG
Aneika Leney University of Birmingham
The BMSS Biomacromolecular Structure Special Interest Group (BMS SIG), starting from 2020 and for a 5 year period, aims to provide training and a forum for discussion on Mass Spectrometry based methods for the elucidation of the structure of peptides, proteins, nucleic acids, polysaccharides, and other biomacromolecules.
During the COVID-19 pandemic, the BMS SIG meetings were held biweekly via zoom. Please click (here) to view our past schedule of events.
We have yearly meetings where the BMS SIG delivers training on techniques used for Biomacromolecular Structure determination by mass spectrometry, with a focus on guidelines and best practice in the collection of data, and its interpretation & reporting. The BMS SIG also provides a forum for the dissemination of the latest research in the field
and proactively seeks to foster collaboration within the British & Global Mass Spectrometry communities.
Early Career Scientist Inclusion
The BMS SIG (and its predecessor Ion Mobility SIG) has a resolute commitment to welcoming Early Career Scientists (ECSs) to its meetings. The BMS SIG invites all ECSs to participate at its events for a nominal charge (N.B. BMSS student members will enjoy enhanced discounts).
BMS SIG Meetings
The 5-year program of BMS SIG meetings will be deployed at five geographically diverse locations. The first meeting in-person meeting was held in 2022 at the University of Leeds with the training elements will focus on hydrogen-deuterium exchange mass spectrometry and native mass spectrometry. Please see more details on
Please see more details on this event here.
BMS SIG Meeting Format
The BMS SIG team facilitates in a 1-2 day informal meeting format. The opening day focuses on training, covering background theory, introduction to specific instrumentation, software tools, and best practice. The specific themes will vary from year to year and will be defined by the SIG team and incorporate feedback from the community.
The second day (i.e. the classic BMSS SIG Meeting format) will include opening and closing keynotes delivered by experts in the field. The majority of the presentations will be solicited from ECRs.
Our aim is to make the BMS SIG an environment where ECRs are able to give their “first presentations” in an enabling environment to an expert audience - and expose all of our delegates to the freshest emerging science from worldwide biomolecular
mass spectrometry orientated research groups.
BMS SIG Co-Leaders
Anton Calabrese (University of Leeds), Joe Gault, (AstraZeneca) and Aneika Leney (University of Birmingham) are all active researchers committed to the mass spectrometry community in the United Kingdom.
We invite contributions from ECR & ECS colleagues. BMSS grants are available for quaalifying membeers.
Stable Isotope Tracer Mass Spectrometry SIG
Daniel Wilkinson University of Nottingham
The field of stable isotope tracers has historically progressed hand in hand with the technical development of mass spectrometry from their initial use by Urey, Schonheimer and Rittenberg in the 1930s up until the present day. In recent decades, these previously niche techniques have been adopted by a large number of non-analytical research labs across the world, and this has been thanks in large due to the technical developments in hybrid MS, such as GC-Pyrolysis-IRMS and LC-high resolution MS. This has allowed the expansion of the application of these tracers within studies of mammalian and human metabolism which wouldn’t have been possible previously. The primary aim of this Stable Isotope Tracer MS special interest group is to provide the link between the application and the analytics. While numerous labs utilise these techniques, many are unaware or unable to access the information about the MS technologies which drive their data. We as a research group have been at the forefront of developing information and training to fulfil this gulf in knowledge, through the use of theoretical and practical stable isotope tracer training course. We believe that there are a great deal of both wellestablished Senior Researchers and Early Career Scientists (ECS) that although know of these techniques, do not have the insight and knowledge to apply and take them further within their research, and would greatly benefit from a group that can provide this necessary
knowledge and training. The primary principles of this SIG will be to provide introductory knowledge and insight into not just the use and application of stable isotope tracers within a number of diverse research fields, but also introduce the MS technological considerations that need to be considered when designing, running and analysing stable isotope tracer projects. We also believe that it is important to get like-minded researchers within the UK to interact and collaborate to enable the UK to maintain our world-leading expertise within the field of stable isotope MS in future years. One of the most important ways in which this can be achieved is through the promotion of stable isotope tracer MS to PhDs and early career researchers through mouthpieces like BMSS and SIGs such as this one.
Primary Structure
During the first year of this SIG we will provide a series of webinars providing an introduction to the techniques and technologies involved in stable isotope tracer MS, before moving on in later webinars to specific applications within diverse research fields. These online webinars (where possible) will consist of two talks, one by a senior researcher in the field alongside a talk by an ECS. These will give attendees the opportunity to learn and understand the basics of both application and technologies involved with Stable Isotope Tracer MS.
