Spectronaut 16: Reaching New Depths in DIA Proteomics with AI

High-throughput DIA Analysis of Ischemic Kidney Injury and Therapeutic Interventions Using Multiple MS Platforms

Birgit Schilling (The Buck Institute)

Post-translational modifications (PTMs) dynamically regulate proteins and pathways through immediate signaling cascades and resulting changes in protein function or activity. Here, we are investigating the protective role of lysine succinylation during ischemia- and -reperfusion-induced acute kidney injury (AKI) which typically would cause kidney dysfunction and oxidative stress. Recently, we examined the succinylome profile during acute kidney injury and the effects of sirtuin-5 knockout, and alternatively the use of 2 different nutritional supplements on AKI injury recovery in mice. Using data-independent acquisitions, we identified and quantified 3,666 succinylated sites in total, and determined that the diet induced significant hypersuccinylation of 1,085 sites which remodeled the succinylome completely. However, this treatment barely affected the proteome itself. Upregulated succinylation sites were related to lipid and fatty acid oxidation, and peroxisomal metabolic pathways. We hypothesize that the increased succinylation levels promote a switch of the fatty acid oxidation from mitochondria to peroxisomes which greatly reduces oxidative stress and thereby protects against AKI. Our spectral library free directDIA workflows using the Orbitrap Eclipse are highly efficient and have allowed us to optimize and increase throughput for our PTM-DIA quantification workflows. We also investigated changes on the protein level with AKI (using a ZenoTOF 7600 system) which showed significant and broad protein level changes even with short ischemia reperfusion injury times. The novel Spectronaut 16 allowed us to improve our identification and quantification rates searching our data-independent acquisitions by 20-25 %. Altogether, our work demonstrates the promising translational and therapeutic applications of nutritional supplements to rescue mammalian (mouse, non-human primate and human) kidney injury phenotypes, and in addition to provide alternative energy sources for patients with genetic disorders affecting mitochondrial metabolisms.

Short Bio Birgit Schilling

Dr. Schilling is an Associate Professor and the Director of the Mass Spectrometry Core at the Buck Institute for Research on Aging in California, and she is also an Adjunct Professor at the University of Southern California (USC). The Schilling lab develops and implements advanced innovative protein analytical technologies (including quantitative proteomics, posttranslational modifications, protein dynamics, and biomarker discovery) to advance basic biology and biomedical research related to aging research. Several research projects include the investigation of protein phosphorylation, acylation, and other posttranslational modifications, as well as differential expression of proteins during disease and aging processes. We are particularly interested in deciphering underlying mechanisms of senescence during aging, and we have developed MS methodologies to quantitatively analyze protein secretomes, secreted exosomes and to perform accurate quantitative protein expression workflows. The Schilling lab has adopted several novel proteomic technologies with comprehensive and extremely sensitive quantification capabilities. We are using proteomic data-independent acquisitions (DIA), or SWATH which allows us to accurately determine changes in relative protein expression levels between multiple different conditions. A key interest is in finding senescence-derived biomarker candidates for aging.