MASSMATTERS
MEETING SUPER
Editor’s letter
Welcome to the latest edition of Mass Matters!
Spring is in the air! In this edition we look forward to our ‘Super meeting’ in September in partnership with the BSPR as well as looking at some of the demographics of the BMSS membership and welcoming the new BMSS Lecturer!
For me, this is a bittersweet edition as it is my final one in the role of Publicity Secretary after over 3 years. I will be passing the reins to Krisztina Radi, who has the enviable job of putting together our 100th issue this autumn!
I 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 always 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, these include the Imaging and MALDI SIG meeting and the Symposium in memory of Jim Scrivens, both in May.
The 27th of February saw the exciting announcement of £50M Government funding for the C-MASS mass spectrometry infrastructure project, which will be led by EPSRC and MRC, with support across the UKRI remit. As many of you will know, the BMSS initiated a consultation exercise 10 years ago, which resulted in a Statement of Need (SoN) document. Following further consultation with UKRI, learned societies, industry, HEIs and other stakeholders, a modified proposal termed Critical Mass UK (C-MASS) emerged. Although no longer a BMSS project – per se – from this point, the BMSS continued to act as a key conduit to the UK MS community, with briefings and feedback sessions at BMSS annual meetings. Many individuals from our community have contributed to this bid, which is driven by the need to maximise our capability and capacity in mass spectrometry. Crucially, it is also designed to act as a catalyst for further investment in UK MS, including areas such as instrument development, with the goal of addressing all aspects of BMSS’s SoN. Spending on C-MASS is scheduled to begin in 2026/7,
and BMSS will endeavour to keep its members updated as operational details emerge. On other important news: this year’s BMSS Annual Meeting will take the form of a ‘SuperMeeting’ together with the British Society for Proteome Research (BSPR) at the Warwick Arts Centre on the University of Warwick campus 4th-6th September. 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. Indeed, we did plan to hold a ‘SuperMeeting’ back in 2020 but had to cancel due to the pandemic. As I mentioned in the last issue of MaMa, 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.
To make the point, there will be sessions on general “Applications” (two of these), “Small Molecules”, “Instrumentation and Fundamental MS”, “Data Processing and Informatics”, “Ambient Ionisation and Imaging”, to name a few, as well as posters covering the full range of MS research.
Our Plenary Speakers will be Kevin Pagel (Berlin), who works on fundamental ion spectroscopy, as well as glycan MS, Corinne Spickett (Aston), whose research chiefly lies in lipidomics and oxidative stress, and Bernhard Küster (Munich), who is a leader in proteomics. A big thanks goes out to Lindsay Harding, our Meetings
Secretary, who has headed the ‘Meetings Team’, for all the hard work she, and they, are putting in to organise the annual meeting. I’d like to welcome Liam Heaney to his new role as Papers Secretary and thank him for his hard work in putting together the Scientific Programme together with the Papers Team. I would also like to welcome Kat Hollywood, Niklas Geue and Sarah Hart onto the BMSS Committee as Co-opted members, and welcome back Patrick Sears as a Casual Member. Kat is helping with organisation of the annual meeting, whilst Patrick and Niklas are part of the Papers Team. Sarah has kindly agreed to join the Education sub-committee, bringing her experience as a former Education Officer. She is also a member of the BSPR Committee, which gives a very useful link between the Societies ahead of the joint meeting. All their help is greatly appreciated.
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
Remember your 2024 BMSS membership subscription!
Renew your BMSS membership subscription for 2024 to not miss out on another year of member benefits!
You can renew quickly and easily on-line by visiting the BMSS Website: www.bmss.org.uk.
Why not also encourage colleagues to join the BMSS? As a member of the BMSS they will:
• Be part of a community of people with similar interests
• Have the opportunity to keep up to date with recent technological developments, learn more about MS and share knowledge and experience e.g. by joining Special Interest Groups (SIG’s)
• Be eligible to apply for grants towards small items of equipment, summer studentships conference travel,
carer's support, and outreach support - subject to eligibility terms and conditions
• Be entitled to discounts for BMSS meetings
• Benefit from reduced subscription fees to the European Journal of Mass Spectrometry
• Receive copies of Mass Matters, the official benefit from 20% off relevant book titles when purchased via Wiley Publishing (discount code required) publication of the BMSS, published three times a year
Students: Can benefit from our education programme by applying for travel grants, presenting research at meetings and by taking part in the Barber, Bordoli, BN Green and Delegate's competitions. All student members also receive a general mass spectrometry text book upon joining.
New & Established spectrometrists: Can benefit from courses, careers events, links with other societies (such as ChromSoc. & RSC) and networking via the BMSS Annual Conference and Special Interest Group Meetings.
Any membership queries should be directed to: Lisa Sage, BMSS Administrator
T: (01606) 810562
E: admin@bmss.org.uk
Recent publications by BMSS members...
Precursor Ion Connectivity of Different Charge States to Improve Peptide and Protein Identification in MS/MS Analysis
Adair, L. R.; Jones, I.; Cramer, R. Utilizing Analysis. Analytical Chemistry 2024, 96 (3), 985-990. DOI: 10.1021/acs. analchem.3c03061
A pervasive problem in MS/ MS analyses, especially in top-down proteomics, is the occurrence of chimeric spectra. Herein, we present a novel workflow that utilizes precursor ion connectivity, collating MS/MS data obtained from different charge states within an individual sample to produce a single peak list. Subsequently, protein sequence coverage and database search scores are improved, providing more confident peptide and protein identification.
