Erin Humphries reports from the 27th Annual Lorne Proteomics Symposium
Written by Erin Humphries, Cancer Proteomics Researcher at the Children's Medical Research Institute, NSW and recipient of the RACI 2021 RACI Student Travel Award
Published 18 March 2022
With RACI's 2022 Postgraduate Student Travel award opening today, it was perfect timing that Erin Humphries, utilising her 2021 RACI Student Travel Award, provided this insightful report from the 27th Annual Lorne Proteomics Symposium.
Back in February, I attended the 27th Annual Lorne Proteomics Symposium in Victoria thanks to the MRACI Postgraduate Student Travel Bursary. The travel bursary assists postgraduate RACI members to travel to a conference by providing free registration and funds towards travel costs. The symposium showcased the latest proteomics and lipidomics research from across the Australia and New Zealand regions including developments in ‘omics’ technologies and tools for the interpretation of proteomics/lipidomics outputs, and their application toward answering fundamental questions in biology, and in clinical and translational medicine.
At the symposium, I presented a poster investigating the performance of different trypsin sources on the preparation of formalin-fixed paraffin-embedded tissues (FFPE) for mass spectrometry. Efficient digestion of proteins is needed for reproducible identification and quantitation in mass spectrometry. Trypsin is the gold standard enzyme for shotgun proteomics but does not efficiently digest FFPE tissues due to protein crosslinking.
I compared three commercial sources of trypsin using overnight benchtop digestions and 30-minute pressure-cycling technology digestions. The samples were analysed using microflow liquid chromatography-tandem mass spectrometry on a TripleTOF6600 in Information Dependent Acquisition mode and peptides were identified using the Paragon algorithm in ProteinPilot.
Digestion efficiency was highest in the benchtop method using Promega’s Trypsin Platinum however, Promega’s Rapid Digestion Trypsin/LysC performed best in the pressure-cycling technology digestion. Peptide identifications were up to 42% higher in samples digested using pressure-cycling technology compared to benchtop methods.
A shift in the ratio of C-terminal residues was observed when digesting tissues using pressure-cycling technology with Rapid Digestion Trypsin/LysC and was not observed with the other trypsin products. The change from 55:45 arginine and lysine observed in the benchtop method to 50:50 in the pressure-cycling method suggests enhanced LysC activity as the other two trypsin products compared did not contain the serine protease.
The poster concludes that the commercial source of trypsin and the experimental protocol has a significant impact on digestion efficiency and peptide identification. Fortunately, the experiments demonstrated that the current trypsin product I was using was still the best enzyme, so we didn’t have to modify the workflow.
The symposium covered many topic areas including disease proteomics, microbial/plant proteomics, bioinformatics, as well as glycoscience and lipids. My favourite session was on post-translational modifications and signalling with A/Prof Melissa Davis and Dr Sean Humphrey as my master’s project is on phosphorylation. My project has a clinical focus, and it was great to hear A/Prof Matt Dun talk about clinical profiling of the posttranslational architecture of paediatric high-grade gliomas. I enjoyed listening to A/Prof Ralf B Schittenhelm talk about Phospho-Analyst as well as A/Prof Nicholas Williamson and Dr Michael Leeming talk about Phosphomatics. These are two web based interactive platforms which can analyse quantitative phosphoproteomic data.
I would like to thank the RACI for the opportunity to travel to this symposium in-person! It was an amazing experience networking with others in the proteomics field during the session breaks, poster sessions, welcome function, student dinner and conference dinner. I highly encourage other postgraduate students to get involved in RACI activities and apply for the travel award later this year.
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About the RACI Postgraduate Student Travel Award
This year the Postgraduate Student Travel Award nominations open earlier on 18 March 2022 and close 22 May 2022. The award includes free registration to the RACI 2022 National Congress and $500 towards travel & accommodation. The RACI 2022 National Congress is 4-7 July 2022 in Brisbane. For more information go to raci.org.au/PGStudentAward