Spectronaut User Seminar II

“Data-independent Acquisition Strategies to Gain Deep Coverage into Human Plasma Exosomes for Biomarker Research”

Prof. Dr. Birgit Schilling (The Buck Institute for Research on Aging)

 

Abstract: Exosomes facilitate various cell-to-cell communication and thus have a remarkably diverse implication in cellular signaling ranging from cancer to aging. Plasma exosomes carry an immense potential to serve as biomarkers. However, isolation of ‘pure’ exosomes from plasma has been challenging due to contaminations from soluble plasma proteins. We developed a high-throughput method to isolate exosomes from human plasma employing sequential size-exclusion chromatography and ultrafiltration (SEC/UF). Quality control of isolated exosomes (e.g., exosome surface markers, particle-size, protein profiles comparisons with ‘Exocarta’) confirmed high ‘exosome purity’. Using an Orbitrap Exploris 480 mass spectrometer we acquired data-dependent acquisitions (DDA) from 22 exosome fractions (generated by off-line high pH reversed-phase peptide fractionation=HPRP). In combination with additional data-independent acquisitions (DIA), we generated a deep proteomic plasma exosome spectral library (~2,100 proteins). Subsequently, for a Pilot DIA-MS study we compared plasma exosomes from young (n=5; 20–26 yrs) and old (n=5; 60–66 yrs) individuals.  We identified and quantified 1,208 total proteins, and of these 197 proteins showed significant changes between young and old plasma groups (with Q<0.05, >1.5-fold change). The use of plasma exosome biomarkers carries great potential for translational studies investigating biomarkers of aging and aging-related diseases.

 

Short-Biography: 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 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, and these are particularly applicable for the proposed project. We are using proteomic data-independent acquisitions (DIA), or SWATH which allows us to accurately determine changes in relative protein expression level between multiple different conditions. A key interest is in finding senescence-derived biomarker candidates for aging.

 

 

“Quantifying the Expression and Turnover Control of Proteoforms”

Prof. Dr. Yansheng Liu (Yale University - School of Medicine)

 

Abstract: The term 'proteoform' is now used to designate different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications. We will present our work using data independent acquisition mass spectrometry (DIA-MS), pulse-chase stable isotope-labeled amino acids in cells (pSILAC) approach, and genome-wide correlation analysis for quantifying both abundance and turnover rate of proteins in genome aneuploidy models. We will further discuss the results of two major proteoforms, namely alternatively spliced protein isoform groups, and site-specific protein phosphorylation, and reveal their genetic and non-genetic quantitative determinants in aneuploidy.

 

Short_Biography: Yansheng Liu is an Assistant Professor in the Department of Pharmacology at Yale University School of Medicine. During 2011-2017, He accomplished his postdoc training in the Ruedi Aebersold lab, at ETH Zurich, where he contributed to the development of SWATH-MS, a data-independent acquisition mass spectrometry (DIA-MS) technique. In December 2017, he joined the faculty at the Yale Cancer Biology Institute and started his own group. Dr. Liu has a long-term research interest in studying human “genotype-phenotype” association. His current research group aims to contribute to the development of multiplexed, high-throughput DIA-MS technique and other proteomic methods, as well as their applications in protein turnover, cancer aneuploidy, and signaling transduction.

 

 

  

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