Combining Thermal Desorption with Selected Ion Flow Tube Mass Spectrometry for Analyses of Breath Volatile Organic Compounds
Ilaria Belluomo, Sophia E. Whitlock, Antonis Myridakis, Aaron G. Parker, Valerio Converso, Mark J. Perkins, Vaughan S. Langford, Patrik Španěl, and George B. Hanna
Analytical Chemistry 2024 Jan 30;96(4):13971401. doi: 10.1021/acs. analchem.3c04286. Epub 2024 Jan 20.
Selected ion flow tube mass spectrometry was coupled with thermal desorption for the measurement of volatile organic compounds in exhaled breath. The new instrument has been designed to have a high throughput in multi-centre, large-scale clinical trials. A method for the quantification of twenty-one breath compounds was validated in parallel at Imperial College London and at Syft Technologies, New Zealand.
Characterizing the proteinprotein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization
Jake A Ward, Beatriz Romartinez-Alonso, Danielle F Kay, Jeddidiah Bellamy-Carter , Bethany Thurairajah , Jaswir Basran , Hanna Kwon , Aneika C Leney , Salvador Macip , Pietro Roversi , Frederick W Muskett , Richard G Doveston. J. Biol. Chem. (2024), 300(2) 105651. DOI: 10.1016/j. jbc.2024.105651
Protein-protein interactions are vital for normal cellular function but can become disrupted in disease. Gluing proteins together to treat disease is becoming an increasingly popular therapeutic avenue. However, difficulty arises when finding techniques to discover new drug candidates. Here we highlight native mass spectrometry as a powerful technique to uncover the gluing
capability of FC-A for the protein-protein complex; 14-3-3 and MDM2.
Ambient ionisation mass spectrometry for drug and toxin analysis: A review of the recent literature
Henderson A, Heaney LM, Rankin-Turner S. Drug Testing and Analysis 2024; doi: 10.1002/dta.3644
Ambient ionisation mass spectrometry (AIMS) has the potential to benefit a range of applications through rapid analysis. Specifically, the use of AIMS both within and outside the laboratory can be advantageous for drug and toxin analysis. This review showcases publications within the different fields of which AIMS has been used for drug and toxin analysis, highlighting the current outlook as well as the future for AIMS to grow within drug testing.
A comparison of cannabidiol (CBD) concentrations in venous versus fingertip-capillary blood
Johnson DA, James LJ, Heaney LM. Clinical Chemistry and Laboratory Medicine 2024, 62, e90-e93. DOI: 10.1515/cclm2023-0928
This work demonstrated that plasma CBD can feasibly be quantitated in fingertipcapillary samples to confirm entry of CBD into the bloodstream. This could, for
example, allow monitoring of the bioavailability and adherence to CBD-containing medications. However despite a strong correlation between the two techniques , increased concentrations in capillary vs. venous plasma suggests that absolute concentrations should not be used interchangeably.
Diary Dates
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British Society for Proteome Research
In advance of this September’s ‘Super Meeting’ between the BMSS and the BSPR, we invited the BSPR to say a little about themselves.
Sarah Hart University of Liverpool School of Medicine and Institute of Life Course and Medical Sciences
Charlotte Hutchings (PhD Student, University of Cambridge)
The British Society for Proteome Research (BSPR) aims to advance the science of proteomics, further public education therein and promote
study and research work in proteomics and related subjects for the public benefit.
The BSPR represents both the Human Proteome Organization (HUPO) and the European Proteomics Association (EuPA) within the UK and is also affiliated to the Royal Society of Biology.
This gives us an ear into the wide structures of proteomicsrelated science on the world stage. We co-hosted the annual EuPA meeting in Newcastle upon Tyne in 2023, a meeting attended by over 470 proteomics professionals from both Industry and Academia.
The objectives of the British Society for Proteome Research (BSPR) are to advance the science of proteomics and to promote the study and research work in proteomics and related areas for the benefit of all.
In addition to hosting our own meetings we have also supported regional groups, and support students and ECS attendance at a range of events via a range of member bursaries (Bursaries and fellowships - (bspr.org)). We offer free student membership with proof of student status for the duration of registration on under- and postgraduate courses, with access to our bursaries as part of the
host a BSPR Lecturer, currently this is Professor Sara Zanivan, who is available to speak at your local departmental, mass spectrometry, proteomics or cancer biology seminar series Sara Zanivan -Tumour Microenvironment and Proteomics (crukscotlandinstitute.ac.uk).
Photos: BSPR-EuPA 2023
Newcastle – conference dinner, Wylams Brewery; EuPA awardees 2023; snapshot of a session; post-dinner mixer.
The British Society for Proteome Research (BSPR) is a registered incorporated charity (Registered Company Number 6319769, UK Charity Number 1121692).
Do your caring needs prevent you from attending a conference? BMSS can help!
BMSS Carers Support Fund
Attending conferences and activities can be hectic for many of us, and juggling that with caring commitments can be preventative, especially when needing to stay overnight. It’s not just single-parent families that are affected, but families with two adults working full time, or even those of us who have responsibilities for adult relatives.
This is not unique as a recent survey by BMSS showed that 1 in 5 members had caring responsibilities for young children, while 1 in 20 had caring responsibilities for adult relatives. Many members with these responsibilities are usually in the middle of their careers.