ITMS SIG Co-Leaders
Dr Daniel Wilkinson –University of Nottingham –Daniel.Wilkinson@nottingham. ac.uk
Prof Ken Smith – University of Nottingham – Ken.Smith@ nottingham.ac.uk
Dr Matthew Brook – University of Nottingham – Matthew. Brook@nottingham.ac.uk
IN PICTURES BMSS 43 in Manchester
Barber Prize
Bordoli Prize
B.N. Green Prize
Delegates Choice Prize
Colleen Maxwell, University of Leicester
Cara Jackson University of Strathclyde
Rebecca Nash National Measurement Lab
Anastasia Kontiza University of Surrey
FEATURE: ASTON MEDAL WINNER
ASTON MEDAL WINNER Alexander Makarov
Krisztina Radi Associate Editors, BMSS
At the 43rd Annual Conference of the British Mass Spectrometry Society in Manchester, Professor Alexander Makarov was awarded the Aston Medal for his pioneering work in mass spectrometry that brought us the Orbitrap.
The Aston Medal was established by the British Mass Spectrometry Society in 1987. As the Society's prestigious scientific award, it is given to individuals deserving special recognition for their outstanding contributions to mass spectrometry.
Responsible for bringing us technology that so many of us use day-in, day-out (and take for granted) it is entirely fitting that this award be presented to Professor Makarov, and to do so just down the road in Manchester from where it all began. An inspiring story that tells of a true passion for the science, determination to meet all the challenges faced headon, and a resilience for when the journey is hard.
Prof. Makarov kindly gave us his thoughts about his award.
So, Alexander – tell us how do you feel about receiving the Aston Medal?
This medal has a special meaning for an instrumentation designer like me. Aston received his Nobel prize 100 years ago for discovery of isotopes but in effect he was the first creator of mass spectrometry instruments that made this discovery possible. This makes receiving the Aston medal especially meaningful
and important for me! He was also a polymath with broad interests in many aspects of life, be it sports or music or technology- so this serves as a nice inspiration!
What does the British mass spec community mean to you and how did you feel being back to the UK for this year's conference? Did you attend many past BMSS conferences?
I am glad to be a member of BMSS although I live and work in Germany - after all, UK was the place where my professional career in mass spectrometry started following PhD on time-of-flight analysers in Moscow. As the annual conference falls on one of the busiest months of the year, my previous attendances date back to late 2000s, but I hope I will come more often in future.
This year’s conference was so important for me not only due to the Aston medal but also due to its location in Manchester where we lived for so many years. Even the Northern College of Music has been a part of this life, as my wife, Anna was a compère there for several charity concerts.
The BMSS is proud to be focusing a lot on supporting students and early career researchers to establish their careers in MS - What are your thoughts on prospects for young people starting out in mass spec?
I believe that mass spectrometry offers a tremendous variety of opportunities and ways to realise oneself, especially in the UK with its highly developed pharma, biopharma, academic
and analytical sectors. There is of course growing competition from other techniques, not the least next-generation sequencing, but the latest advances give us hope that MS will be able to hold its ground!
Clearly, mass spec has been adopted into many areas – but what do you think about new application areas for MS?
I think that we are going to witness great advances in several very different areas such as high-throughput translational -omics analysis (although not necessarily clinical), single-cell -omics and imaging, combination of MS with structural biology, routine analysis and field applications using miniature MS detectors, etc. Each of them will require numerous improvements of all aspects of MS and further expansion of the already amazing variety of professions that need to work together.
Tell us your Orbitrap secrets! Joking aside, are there still new directions/ improvements to be made in Orbitrap technology that you could share?
Even though the new Astral analyser clearly outshines everything else by its combination of speed with resolution and sensitivity, there is still a number of advances that could be achieved for the good old Orbitrap analyser.
This includes improvements in charge-detection, highresolution top-down/ middledown/ complex-down analysis, extraction of collision cross-sections from standard mass measurements, further acceleration of data acquisition – but also continuation of the hard-fought push towards making Orbitrap technology as robust, affordable, and simple to use as quadrupoles. The latter is a pretty difficult and unthankful task, but it still needs to be done!
Aside from mass spec, what else occupies your time?
Well, the photo (our cover image) is from our short vacation on Iguassu falls following the 3rd IberoAmerican Mass Spectrometry Conference in December 2022 in Rio-de-Janeiro, Brazil. Traveling is one of my hobbies - although we do it in a pretty conventional way and without any extremes…!
Other hobbies (outside of inventing at work!) are maybe pretty timid: but I enjoy mountain skiing, inline skating, volleyball, hiking, new scientific theories, and history…
Thank you so much for your time, Alexander – we are thrilled that you are now an Aston Medal holder!
