In our next Researcher Live series this summer, we will be focusing on ‘Applications of Mass Spectrometry’ – bringing you four fantastic speakers over the course of July and early August.
Join our second episode on 21st July at 4pm BST / 3pm GMT with Dr Aaron Bailey, The University of Texas Medical Branch. Sign up here to receive email reminders for this series.
What are we going to talk about in this episode?
Protein primary structure characterization is a basic requirement for understanding structure/function relationships in all application areas of protein engineering and basic research. Purified protein samples are mixtures of specific protein isoforms, or “proteoforms”, which are based on a common amino acid sequence with various possible post-translation modifications (PTMs) and/or sequence changes. Mass spectrometry, often combined with separation by liquid chromatography (LC−MS) is the predominant means for characterizing proteins, minimally achieved using a combination of two essential assays: LC−MS/MS peptide mapping analysis (akin to bottom-up proteomics) and LC−MS intact mass analysis. Reversed-phase LC−MS/MS peptide mapping allows for determination of amino acid sequence and PTM site data. Intact mass analysis offers semi-quantitative profiling of protein isoforms subjected to either denaturing or non-denaturing (“native”) MS conditions. To address issues of protein overcharging of unstructured proteins under acidic, denaturing conditions and sample heterogeneity (macro- and micro-scales) which often confound denaturing intact mass analysis of a wide variety of protein samples, we demonstrate the use of broadband isolation of entire charge state distributions of intact proteins followed by ion-ion proton transfer charge reduction, which we have termed "full scan PTCR" (fsPTCR). Using rapid denaturing size exclusion chromatography coupled to fsPTCR-Orbitrap MS and time-resolved deconvolution data analysis, we demonstrate a strategy for method optimization, leading to significant analytical advantages over conventional MS1. We show that the increased sensitivity and dynamic range afforded by fsPTCR denaturing intact mass analysis has significantly improved our ability to confidently integrate the multi-assay LC-MS characterization efforts of a wide variety of protein samples.
- 20th July, 10am BST / 9am GMT - ‘A new method for the analysis of short-chain fatty acids (SCFA) and other polar metabolites in microbiome-related samples by ion-exchange chromatography-mass spectrometry (IC-MS)’ with Dr Mariya Misheva, University of Oxford
- 21st July, 4pm BST/ 3pm GMT - ‘Building a Versatile Intact Mass Toolkit to Improve Integrative Protein Structure Characterization for Drug Development and Basic Research’ with Dr Aaron Bailey, The University of Texas Medical Branch
- 28th July, 10am BST/9am GMT – ‘Mass Spectrometry-based bottom-up proteomics and its applications in cancer research: an overview’ with Dr Roopesh Krishnankutty, Senior Mass Spectrometry Researcher at the University of Edinburgh
- 2nd August, 10am BST/9am GMT – ‘Determining mass, why it is important and why it matters?’ with Dr Gillian Taylor - 2nd August 10am BST, University of Newcastle-upon-Tyne
Please follow Researcher Live’s profile ‘Applications of Mass Spectrometry’ for updates on the topic and to keep up with future event series!
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Dr Aaron O. Bailey is currently a Senior Research Scientist and Associate Director of the Mass Spectrometry Core Facility at the University of Texas Medical Branch. After receiving an undergraduate degree in Molecular, Cellular, and Developmental Biology at the University of Colorado in 2003, Dr. Bailey worked as a Research Technician in the laboratory of Dr. John R. Yates III at The Scripps Research Institute, where he began learning about how multidimensional liquid chromatography (LC) and proteomic mass spectrometry (MS) can be used to perform large scale studies of proteomes and purified protein complexes. He completed his doctoral studies under the joint mentorship of Dr. Donald F. Hunt in the Department of Chemistry and Dr. Daniel R. Foltz in the Department of Biochemistry and was supported by a fellowship from the NIH Biotechnology Training Program. After graduate school, Dr. Bailey joined the Life Science Mass Spectrometry group at Thermo Fisher in San Jose, CA and worked closely with both Research & Engineering and Marketing groups by testing prototype HPLC and MS instrumentation and developing new methods for LC-MS characterization of intact proteins, including novel methods for in-line coupling of novel non-denaturing separations to non-denaturing mass spectrometry using newly compatible Orbitrap mass spectrometers. Dr. Bailey returned in late 2019 to academic research, joining the Mass Spectrometry Facility at the University of Texas Medical Branch in Galveston, Texas, USA. Under the leadership of Mass Spectrometry Facility Director, Dr. Bill Russell, Dr. Bailey continues to focus on developing new LC-MS technologies for protein characterization, leveraging combined efforts of bottom-up proteomics approaches and LC-MS/MS peptide mapping methods, as well as multiple LC-based protein separation strategies enabling intact mass profiling in both denaturing and native conditions
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