The BMSS equality,
diversity, and inclusion (EDI) subcommittee recognized in 2020 that this can be a barrier for members in attending conferences or activities and wanted to address this issue. As a result, the BMSS introduced its Carers Support Fund, open to any member to help with additional expenses such as home help, respite care or extended hours for a childminder.
The chair of the EDI subcommittee, Dr Rhodri Owen said “The BMSS is committed to ensuring equality, diversity, and inclusion in our activities. As a Society, we want to ensure that all members can participate as fully as they wish which is why we introduced the Carers Support Fund to help members
The BMSS welcomes applications to the fund continuously and is available to all members irrespective of career stage and can demonstrate that their professional development as mass spectrometrists will be enabled through such support by the Society.
Applications are handled discreetly and confidentially and are accessed on the BMSS website https://www.bmss.org. uk/grants/.
with caring obligations.”
The faces of BMSS
A look at our Society’s demographic data prepared by the Equality, Diversity and Inclusion Sub Committee*
Where are BMSS members from?
More than 79% of members are from the UK Gender Demographics Just under two in five members are female
The British Mass Spectrometry Society (BMSS) is committed to ensuring equality, diversity and inclusion for its members, employees, scientific partners and the Society’s service providers The BMSS EDI subcommittee, formed in September 2019, has a remit to systematically understand and calibrate the current practice of equality, diversity and inclusion within our Society
R Recent Initiatives
The first initiative of the EDI Sub-Committee was to champion the adoption of the BMSS C Carers Support Fund We have identified caring responsibilities as a key barrier to peer retention and career progression in mass spectrometry The Society has introduced the BMSS Carers Support Fund to facilitate attendance at scientific events, including but not limited to BMSS meetings, by our members with caring commitments At the annual BMSS conference in 2019 we held the first BMSS L LGBTQ+ social event This event was held following the specific request of one of our members This event was positively received and is now a regular part of the annual conference social programme We recognise that it is important to better understand the people we represent With this in mind, we have designed an anonymous Questionnaire Here we present 21/22 results 8 Caring Commitments
Carer of an adult (18 or over);
Carer of child/children (under 18); Carer of child/children (under 18);Carer of an adult (18 or over); Carer of disabled or seriously ill child/children (under 18);Carer of child/children (under… No caring responsibilities; Female Male
*Data derived from 118 responses to a questionnaire,
Targeted Quantitative MS Imaging by Triple Quadrupole DESI-MS/MS and Standard Additions
Georgia Millard Supervisors; Laura Cole, Robert Bradshaw and Malcolm Clench
Sheffield Hallam University
Introduction
Mass spectrometry imaging (MSI) is widely known as a qualitative technique that provides intensity distribution maps of varies species across a tissue section. 1 2 Spatial distribution maps can be helpful to determine the biological functions and phenotypes within a sample. 3 Matrix Assisted Laser Desorption Ionisation (MALDI) or Desorption Electrospray Ionisation (DESI) are both soft ionisation techniques used for MSI and both have benefits over one another. DESI does not require any sample preparation and hence there is no signal suppression due to matrix. Whereas MALDI can currently achieve a higher spatial resolution. 3
Although MSI shows relative quantification, it also has absolute quantification applications when a calibration curve is used. For MSI analysis the quantity of a species is measured within a tissue, yet this can induce the possibility of ion suppression effects from the tissue which can change across the tissue due to histological frameworks and compound competition. Ion suppression effects can also come from the MALDI matrix
as mentioned previously. Russo et al, used a label free method in which they used a robotic spotter to apply a calibration curve, spiked with internal standard, to lab skin for MALDIMSI analysis. 2 The internal standard must be a molecule with similar properties to the analyte of interest, often this is the isotopic or deuterated form of the analyte but these can be expensive. Russo found that incorporating the internal standard into the calibration standards for spotting prevented migration of the analyte which occurs when the internal standard is sprayed on top of the calibration curve. 2 As a result the standard addition technique was used. Calibration curves are essential to accurately determine the concentration of a sample. It is also important to use an internal standard for calibration to enable normalisation within the calibration spots themselves. 4
To generate highly accurate and precise spots, a robotic spotter can be used to apply a set volume, in turn this ensures that all calibration spots have a reproducible size and diametersomething that is difficult to achieve with manual spotting. 2 4 This work used porcine liver as the tissue source due to its homogeneity and focused on quantifying the concentration of glutathione within the tissue. Glutathione is present in virtually all mammalian cells and is very abundant in
the liver due to role in against oxidative stress. It is found in the liver at concentrations between 1-10 mM. 5 6
Previous works shows the use of matrix assisted laser ablation ionisation (MALDI) as the ionisation source for quantitative mass spectrometry imaging but the MALDI matrix can often have a suppressive effect on low mass, small metabolites such as glutathione.4 Many different software packages have been produced for analysing quantitative mass spectrometry data such as MSI Quantify from Waters Corporation (Manchester, UK) which has the capability to visualise images, select specific regions, apply calibration curves and estimate error. 7
The aims and objectives of this project were to use targeted MSI to quantify common metabolites in liver (glutathione, glutamate, cysteine, glycine, 5-oxoproline and cysteinylglycine) using Waters new micro application- MSI Quantify. Initially this would involve using the biospotter to spot controlled volumes of standard onto liver samples, acquire data using the DESITQ-MSI in multiple reaction monitoring mode and then use MSI Quantify to analyse the data.