Thank you very much!
Identification of protein variants in ATTR amyloid by proteomics. Is the p.H110D TTR an artefact or biologically generated?
Simran Raheja Supervisor: Diana Canetti, University College London
Introduction
Amyloidosis is a heterogeneous group of diseases caused by misfolding of normal circulating proteins and their subsequent deposition of insoluble cross-beta-sheets fibrillar protein aggregates in various tissues including the heart, kidneys and nervous system. This leads to organ dysfunction, morbidity and mortality.(1) Currently, over 30 human proteins can form amyloid deposits in vivo, resulting in significant differences in pathogenesis, clinical progression, prognosis and treatment.(2) Transthyretin amyloidosis (ATTR) is one of the most common forms of amyloidosis, resulting in cardiac, renal, and neurological impairment.(3)
Proteomics is recognised as an important method to identify amyloid protein type in patients’ samples, championed as the goldstandard, replacing immunological typing methods like
immunohistochemistry.(4) Laser Capture Microdissection(LCD) coupled to Liquid chromatography(LC)Mass Spectrometry(MS) (LCDMS) is currently employed clinically for amyloid typing at National Amyloidosis Centre (NAC)(London, UK). It is applicable to formalinfixed, paraffin-embedded (FFPE) samples and fresh tissue.(4) LCD microscopes with a fluorescence module are used to visualise Congo-red positive areas. This allows amyloid-enriched deposits to be dissected away from nonCongo-red material, thus enhancing specificity of the MS analysis.(5) The dissected sample is treated with detergent to solubilise the proteins, digested with trypsin, and reduced with dithiothreitol(DTT). The resulting mixture is analysed by LC-MS/MS using TopN Data Dependent Acquisition (DDA) method.(6) The MS raw data are analysed using the Mascot search engine.(7) In
addition to DDA, which preferentially targets highabundance precursors with limited sampling of lowabundance peptides, Parallel Reaction Monitoring (PRM) may be employed. PRM is a targeted method which detects with high selectivity and sensitivity targeted peptides in complex samples, such as tissue biopsies and biological fluids.(6)
By altering the underlying database, the approach can be extended to search for protein variants and post-translational modifications. Variant searches are central to identification of fibrinogen A amyloidosis at the NAC. This has been extended to other amyloid types, leading to the observation that the rare pathogenic p.H110D TTR variant was unusually identified in ~70% of WT-TTR FFPE tissues biopsies.
The aim of this research project is to investigate the cause of this apparent histidine-aspartic acid change observed in WT-TTR amyloid. One hypothesis is that it may be generated in vivo from normal circulating WT-TTR; alternatively, it could
be an artefact produced nonenzymatically on storage from WT-TTR by in vitro oxidation of histidine (p.110H) to hydroxy histidine followed by hydrolysis to asparagine(Asn)/aspartic acid(Asp).
Materials
and Methods
1. Confirming the presence of p.H110D TTR is real by proteomics analysis of FFPE tissue biopsies 1.2 Standard peptide mixture (SPM) preparation
(Comparison of chromatographic profiles and MS/MS spectra of authentic p.H110D TTR patient with WT-TTR patient and authentic standard peptides)
1.2 Processing of clinical samples
Clinical samples from WT and p.H110D patients were examined for amyloidosis by Congo-red staining and immunohistochemistry. Congored FFPE samples were microdissected and captured into the lid of a microcentrifuge tube using a Leica LDM7 laser capture microscope. Samples were digested with trypsin
following the standard NAC protocol(4) MS analysis was conducted using a Thermo Fisher Scientific Q-Exactive Plus Orbitrap mass spectrometer connected to an Ultimate 3000 nanoLC system, and the subsequent data analysed using Mascot software, using the Swiss-Prot database and in-house amyloid variants database.
The SPM, provided by ThermoFisher Scientific, was prepared by using a final concentration of 30 fmol/μL of each peptide (WT, p.H110D, p.H110N): p.101-123(81103): ALGISPFHE{H/D/N} AEVVFTANDSGPR.(4)
2. Investigating whether p.H110D can be generated in vitro, treating a recombinant WT-TTR in non-enzymatic oxidation
TTR 100μg was treated using the following conditions:
A. standard clinical proteomics protocol-high temperature
B. low temperature condition-control
C. low temperature and oxidative conditions using
Cu2+ with arachidonic acid (AA)
Sample A was treated following the standard clinical proteomics protocol, solubilising at 99°C for 1.5 hours.(4) Sample B was treated with 10mM DTT in 50mM Ammonium Bicarbonate (AMBIC) pH 8 and incubated for 20 minutes at 37°C followed by treatment with 55mM iodoacetamide (IAM) in the dark at 37°C for 20 minutes. Excess reagents were removed using Bio-Spin, and the sample was incubated with trypsin(100μg) overnight at 37°C.
