Tom Lin, Ph.D., got his B.S. in Pharmacy in 2009 from Zhengzhou University, M.S. in Bioanalytical Technology in 2012 from Peking University, and Ph.D. in Medicinal Chemistry in 2017 from University of Tennessee HSC. He has been a postdoc for 4 years in Megan Matthews group at University of Pennsylvania working on protein-hydrazine covalent chemistry using Activity-based Protein Profiling (ABPP). Since 2021, he has been a Staff Scientist in Benjamin Garcia lab at Washington University in St. Louis working on discovery and biological functions of posttranslational arginylation.
Posttranslational arginylation installed on proteins by arginyltransferase (ATE1) is a critical modification for mammalian cellular homeostasis and development. Absence of this modification in ATE1 knockout mice was embryonically lethal with various signs of cardiovascular defects. Proteomic profiling of arginylation sites is extremely difficult since mass spectrometry cannot distinguish the same mass of translational and posttranslational arginine (+156 Da), and arginylated proteins go through Arg/N-degron pathway for ubiquitin-mediated degradation. Existing methods have proposed plausible arginylation sites on a handful of proteins (<20), most of which have not been further validated or functionally investigated. Therefore, protein arginylation remains an understudied field.
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In this work, we have developed a deep proteomic profiling approach using high-pH fractionation and proteomics for unbiased identification of arginylation sites from complex whole proteomes. By performing the ATE1 enzyme reaction ex vivo, we incorporated isotopic Arg (Arg10 and Arg0) to proteome-wide arginylation sites under ribosome-free condition. Labeled proteomes were mixed 1:1, prepared, and fractionated for proteomics analysis. Raw data were searched to give peptide IDs. Using Skyline, we were able to determine the co-eluting behaviors and heavy/light ratios (~1.0) of peptides containing both Arg10 and Arg0 modifications. In addition, a customized R script “ArginylomePlot” was written (publicly available at GitHub) to 1) identify co-eluting peptides modified by isotopic Arg, 2) extract their MS1 pairs in sextets (3 heavy and 3 light peaks), 3) calculate H/L ratios and statistics, 4) export all scans/results in PDF. To increase the throughput of the discovery rate by avoiding peptide fractionation, we have developed a data-independent acquisition (DIA) method for analyzing the same samples. A wide m/z window was used to send isotopic MS1 pairs to MS2 for fragmentation to generate b/y ions including those containing Arg10/Arg0 pairs. The DIA data was searched for peptide IDs, among which paired b/y ions with Arg10/Arg0 were analyzed by Skyline to generate H/L ratios at MS2 level first. Peptides with a series of paired b/y ions will be further quantified by Skyline for their H/L ratio at MS1 level. Using Skyline and DIA, we were able to detect all arginylation sites in a high-throughput manner (1 raw/sample). The established approach allows to discover a set of 64 high-confidence targets containing 86 arginylation sites from four cell lines. We have validated 7 of the newly discovered sites using synthetic peptides. As external validation, our identified sites of CALR and PDIA1 are consistent with literature reporting their arginylation sites at E18 and D18, respectively. The developed platform is applicable to any biological lysates and paves the way for functional studies of posttranslational arginylation.
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