Materials and Methods
Sample Preparation
Liver was cryo-sectioned (Leica,
Wetzlar, Germany) at 10 µm, freeze thaw mounted onto polysine glass slides (Eprendia, Runcorn, UK) and subsequently dried with nitrogen air. Where analysis could not be completed immediately after sectioning, samples were vacuum packed and stored at -80°C.
Two different preparation methods were tested. Either a glutathione dilution series (0-15 mM, Merck, Gillingham, UK) was prepared in milli-Q water using glutathione ethyl ester (GSHEE) (9.1 mM, Merck, Gillingham, UK) as the internal standard or prepared with glutamate (9.5 mM) as the internal standard. Samples were allowed to reach room temperature before analysis to avoid condensation.
For samples requiring the GSHEE calibration line, samples were sprayed with GSHEE using the HTX M3+ sprayer (HTX Imaging, NC., USA) before calibration line application. The glutathione dilution series was applied to both blank polysine glass slides and the liver sections using the Biospotter (BioFluidiX, Germany) on the day of analysis. To achieve a circular drop, the stroke and stroke velocity was calibrated for each channel. The stroke volume was 39, stroke velocity was 45 giving a 30 nL drop size. Spot location was also calibrated for each channel. The Nanodispenser in the Biospot dispensed 30 nL droplets on
to the blank glass slides and liver sections in a triplicate formation.
DESI-TQ-MSI
MSI analysis was performed on the TQ-XS MS (triple quadrupole mass spectrometer) with desorption electrospray ionisation (DESI) source (Waters Corporation, Manchester, UK) parameters. Data was acquired in negative V-mode. The electrospray solvent was 100 pg/µL leucine enkephalin (Waters Corporation) in 95% methanol (Fisher, Loughborough, UK) at a flow rate of 2 µL/min. A spatial resolution of 50 µm2 was achieved. The MS conditions were set as follows: capillary voltage 0.7 kV (ESI+), cone voltage 10-15, source temperature 100 °C, and collision energy 10-20 V. Low mass resolution was 3.5 and the high mass resolution was 13.8. Data acquisition was performed in multiple reaction monitoring (MRM) mode. Cone voltage and collision energy optimisation was performed with the imaging function (Table 1).
Processing
Data was viewed in Mass Lynx 4.2, HDI 1.7 and MSI Quantify 1.2.1 all of which are Waters software. Data analysis was also performed in microsoft excel. Data was inputted into MSI Quantify as a txt imaging file at 10 µm sample thickness. Regions of interest (ROI) were selected with consistent pixel numbers, although the software should account for slight differences in selected area size. It would be highly beneficial if the software had a ROI cloning tool. Data was normalised to the internal standard which is performed on a ‘per pixel basis’. The calibration curve was formed using the ‘quantity' option (the normalised intensity values were fit to the dose values normalised per
a) LM; 3.0, HM: 14.9
b) LM; 3.9, HM: 14.0
c) LM; 3.2, HM: 13.8
d) LM;3.5, HM; 13.8
e) LM; 3.5, HM; 13.5
f) LM; 3.8, HM; 13.5
g) LM; 3.5, HM; 13.7
Figure 1: Altering the low mass resolution (LM) and high mass resolution (HM) has a large e:ect on the spectra. spectra have the same following parameters; source temperature 100°C, cone voltage 20 V, capillary voltage 0.7 collision energy 0 V. Sample is 50mM glutathione on a polylysine glass slide.
figure 1, decreasing the high mass resolution (HM) decreased the amount of peaks above m/z 306 being detected. The low mass resolution (LM) needed to be increased slightly,
unit area). Anomalies were discarded as required.
Results and Discussion
Glutathione has a mass of 307.323 Da, which when analysed using DESI-MSI in negative mode is detected at m/z 306.076. The product
ions were predicted using the structure of glutathione and basic mass spectrometry knowledge. 8 The DESITQ-MSI instrument method was first optimised to detect glutathione (50 mM) on a glass slide and using plain liver. The initial results showed possible in source collision induced
dissociation for 0V collision energy (Fig. 1a). As shown in figure 1, decreasing the high mass resolution (HM) decreased the amount of peaks above m/z 306 being detected. The low mass resolution (LM) needed to be increased slightly, but not too much as this also seemed to have a negative effect on
the spectra (Fig. 1 a, d and g). Upon reflection, I should have done this optimisation step more systematically by changing the HM first and then the LM.
The method was optimised for the transitions of glutathione (m/z 306 to m/z 128 and m/z 143) glutathione ethyl ester (m/z 334 to m/z 282 and m/z 300) and glutamate (m/z 146 to m/z 59 and m/z 129). For m/z 128 cone voltage 10 V was to analyse glutathione on a glass slide. However, for glutathione in liver 20 V cone voltage was required. Overall, in MRM mode, a collision energy of 20 V gave the best intensity and HDI image for m/z 128 (Fig. 2c and Table 2c), these parameters varied for each product ion. HDI imaging data is shown in both log scale and linear scale (Fig. 2). The log data (left) shows a lot more detail than the linear scale is acceptable
for this project as it simply needed to show which collision voltage gave the most intense signal. The HDI data (Fig. 2) also shows a higher intensity of the glutathione at the outer rim of the sample. This is due to the Marangoni or ‘coffee-ring effect’ which does not happen with the biospotter due to the small volume, the entire drop dries almost instantly. 9
The Biospotter method was optimised using water and moisture sensitive paper to ensure the instrument was in full working order. The instrument had to be calibrated for each concentration to ensure a circular spot was produced every time, this included calibrating the stroke volume and stroke velocity to give a 30 nL spot for each calibration point. The calibration curve was repeated in triplicate.