Sample C was treated with DTT followed by IAM (20 minutes each at 37°C), after which reagents were removed using a Bio-Spin. AA(10μL of 1.6mM) and CuCl2(1.2μL of 10mM) were added, and the sample was incubated at 37°C for 60 minutes and stopped using BioSpin. Overnight digestion with trypsin at 37°C followed. The samples were centrifuged and placed in the SpeedVac after overnight incubation and solubilised in 0.1% trifluoroacetic acid and 1%
acetonitrile.
Peptide mixtures were analysed on LC-MS/MS using a C18 column.
3. Investigating whether p.H110D can be generated ex vivo treating purified WT-TTR fibrils.
A fibril extraction protocol with collagenase digestion, based on that described by Annamalai et al.(8)
0.5g of previously frozen tissue were cut in small (1-2mm3) pieces and washed 5 times with 1mL Tris calcium buffer. This was followed by centrifugation, discarding of supernatant and overnight digestions with Clostridium histolyticum collagenase type I. On the following day, pellets were manually homogenized in 1mL of Tris EDTA buffer. The homogenate was centrifuged for 5 minutes, and the previous homogenization step was
repeated 9 more times. The remaining tissue pellet was homogenized in 1mL of icecold water, centrifuged, and its supernatant retained. This was repeated 5 additional times. Immediately after the protocol was completed, complete protease inhibitor cocktail was added to the supernatants.
Following the fibril extraction, an estimate of protein concentration was carried out by BCA assay. 50μg of TTR fibrils were used for each treated and untreated conditions. Reduction and alkylation were conducted. The previously conducted copper(II) and AA treatment experiment followed.
4. Examining serum from WT and p.H110D patients to determine whether the pathogenic p.H110D is biologically generated.
Immunoprepcitation was
conducted(9) and the eluted samples were dialysed against PBS for 2 days. The immunoprecipitated samples were then treated with the standard clinical protocol as reported before and analysed by mass spectrometry using both DDA and PRM methods targeting the peptides of interest.
Results and Discussion
At the UK-NAC, the rare pathogenic p.H110D TTR variant has been identified by proteomics in in ~70% of WT-TTR FFPE tissues biopsies, including FFPE bone marrow, bladder, cardiac, fat aspirate, carpal-tunnel, but interestingly not in unfixed fresh fat aspirates; this raised the question regarding the source of this potentially pathogenic variant. One possibility is that it may be generated in vitro from WT-TTR oxidation of histidine(p.110H)
to hydroxy-histidine followed by hydrolysis to Asn/Asp, as has been reported previously in immunoglobulins(10) and can occur on sample handling and storage leading to a false identification and misdiagnosis. A similar issue has arisen where formalin-mediated lysine methylation of WT-TTR has resulted in the misidentification of the V122I TTR variant.(11) This could occur in vivo from normal circulating WT-TTR, on sample storage, or alternatively could arise on sample handling and storage.
Confirming presence of p.H110D TTR is real by proteomics analysis of FFPE tissue biopsies
In order to confirm the identification of p.H110D TTR, proteomics analysis was conducted on a biopsy from an authentic p.H110D TTR patient and compared to a
WT-TTR patient, using a SPM as a further control. MS data was processed using the Mascot software, which employed the SwissProt database, and subsequently confirmed through manual analysis of the raw data.
The extracted-ion chromatogram(XIC) from the SPM showed the triplycharged p.101-123(81-103) peptide(810.3918 m/z) of the p.H110D variant, with a retention time (RT) of 18.72 min. This information, produced from the SPM, was used as a control and provided the reference for RT and the MS/ MS spectrum for analysis of clinical samples. Manually examined MS/MS spectra, showed 2 and 5 variantdiscriminating signals in the y+ and b+series, respectively; this confirms presence of the p.H110D variant. Similarly, the p.H110N presence was confirmed manually, identifying 3 (y+series) and 4 (b+series) variant-discriminating signals.
The p.H110D/p.H110N variants were identified in a clinical sample from a WT patient (Figure 1A). A triply-charged p.101-123(81-103) tryptic peptide (810.7395 m/z) eluted at 18.96 minutes with an MS/ MS spectrum consistent with p.110D (Figure 1B). Similar manual analysis of p.H110N, showed identification of 4 variant-discriminating signals in the b+series of the triplycharged peptide to confirm its presence in the WT patient.
