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Mass spectrometry Proteomics: Depth of Sequencing vs. Breadth of Identifications

Biology Colloquium | Dr. Sabyasachi Baboo | Oct 3rd, 2023

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Proteomics is the study of proteomes, where the proteome is defined as group of all proteins expressed in an organism or species. This definition of proteome has been diluted to include sub-groups of proteins based on tissues, cell-types, protein-age and sub-cellular or extra-cellular localization of proteins.  However, the defining factor remains a large and complex mixture of proteins. Identification and quantification of as many proteins as possible, in such a complex mixture is challenging and determines the potential of a proteomics approach. Bottom-up proteomics by mass spectrometry (MS) provides the best-in-class breadth of protein identifications, and over the last few decades, this has been tremendously enhanced owing to increased sensitivity of mass spectrometers, better chromatographic separation, and improved computational tools for data analysis. The fantastic mass resolution provided by modern MS is also able to accurately identify post-translational modifications (PTMs) on proteins, that play an essential role in critical protein functions. Evaluating them by MS can be limited owing to loss of sequence coverage by “amino acid”-specific cleavage by trypsin, where in missing sequence coverage could result in information loss for any PTMs hosted on these missing sequences. I use the unconventional non-specific protease – Proteinase K, at sub-optimal conditions and simplified digestion reactions to yield complete sequence coverage of purified proteins. Though applied for vaccine development to evaluate the complicated PTMs that are N-glycans, this method named DeGlyPHER, can be applied for any PTM, generating abundant data at each PTM site, significantly improving confidence and sensitivity in its quantitation.

About the Speaker:

Sabyasachi Baboo is a Staff Scientist in Prof. John R. Yates III’s lab at Scripps Research, California – a lab known for its pioneering contributions and as one of the leaders in the field of mass spectrometry-based proteomics since its inception a few decades ago.

After graduating with Botany major, topping the Visva-Bharati University and completing his Masters in Genetics from University of Delhi, Sabyasachi pursued his DPhil/PhD from the Sir William Dunn School of Pathology in University of Oxford, UK, on a prestigious Felix Scholarship. Here, under the mentorship of Prof. Peter R. Cook, he generated highly persuasive evidence that some translation occurs in eukaryotic nucleus and proposed that majority of translation initiated is abortive, like transcription.

Sabyasachi joined for his postdoctoral research in Prof. John R. Yates III’s lab at Scripps Research, California, and has been continuing here for 6 years. Since last 4 years, he is mostly invested in leading an initiative for analyzing site-specific N-glycosylation on viral spike proteins – especially on HIV Env, as part of the NIH/NIAID funded international Consortium for HIV/AIDS Vaccine Development (CHAVD) led by Scripps Research. Using the DeGlyPHER method that he developed in Yates lab, he has been assisting CHAVD collaborators in analyzing the N-glycan cover on HIV immunogens that they are designing and testing in human clinical and non-human primate trials. In addition, he collaborates exhaustively with scientists on evaluating protein turnover, protein-protein interaction, and post-translational modifications (PTMs) on proteins, across range of applications in protein quality control, vaccine design, subcellular biology, and animal physiology. He has presented his work at prestigious international conferences such Keystone Symposia, Human Proteome Organization (HUPO) Congress and US chapter, and American Society for Mass Spectrometry (ASMS) Annual Meeting. Recently, he has been honored with Young Investigator Award from the Scripps CHAVD for his contributions toward HIV vaccine development.

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