Following DESI-TQ-MSI and biospotter optimisation, liver samples with glutathione concentration arrays were created for data collection. When the sample is too close to the top edge of the slide for acquisitions, it means that when the DESI is acquiring data the capillary tube is not on the slide. As a result, the distance between the slide and capillary tube varies, as shown in figure 4 when the capillary tube is not over the sample (above the white line) the intensity is greater than when the capillary tube is over the sample (below the white line). Due to these findings, following this analysis I ensured that my samples and calibration lines were towards the lower half of the glass slide, or I started my analysis lower on the glass slide.
Initially a glutathione array with GSHEE was spotted straight onto liver as a control.
This confirmed that there is no GSHEE in the liver and prompted the spraying of GSHEE onto the liver. There is a large difference for the R2 value (Fig. 4 a vs 4c/d), but this could be due to the fact that the MSI data is normalised per unit area. The software generated a calibration curve from the three repeats and a line of best fit all normalised to the internal standard (glutamate m/z 300) (Fig. 4a). After creating a calibration curve, the software used its line of best fit to calculate the unknown region concentrations (Fig. 4 b, and Table 3a). To confirm the reliability of the software, manual calibration lines were also created. Usually, the calibration curve should cross the x-axis in the negative. However, this data set when normalised to the internal standard, does not. Therefore further work is required. HDI allows for ROIs to be cloned,
Table 1: Tabulated data displaying the optimisation of the collision voltage for m/z 128. 1mL of 50 mM glutathione was dried onto a clean glass slide using the vacuum desiccator for drying the slide. This table corresponds with figure 2.
Figure 2: This data indicates optimisation of the collision voltage for m/z 128. 1mL of 50mM glutathione was dried onto a clean glass slide using the vacuum desiccator for drying the slide. The HDI images correspond to the values in table 1. The HDI images are displayed in both log form (left) and linear form (right).
Glutathione m/z 128
Glutathione m/z 143 Glutathione m/z 210
Figure 3: HDI images of glutathione product ions a) m/z 128, b) m/z143 and c) m/z 210 to enhance how the positioning of a sample on a slide is important. Above the white dotted line, the capillary tube was not on top of the sample slide, below the white dotted line, the capillary tube was on top of the sample slide.
yet MSI Quantify does not, therefore ROIs were drawn as similar as possible. It would be beneficial if ROI’s could be cloned in MSI Quantify. MSI Quantify creates the calibration line according to the area of the ROI selected, hence slight size variations should not matter.
When evaluating unknown data in MSI Quantify, there is the option to analyse it ‘per unit area’ (Table 2a), but this creates data that is unrealistically high compared to the manual calibration line and not ‘per unit area’ data (Tables 2b and 2c).
To consider the possibility of using an internal standard which is endogenous within the tissue, glutamate was used. HDI displayed that although glutamate is present throughout the liver, it is not completely consistent. The ROIs for both methods are
as expected, the values are all similar (approximately 1 mM) with slight variation (Tables 3a and 3b). ROIs were placed in the same place for both methods. Although the liver is homogenous, it is not perfect. This data set produced calibration lines with
Figure 4: Calibration lines for the same set of data- liver, sprayed with glutathione using the HTX M3+, spotted with glutathione standards (0-15 mM) containing glutathione ethyl ester (9.1 mM) as the internal standard. A) The data was put into MSI Quantify to generate a calibration curve; ROIs were approximately 70 pixels +/- 3 pixels. B) The unknown ROIs inputted in MSI Quantify on calibration curve A. C) The data was extracted from cloned ROIs in HDI and put into excel. D) The data was extracted from cloned ROIs in HDI and put into excel to be normalised against the internal standard.
Table 2a: Extrapolated values for the unknown regions of interest corresponding to Figure 4a. Table 3b shows the unknown ROI concentration values associated with Figure 4b when the data isn’t extracted ‘per unit area’. Table 3c shows the unknown ROI concentration values associated with Figure 4b when the data is extracted ‘per unit area’.
more similar R2 values and more similar unknown ROI concentrations than the GSHEE calibration line. Therefore, this data set seems more reliable, but more repeats are required.
Conclusion
MSI Quantify alongside the DESI-TQ-XS and biospotter is a promising tool for absolute quantification but further work is required to fully optimise the method. The project has allowed me to further develop my mass spectrometry understanding. It has allowed me to learn how to use and troubleshoot the DESI-TQ-XS, biospotter and MSI Quantify. It has taught me the importance of keeping detailed notes; excel is very useful for recording mass spectrometry parameters. In future I wish to learn how to use the method function on the biospotter and learn how to do 2nL spots. For MSI Quantify I wish to continue to work with the software developers to get the function of ROI cloning. Overall, this project has taught me valuable skills that could be used in my PhD work.