The TTR variant protein was confirmed in the authentic p.H110D ATTR patient (Figure 1C) showing presence of WT-TTR and p.H110D among others. Manual analysis was repeated to validate Mascot’s identification of WT-TTR and the p.H110D variant, the latter of which generated 5 variant-discriminating signals
in the b+series for the triplycharged peptide (Figure 1D). Although Mascot revealed the absence of p.H110N, this was further confirmed by manual interpretation
This experiment allows confirmation that p.H110D TTR is identifiable through proteomic analysis. Additionally, it establishes that the biopsy of the WT-TTR patient, classified as such through genetic screening, demonstrates presence of the p.H110D variant.
Investigating whether p.H110D can be generated in vitro treating recombinant WT-TTR and in non-enzymic oxidation condition.
To investigate the cause of histidine’s substitution by aspartic acid, the effect of high temperature used in standard clinical proteomics protocols was examined and compared to oxidative (CuCl2+AA+H2O2) and low temperature conditions.
AA is oxidised to hydroperoxy lipids by Cu2+/H2O2 under non-enzymic oxygen radicalmediated conditions: this process, which occurs on storage of plasma to generate isoprostanes(12), could act as a mechanism for oxidising histidine in biopsies.
Recombinant human WT-TTR purified from E.coli was used. Data for these experiments was analysed using Mascot software, employing SwissProt and TTR variant databases. To prevent reformation of the disulphide bonds and protect cysteine residues from oxidation, iodoacetamide as an alkylating agent was used. This is accounted for on Mascot, using cysteine carbamidomethyl CAM(C) as a fixed modification(13).
For the low temperature protocol, the presence of WT-TTR in the 81-103 and 81-104 peptides was identified, with a protein Mascot score of 916 and a high sequence coverage of 69%. p.H110D and p.H110N variants were absent, while WT-TTR was present. Manual analysis confirmed its presence, detecting a quadruply-charged 81-103 peptide with an RT of 17.10 minutes and 613.8076 m/z. Mascot-identified WT-TTR for the standard protocol was associated with a high TTR score (4089). WT-TTR presence was validated with high confidence through manual analysis of the MS/MS spectra of the quadruply-charged ion. p.H110D/N variant signals were absent, suggesting the high temperature did not generate oxidation of histidine to aspartic acid/asparagine.
In order to investigate a possible cause of histidine oxidation at position p.110, we introduced 1 hour-treatment with CuCl2, H2O2, and AA. Histidine is particularly susceptible to metal-catalysed oxidation and has previously shown to produce 2-oxohistidine as an oxidation product upon incubation with the O2/Cu2+/ascorbate system. After undergoing further oxidation, generation of ring-opened products such as aspartate, aspartylurea, and formylasparagine was demonstrated.(14) The low temperature conditions coupled with copper, AA, and H2O2 treatment induced oxidation on histidine, generating p.110D. This is demonstrated by the presence of multiple (33) D90 peptides identified with a high score(106) under oxidative conditions compared to only one p.110D peptide with a low Mascot score (41) in the control (Figure 2).
Investigating whether p.H110D can be generated ex vivo treating purified WT-TTR fibrils.
Oxidation was successful in treatment of WT-TTR fibrils;p. H110D was identified with 2 peptides in the control and 7 peptides in the sample under oxidative conditions. The difference is less extensive than that observed in recombinant TTR, which could be due to purified ex vivo fibrils being protected from oxidation and digestion. However, this nonetheless demonstrates some oxidation occurring to generate the p.H110D variant.
Examining serum from WT and p.H110D patients to determine whether p.H110D is biologically generated.
To investigate whether the p.H110D variant is biologically generated in vivo, serum from a WT ATTR and a p.H110D patient was examined, comparing them to two healthy controls. The “healthy control 1” chromatogram (Figure 3) demonstrates presence of WT-TTR, labelled “110H”, with an RT of 17.34 minutes. WT-TTR was also identified in “healthy control 2”, “WT patient”, and “p.H110D patient”, demonstrated by peaks at a similar retention time. Mascot reported the presence of the p.H110D variant, with 86 matches and a high overall p.H110D TTR score (2634) through the DDA method. Manual analysis confirmed presence of p.H110D in the heterozygous p.H110D patient, associated with an RT of 19.71 minutes (Figure 3). Searching for peaks at this RT in “healthy control 1”, “healthy control 2”, and “WT patient” confirmed the absence of p.H110D (Figure 3).
This demonstrates that in vivo the WT patient has WT-TTR and the heterozygous p.110D patient has circulating levels
of the expected variant. The absence of p.110D TTR in controls suggests there are not measurable circulating levels of the variant in these two control samples. The high proportion of p.H110D in tissue samples may therefore have arisen through histidine oxidation on storage.
