MSACL 2022 Abstract

Mapping the 3 D Proteome Using Surface Accessibility in Animal Models of Disease

Ahrum Son, Casimir Bamberger, Sandra Pankow, John R. Yates III
Departments of Molecular Medicine and Neurobiology, 10550 North Torrey Pines Road, SR302B, The Scripps Research Institute, LaJolla, CA 92037

John Yates, PhD (Presenter) Scripps Research Institute

Presenter Bio: John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. He received a B.A in Zoology and an M.S. in Chemistry from the University of Maine at Orono. He obtained his Ph.D. in Chemistry at the University of Virginia in the laboratory of Donald F. Hunt with a dissertation entitled Protein Sequencing by Tandem Mass Spectrometry. He performed postdoctoral research in the laboratory of Leroy E. Hood at California Institute of Technology. At the University of Washington, he obtained the rank of Associate Professor with tenure before moving to The Scripps Research Institute in LaJolla, CA. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award, 2015 The Ralph N. Adams Award in Bioanalytical Chemistry, the 2018 Thomson Medal from the International Mass Spectrometry Society, the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS, the 2019 HUPO Award in Discovery, and the 2024 Pittsburgh Society Award in Analytical Chemistry. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List of Most Highly Influential Biomedical Researchers, 1996-2011 (European J. Clinical Investigation 2013, 43, 1339-1365) and the Clarivate List of Highly Cited Scientists in 2015 and 2019-2024. He has published over 1000 scientific articles with >183,000 citations, and an H index of 211 (Google Scholar). Dr. Yates served as an Associate Editor at Analytical Chemistry for 15 years and is currently the Editor in Chief at the Journal of Proteome Research.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) Grant/Research Support NIH Committee/Board/Advisory Board OMASS, 908Devices, Yatiri Royalty / IP / Other Income Patent royalties from University of Washington Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Methods to determine the surface accessibility of amino acids on proteins can provide information about conformational changes to proteins because of mutation, drug treatment, or other perturbations. These methods such as FPOP, SPROX or LiP are collectively described as protein footprinting and when integrated with proteomic methods can provide global information on the status of protein folding.

OBJECTIVES:
To develop a method to perform protein footprinting in animal models of disease.

METHODS:
We describe an approach using formaldehyde to label accessible lysine residues in intact cells which are then converted to dimethyl tags using cyanoborohydride. This method has been dubbed Covalent Protein Painting (CPP). As has been used in quantitative proteomics, different stable isotope elements can be used in formaldehyde and cyanoborohydride to create different weight tags and thus a secondary labeling after proteolytic digestion of proteins using different weight reagents labels lysine residues that were not labeled. When performed in vivo this method in combination with advanced mass spectrometry/proteomic methods can provide a measure of how surface accessibility changes in proteins based on a perturbation. Recent efforts to advance this method to animal models of disease will be discussed.

RESULTS:
By using our perfusion method proteins were uniformity labeled and reproducibility between biological replicates was good. Protein conformation differences were measured in mice using age as a perturbation. About 5,000 proteins were identified in 8 different tissues in 3- and 9-month-old mice to compare how protein conformations change as mice age. The surface accessibility of some proteins was changed as a function of age.

CONCLUSION:
We applied a cardiac perfusion method to covalent protein painting (CPP) to measure the changes in the structure of proteins while maintaining their in-vivo conformation as much as possible. We observed reproducible labeling performance between biological replicates
consistent labeling efficiency across organs overall. Improved methods for mass spectrometry analyses to increase sequence coverage would help improve reproducibility. In the brain data, we observed changes in several age-related pathways where accessibility was altered more than 1.5-fold in older mice (9 months in age).