1 N. Sun and A. Walch, Histochem. Cell Biol., 2013, 140, 93-104 (DOI:10.1007/ s00418-013-1127-4).
2 C. Russo, N. Brickelbank, C. Duckett, S. Mellor, S. Rumbelow and M. R. Clench,
Quantitative Investigation of Terbinafine Hydrochloride Absorption into a Living Skin Equivalent Model by MALDIMSI, American Chemical Society (ACS), 2018, 90(16), 10031-10038 (DOI: 10.1021/ acs.analchem.8b02648).
3 L. E. Flint, G. Hamm, J. D. Ready, S. Ling, C. J. Duckett, N. A. Cross, L. M. Cole, D. P. Smith, R. J. A. Goodwin and M. R. Clench, Anal. Chem. (Wash.), 2020, 92, 1253812547 (DOI:10.1021/acs. analchem.0c02389).
4 C. Russo and M. R. Clench, in Imaging Mass Spectrometry, ed. L. M. Cole & M. R. Clench, Springer Science + Business Media, New York, USA, 2nd ed., 2023, ch. 3, pp.27-40.
5 Å Florholmen-Kjær, R. A. Lyså, O. Fuskevåg, R. Goll, A. Revhaug and K. E. Mortensen, A sensitive method for the analysis of glutathione in porcine hepatocytes, Informa UK Limited, 2014.
6 M. Vairetti, L. G. Di Pasqua, M. Cagna, P. Richelmi, A. Ferrigno and C. Berardo, Changes in Glutathione Content in Liver Diseases: An Update, MDPI AG, 2021.
7 MSI Quantify waters poster, 2023asms_trinkle_msiquantify. pdf (waters.com) (01/04/24)
8 Z. Wang, Y. Fang, D. Rock and J. Ma, Rapid screening and characterization of glutathionetrapped reactive metabolites using a polarity switch-based approach on a high-resolution quadrupole orbitrap mass spectrometer, Springer Science and Business Media LLC, 2017.
9 R. G. Larson, Nature, 2017, 550, 466-467
BMSS Lecturer: Tony Bristow
We are very excited to announce that Tony Bristow is to be the BMSS Lecturer!
Krisztina Radi Associate
Editor
The role of the BMSS Lecturer is to promote mass spectrometry and the activities of the BMSS, to the wider scientific community. This will help develop a greater understanding of the power of mass spectrometry and hopefully generate a greater interest in the subject amongst researchers, students, and the public. In 2023, Tony Bristow agreed to become the next lecturer and will shortly embark on his series of talks.
Tony Bristow is Principal Scientist for Analytical and Measurement Science and Director for Trace Analysis in Chemical Development at AstraZeneca (Macclesfield, UK). In addition, Tony holds the role of technology and capability lead for the Analytical Sciences Leadership Team, within AstraZeneca’s Pharmaceutical Technology & Development organisation. These roles are focussed on the development and delivery of analytical science strategy and currently has a focus on the implementation and application of automation and digital technologies. Prior to this current role, his expertise was the application and development of mass spectrometry (2005 - 2018). He is the Chair of the Industry Advisory Board of the Community for Analytical Measurement Science (CAMS) and a Visiting Professor at the University of Warwick. Tony is a former member of the Royal Society of Chemistry Analytical Division Council (2013-2019) and the former Chair of the
British Mass Spectrometry Society (2012-2014). Tony was awarded a degree in Applied Chemistry in 1992 and his PhD (mass spectrometry) in 1996. Tony previously worked for Kodak and LGC focussed on a variety of analytical sciencebased projects. He is an author/ co-author of 47 peer reviewed publications and regularly presents at national and international conferences.
Tony’s lecture series will
• Mass Spectrometry in the pharmaceutical industry - its amazing impact on the delivery of medicines to patients.
• The challenges in trace analysis of nitrosamines to demonstrate patient safetythe story so far.
• Trust in science in an increasingly polarised and noisy world - a thought experiment for the next generation of scientists.
Read more about the role of the BMSS Lecturer on our website, with some previous talks available to members via the Heritage pages.
Tony Bristow – BMSS Lecturer and Xenomorph seeker.
20 years of Ambient IonisationA Celebration!
Andrew Ray & Patrick Sears
On the 1st February the Ambient ionisation Special Interest Group held a celebratory meeting to celebrate twenty years since the first publications on ambient ionisation. We had our biggest ever meeting with 124 delegates and 12 exhibitors descending on the ThinkTank at Birmingham, this represented a move from our usual universitybased meetings to a commercial venue and the feedback we received suggests this was a great success.
We were very fortunate that some of the leading lights in the ambient ionisation field agreed to come and present. Chip Cody from Jeol started us off by pointing out that ambient ionisation was actually 21 years old as the first spectra were acquired in 2003! He discussed the history and development of DART before looking at the other growth areas within ambient ionisation particularly indicating some of the most interesting application areas, he observed that there was no universal ambient ionisation source and that it is particularly useful to have several techniques available. He highlighted that one area that has restricted the development of ambient ionisation has been a lack of suitable software, this has been eliminated with the development of data analysis software specifically tailored for the data generated by ambient ionization.
Akhila Anjith from the University of Manchester had
The busy lunch time discussions in the exhibition hall at the BMSS Ambient Ionisation Meeting
DESI ion images from an imprint of a young tomato leaf onto a PTFE substrate.
be a practical alternative to autoradiography as was labelfree, user friendly and highly reproducible.