Conclusion
The importance of accurate amyloidosis typing and diagnosis cannot be understated, particularly in guiding treatment. Misdiagnosis and subsequent incorrect therapy could be detrimental to patients, depriving them of the benefits of the required therapy.
• p.H110D/N was identified by MASCOT in ~70% of WT-TTR FFPE tissue biopsies, however it was not detected in unfixed fresh fat aspirates.
• Authentic standards and amyloid samples from a patient with p.H110D TTR amyloidosis were used to confirm the identification of p.110D TTR in stored biopsy samples
• The standard extraction/ digestion protocol used at the NAC did not generate p.110D peptides
• p.H110D TTR was absent in the serum of normal WT-TTR volunteers
• Non-enzymic oxidation of TTR by Cu2+/H2O2/AA generated the p.110D variant, presumably through oxidation of p.110H and subsequent hydrolysis. Other oxidation products were also formed.
• These data are consistent with the formation of p.H110D TTR on storage from WT-TTR through a transition metal ion/AA-mediated mechanism (cf-isoprostane formation in stored plasma). This could result in misdiagnosis of p.H110D TTR amyloidosis.
References
1. Bustamante JG, Zaidi SRH. Amyloidosis [Internet]. PubMed. Treasure Island (FL): StatPearls Publishing; 2020. Available from: https://www.ncbi.nlm.nih. gov/books/NBK470285/
2. Sekijima Y. Hereditary Transthyretin Amyloidosis [Internet]. Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJ, Stephens K, et al., editors. PubMed. Seattle (WA): University of Washington, Seattle; 1993. Available from: https://www.ncbi.nlm.nih.gov/ books/NBK1194/
3. Jain A, Zahra F. Transthyretin Amyloid Cardiomyopathy (ATTR-CM) [Internet]. PubMed. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Sep 2]. Available from: https:// www.ncbi.nlm.nih.gov/books/ NBK574531/
4. Canetti D, Rendell NB, Gilbertson JA, Botcher N, Nocerino P, Blanco A, et al. Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre. Clinical Chemistry and Laboratory Medicine [Internet]. 2020 Jun 25 [cited 2022 Sep 8];58(6):948–57. Available from: https://pubmed.ncbi.nlm. nih.gov/32069225/
5. Vrana JA, Gamez JD, Madden BJ, Theis JD, Bergen HR, Dogan A. Classification of amyloidosis by laser microdissection and mass spectrometry–based proteomic analysis in clinical biopsy specimens. Blood. 2009 Dec 3;114(24):4957–9.
6. Hu A, Noble WS, WolfYadlin A. Technical advances in proteomics: new developments in data-independent acquisition. F1000Research. 2016 Mar 31;5:419.
7. Mascot search engine | Protein identification software for mass spec data [Internet].
www.matrixscience.com. Available from: https://www. matrixscience.com
8. Annamalai K, Gührs K-H, Koehler R, Schmidt M, Michel H, Loos C, et al. Polymorphism of Amyloid Fibrils In Vivo. Angewandte Chemie International Edition. 2016 Mar 8;55(15):4822–5.
9. Canetti D, Nocerino P, Rendell NB, Botcher N, Gilbertson JA, Blanco A, et al. Clinical ApoA‐IV amyloid is associated with fibrillogenic signal sequence. The Journal of Pathology. 2021 Aug 27;255(3):311–8.
10. Miyahara Y, Shintani K, Hayashihara-Kakuhou K, Zukawa T, Morita Y, Nakazawa T, et al. Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies. Scientific Reports. 2020 Apr 14;10(1).
11. Canetti D, Rendell NB, Di Vagno L, Gilbertson JA, Rowczenio D, Rezk T, et al. Misidentification of transthyretin and immunoglobulin variants by proteomics due to methyl lysine formation in formalinfixed paraffin-embedded amyloid tissue. Amyloid. 2017 Oct 2;24(4):229–37.
12. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proceedings of the National Academy of Sciences. 1990 Dec 1;87(23):9383–7.
13. Kuznetsova KG, Solovyeva EM, Kuzikov AV, Gorshkov MV, Moshkovskii SA. Modification of cysteine residues for mass spectrometry-based proteomic analysis: facts and artifacts. Biomeditsinskaya Khimiya. 2020 Jan;66(1):18–29.