Abi Cook from the University of Surrey discussed the analysis of single cells by comparing direct capillary nanospray ionisation where the cells are sprayed directly into the mass spectrometer and LC/MS to profile amino acids identified in cells, efficacy of the two approaches for the analysis of metabolites in single cells were considered. She highlighted the relative advantages and disadvantages of each which included aspects of speed, accuracy and the number of observed species concluding
these techniques to imaging and cancer diagnostics and newer areas such as identification of micro-organisms. He showed how some of the early serendipitous discoveries were developed through the application of scientific principle and have become the backbones of the techniques we use today. He also gave a view towards other ambient techniques and application areas he sees as important in
We then had an excellent lunch, enjoyed talking to all the exhibitors and viewing the 35 posters that were presented (another record). The poster on the “Forensic characterisation of trace contamination” by Andriana Michailidis was awarded the Delegates Prize voted for by everyone attending. We should also highlight the contributions of two undergraduate researchers who both used ASAP for analysis of consumer products,
Ben Campbell (who presented a poster detailing the analysis of counterfeit paracetamol) and Alaric Buckell (whose poster showed the analysis of adulterated edible oils).
Helen Cooper from the University of Birmingham kicked the afternoon session off by discussing the use of native ambient mass spectrometry (NAMS) for in-situ analysis of proteins and their complexes. The combined benefits of NAMS for analysis of protein assemblies and proteinligand complexes include measurement of accurate mass and stoichiometry, identification of both protein and noncovalently bound ligands, together with information on spatial distribution. Recent efforts using liquid extraction surface analysis (LESA) and nanospray desorption electrospray ionization (nanoDESI), have advanced NAMS for the analysis of fresh frozen issue, allowing the
clinical validation of paper arrow-mass spectrometry for salivary acetaminophen analysis; this area is particularly aimed towards bedside monitoring when patients are believed to have overdosed acetaminophen – saliva being a less invasive fluid to obtain. The work identified stimulated saliva as the most promising approach for this analysis although the method for stimulating saliva requires further optimisation based on patient feedback.
Zoltan Takats discussed his journey through ambient ionisation from the discovery of DESI through to REIMS and further to laser-assisted REIMS; highlighting the application of
Andriana Michailidis from the University of Surrey, winner of the Delegate Poster prize.
Professor Graham Cooks at the Ambient Ionisation meeting 2024.
spatial distribution of protein assemblies to be mapped. Endogenous protein assemblies and their constituents (including small molecule ligands) can be identified by top-down dissection of assemblies in the gas phase, potentially allowing the discovery of new proteinligand interactions.
Rachel McGuire and Catherine Welsh from AstraZeneca then did a double act on the use of ASAP for the assay of compounds in pharmaceutical drug development, they demonstrated great data on one compound but much poorer data on another. There were many helpful discussions with them at the coffee break with a variety of different experiments to try – we look forward to seeing how this area develops.
Patrick Sears from the University of Surrey then gave a tutorial on his findings on how to get the best out of coated bladespray – a new and potentially very exciting technique that we will undoubtedly hear more about at subsequent meetings. Patrick showed how this this technique is effectively a combination of SPE and paper-spray and that when the principles of these are followed, a more efficient analysis results.
Continuing the theme of quantitation, Brian Musselman discussed the development of quantitation with DART – in this case towards the analysis of pharmaceutical products in urine. This work highlighted the difficulties associated with using small volume samples, adding and mixing labelled standards into the samples; the use of current state-of-the-art sample preparation and liquid handling technologies were shown to be critical in generating high quality data.
The last presentation was by Graham Cooks who discussed the development of DESI, its applications to tissue diagnostics and
imaging. He then moved on to accelerated reactions in droplets, particularly interesting was amino acid condensation in water to produce peptides which he put in the context of the origins of life. The third part was on high throughput reaction screening, small scale synthesis and bioassays in an automated DESI-based system. Of particular interest here is late-stage functionalization in chemical synthesis, the high-quality enzyme kinetics available from DESI, and progress towards drug discovery using the system.
The awards were then presented but we decided to take advantage of our invited speakers and had a panel discussion. There were lots of interesting points raised in an informative but very amusing session with Graham taking great delight in disagreeing with Chip!
We then decided that the only way to finish an excellent day was a trip to the “Head of Steam” pub, here a good night was had by all (although
mild poured over it!) There were still 20 people still in the pub at closing time. Thanks go out to everyone who helped in the organisation, including the Early Career Scientists who chaired sessions (if you would like to chair a session at a future meeting do let us know). We are already starting to look at venues for next year – if you know of somewhere nearby that you think could be a great place to hold the meeting (whether at a university or a commercial venue) please contact Patrick and let him know.
So to sum up the meeting –great venue, great science, great networking, great fun. The feedback we had suggested this was not just our view so hopefully will stimulate people to come along and present their work next year.
Ambient Ionisation Panel discussion, Helen Cooper (centre) keeping score with Chip Cody (right) and Graham Cooks (left).
Reflections : Running a Mass Spectrometry Facility
Interviewed by Mervyn Lewis, Associate Editor
What persuaded you that mass spectrometry could be a good career option for you?
Rather than me choosing mass spectrometry, I think mass spectrometry chose me! My first degree was in Geology and Geography, and I followed this with a job as a technician at Cardiff University. There I was part of the Earth Science Sedimentary Research Group and completed a part time MSc degree in Environmental and Engineering Geology. I loved field work and took part in a research cruise to the Mediterranean Sea and supported field work activities across South Wales and the Humber Estuary. Back in the laboratory, I maintained the analytical equipment and helped PhD students and researchers analyse their samples. During this time, I did come across mass spectrometry occasionally as I sometimes helped the technician running the departmental ICP-MS system.
How did you find your first position in running a mass spectrometry service?
After nine years at Cardiff, I was looking for a move to Southampton and applied for a fixed-term technical position with Professor John Langley in the School of Chemistry. Even though I had not worked previously with mass spectrometers, I thought
my laboratory and technical skills could be transferable to the role. John had just secured funding for two MALDI-TOF instruments and a technician to develop mass spectrometry methodologies for oligonucleotide analysis and was looking for someone to work with him on this project. I was a bit concerned that being laboratory based the job would be a bit boring for me, but as it was only a fixed-term position I thought I could always move on; 25 years later I am still here! At that time, the facility at Southampton consisted of a VG-70 SE magnetic sector instrument with EI, CI and FAB sources and a Micromass Platform II open access LC-MS system (ESI and APCI sources). Shortly after I started work in Chemistry, the two MALDI-TOF instruments arrived, and I was involved in the installation and training.
How do you handle the wide range of applications and encourage new users to use your service?
The mass spectrometry facility here at Southampton has expanded over the years to meet the demands of research chemists. We consult with users at an early stage to provide the most appropriate instrumentation for their current and future research projects. Funding is always difficult to come by, but we have been successful over the years to purchase a wide range of instrumentation and ancillary equipment. We now offer a variety of chromatographic
Juile Herniman
Southampton University
options including GC, UHPLC and UHPSFC coupled to different ionisation sources and analysers. We also specialise in high resolution mass spectrometry and provide ion mobility options. This means that we can analyse almost any type of sample that passes through the laboratory. Recently we commissioned 2D GC-MS (LECO 4D BT and HRT) and 2D LC-MS (Waters Cyclic) instrumentation for the separation and analysis the most complex of samples. This project was funded by EPSRC in conjunction with mass spectrometrists at the Universities of Bath, Portsmouth, Swansea and Surrey.
To analyse large numbers of samples we provide open access mass spectrometry and were the first lab in academia to follow the open access model developed by Frank Pullen and George Perkins for pharmaceutical chemists at Pfizer1. We achieve this via the RemoteAnalyzer® webbased interface developed by SpectralWorks Ltd. RemoteAnalyzer is vendor independent which means that we do not have to rely on one type of open access provision from a single manufacturer. We can install the most appropriate instrument for the job and the open access interface and output for the chemists is identical. This also means that we do not have to provide
multiple software options for data processing.
How do you attempt to make the service you offer environmentally sustainable?
To make the service as environmentally friendly as possible, we assess the environmental impact of the whole laboratory and every method we currently use. We look at the types of solvent used, the flow rates, column dimensions and the time it takes for each method to run. We also use energy saving devices where we can and ensure that all waste is minimised. We are currently working on a project to compare the environmental impact of methods using reversed phase UHPLCMS compared to ultrahigh performance supercritical chromatography (UHPSFC) -MS and use AGREEprep2 to include an assessment of different sample preparation techniques. Many compounds conventionally analysed using reversed phase UHPLC could easily be analysed using SFC and since this utilises recycled
What is your view on having and keeping good working relationships with instrument manufacturers?
It is essential to have good working relationships with manufacturers. It enables you to find out about new instrumentation at an early stage of development and you can also feed back to the manufacturers any new chemistries being developed by colleagues that may require novel chromatography and mass spectrometry solutions. Sometimes grant applications need to be completed quickly and hence staying connected with a representative from the manufacturer helps with quotes in short time scales! Good relationships with manufacturers also put your laboratory in an advantageous position to beta test new equipment and in the past, we have received instrumentation on loan for specific research projects.
What is your opinion about the impact that Artificial Intelligence will have on mass
ChatGPT to answer. I have not really thought too much about the use of AI and mass spectrometry services although I hope that it will be used in an effective way to streamline some of the methods and hence make them more sustainable. The use of AI to predict the optimum separation or mass spectrometric method from a structure or group of structures can only be a good thing, as long as the need for trained mass spectrometrists does not disappear totally!
Could you sum up your thoughts on career prospects for young people in operating a mass spectrometry service?
As I mentioned previously, I thought I might be bored working in a laboratory all the time, but the variety of instrumentation and applications certainly keeps things interesting. If you work for any University MS service, then the range of applications you will deal with will be huge! There are many opportunities to develop your own research and work with academic teams, I have worked on everything from small natural products,
some of my work at national and international conferences and user group meetings and now have friends working in MS from all over the UK. If you want to learn about different instruments, different software packages and different chemistries quickly, I would definitely recommend working for a mass spectrometry service.
References
1.F.S. Pullen, G.L. Perkins, K.I. Burton, R.S. Ware, M.S. Teague, J.P.Kiplinger. Putting mass spectrometry in the hands of the end user. J. Am. Soc. Mass Spectrom. 1995, 6,394.
2.W. Wojnowski, M.Tobiszewski, F.Pena-Pereira, E.Psillakis. AGREEprepAnalytical greenness metric for sample preparation. Trends Analyt Chem. 2022, 149 116553.
Acknowledgements
Julie Herniman is employed as a senior experimental officer/research technical professional at the University of Southampton. She works alongside Prof. John Langley in the Chromatography and Mass Spectrometry facility within