14. Uchida K. Histidine and lysine as targets of oxidative modification. Amino Acids. 2003 Jul 29;25(3-4):249–57.
Reflection and Skills
The BMSS studentship has truly been one of the most enriching experiences of my academic career and it has been an honour to carry out research under my supervisor, Diana Canetti, at UCL. I began investigation into this topic as part of my bachelor’s research project, during which I was introduced to many of the fundamental concepts and procedures which have central to this work. My bachelor’s research project allowed me to discover my interest in research and so I was eager to gain more experience in the field over the summer. Thanks to the British Mass Spectrometry Society (BMSS) I was given the opportunity to continue my research at the Centre for Amyloidosis. In this project I was able to put into practice and refine my knowledge and skills and improve my understanding of mass spectrometry and proteomics.
Proteomics is a vast subject and its role in amyloidosis diagnosis and research is fundamental, particularly in diagnosis and guiding treatment. During the studentship I solidified my understanding of the principles and implications of mass spectrometry and the technical workings of the instrument – a Q-Exactive plus mass spectrometer. Over the studentship, I became more independent and confident in operating the high-performance liquid chromatographymass spectrometer as I was exposed to the various MS methods, including parallel reaction monitoring and data dependent acquisition others. Data analysis, through various bioinformatics approaches, has undoubtedly been an integral
part of this project. Beginning with a basic understanding of bioinformatics, during the course of this studentship I was taught how to use various different programs and databases to analyse and evaluate data. Such I used Mascot, Skyline, Xcalibur, and Databridge, all of which do not only require an understanding of how to navigate what their outputs demonstrate and, importantly, the limitations of these approaches.
My experience at the Centre also allowed me to learn about the various other projects taking place. I learned to practice a variety of fundamental biochemistry protocols: Western Blot and SDS page, preparing calibration curve in serum, protein purification and fibril extraction procedures, and drug interaction experiments. I also had the opportunity to learn about quantitative mass spectrometry, through a project being undertaken to quantify transthyretin fragments in patient biopsies to allow for stratification of the transthyretin amyloidosis patients.
The studentship has not only equipped me with many technical skills necessary for a future in research/ academia and the confidence to carry out protocols independently, but has also confirmed my deep interest in for medical research, particularly through mass spectrometry and its myriad applications. This experience was largely possible thanks to my supervisor, Dr Diana Canetti, for giving me the chance to work on this exciting project, for explaining the clinical applications of the work, and for mentoring and teaching me with endless patience and the utmost enthusiasm. I am also grateful to Dr Verona for involving me in his own research work, to Dr Graham Taylor for his advice, and his enthusiasm
in supporting me during all stages of the studentship at the Centre, and to Ms Nocerino for her patience and excellent explanations of the Skyline software during our data analysis sessions.
In Memoriam: Richard Sleeman
Remembrance words courtesy of Lance Hiley
Dr Richard Sleeman BSc (Hons), PhD, CSci, CChem, FRSC, MFSSoc, a distinguished English chemist and a pioneer in the field of forensic chemistry, died suddenly on September 8, 2023, at the age of 67. He is survived by his wife Sue.
Richard Sleeman was born in Exeter, England, on December 3, 1955. His family moved to Bristol, where he attended Weston Grammar School. A keen athlete, he supported Exeter City Football Club, played tennis, and had an enthusiastic amateur football career on Bristol Downs throughout his university studies and first career at British Aerospace. He obtained his PhD from the University of Bristol and was a member of the Academy of Experts and a Law Society Checked Expert Witness from 1997 to 2019. Dr Sleeman was one of the founders of Mass Spec Analytical Ltd., and for most of his career he specialized in the detection of trace quantities of drugs and explosives. He published over thirty scientific papers and articles on trace detection, and he was closely involved in the development of the techniques and methodologies employed in the examination of drugs traces on paper currency. He presented his work in court on approaching one hundred occasions, including at the Court of Appeal on three occasions. Richard's work has helped to make
our communities safer and to reduce the crime associated with drug trafficking and terrorism.
In addition to his scientific accomplishments, Dr Sleeman was also a kind and generous person, always willing to help others. He was a passionate educator and mentor, and he helped to train many of the next generation of forensic scientists. He was also a great friend and colleague. He will be deeply missed by all who knew him.
Reflections: Mass Spectrometry as a Career Choice
Interviewed by Mervyn Lewis, Associate Editor
What was your impression of Mass Spectrometry when you were first introduced to it?
RB: I was first introduced to mass spectrometry when I was studying for my BSc in Forensic Biology in 2006. I was fascinated by the theoretical concepts and the broad range of applications which were available. This particularly resonated with me as I was being taught by world renowned experts in this field, making the research applications that were being discussed more relateable. The enthusiasm for the topic shown by my tutors was something that made me very intrigued.
LC: My first introduction to mass spectrometry was as an undergraduate Biomedical Science Student back in 2004. Being only a small part of analytical science modules initially it was a fascinating phenomenon to me! Unlike now, it was not a technique that was associated as a key tool in biomedical science. My lecturers at the time did their best to convince us all of its power!
What persuaded you that Mass Spectrometry could be a good career option for you? Were you attracted by an application of Mass Spectrometry or excited by the prospect of developing it as an instrumental technique?
RB: Due to the research
that was being undertaken in the Biomolecular Sciences Research Centre (BMRC) at Sheffield Hallam University, I was particularly drawn towards applications within mass spectrometry imaging. I saw that this work was being disseminated in journals and in conferences worldwide and this is something I wanted to be involved in.
LC: Imaging work within the Biomolecular Sciences Research Centre (BMRC) at Sheffield Hallam University was the initial fascination for me and was the turning point for my career choice and future research specialism. I was attracted by the numerous applications, its future applicability and advancement as a powerful disease diagnostics tool.
How did you go about finding your first opportunity in Mass Spectrometry?
RB: A sandwich placement opportunity became available during my Undergraduate studies which focused on the use of MALDI MSI for the analysis of latent fingermarks under the supervision of Prof. Simona Francese (who has gone on to make significant advancements in this topic over the past 15 years). During my placement year, I was able to see the power of this technique and the ability to obtain an abundance of information (both chemical and physical) from minute sample quantities. I went on to complete a PhD and Post Doc in the same topic,
Robert Bradshaw
Laura Cole
working closely with potential end-users at the UK Home Office and West Yorkshire Police. I have now gone full circle, teaching the new generation of scientists about mass spectrometry concepts and applications in my role as a Senior lecturer in Analytical Science at SHU.
LC: Mass spec found me I guess! I was looking for a PhD project with a cancer research theme and did not ever foresee a switch to bioanalytical chemistry and mass spectrometry. In fact, I declined when a colleague suggested I apply to a mass spec project! I am so glad I made the plunge for what at first was a steep learning curve (with many peaks and troughs!) but then so rewarding and resulted in finding my niche in scientific research. I went on to complete my PhD with Director of Studies being Professor Malcolm Clench on a CRUK funded project. I then continued working with Malcolm as a Post Doc before being appointed as Course Leader and Senior Lecturer in Biomedical Science at Sheffield Hallam University.
In what application fields do you see new opportunities in Mass Spectrometry?
RB: One of my current research interests lies in environmental chemistry, in particular the transfer of potentially harmful molecules from plastics onto foodstuff. It is evident that the public are becoming more aware about issues surrounding the use of plastics and the impact it has on our world. Modern instrumentation is becoming increasingly more capable in detecting low abundant
analytes in a range of sample types, which makes analysis of leachate more achievable.
LC: I am passionate about mass spec imaging within the clinical setting and encouraging links between pathologists and multimodal mass spectrometry imaging. The multiplexing nature of mass spec offers numerous possibilities in disease diagnostics and especially now where instrument run times are becoming more high throughput maintaining spatial resolution.
What is your opinion about the impact that automation and informatics will have on Mass Spectrometry?
RB: Modern instrumentation can provide an abundance of data which can be extremely difficult and time consuming to interpret manually. Automation for data interpretation will certainly make this process easier and should ultimately maximise the information obtained from datasets.
LC: Automation, informatics tailored with machine learning innovations seem to be driving the forward momentum of data analysis. In the appropriate setting why not? Human decision making, especially involving a patients treatment plan teamed with such advancements could be very beneficial in healthcare.
Could you describe your views on career prospects in Mass Spectrometry for young people?
RB: Mass spectrometry is an exciting field to work in as it
is everchanging and improving. A career in mass spectrometry often means working on new and interesting topics requiring multiple key professional skills such as communication, collaboration, and innovation. The world of mass spectrometry is also so vast and covers multiple analyte types, innovative applications, and instrument types – this opens a world of opportunities for a budding mass spectrometrist!
LC: Mass spectrometry unbeknown to many, is a mainstay technique being employed universally from hospitals to airports; aside from the academic workbench. However, such a powerful, diverse tool still appears to be unfamiliar to many students that I teach during lectures and practical sessions. The analytical team here at Sheffield Hallam University (academic and technical) continue to promote the benefits and inform on the diverse career prospects now available for young people to ensure the mass spec future is in safe hands!
Acknowledgements
Laura and Rob would like to thank Sheffield Hallam University, CMSI and colleagues within the mass spectrometry group and research centre here, headed by Prof. Malcolm Clench. "Enjoy your retirement, Malcolm!". Rob would also like to thank Prof. Francese for her support throughout his progression within academia.
