BOOST channel Tandem Mass Tag (TMT) approach: quantitation of more than 5,000 unique pTyr sites

Quantitative proteomics generates large datasets with increasing depth and quantitative information. With the advance of mass spectrometry and increasingly larger data sets, streamlined methodologies and tools for analysis and visualization of phosphoproteomics are needed both at the protein and modified peptide levels. To assist in addressing this need, we developed ProteoViz , which includes a set of R scripts that perform normalization, differential expression analysis of both the proteins and enriched phosphorylated peptides, and identify sequence motifs, kinases, and gene set enrichment pathways. The tool generates interactive visualization plots that allow users to interact with the phosphoproteomics results and quickly identify proteins and phosphorylated peptides of interest for their biological study. 

The tool also links significant phosphosites with sequence motifs and pathways that will help explain the experimental condition and guide future experiments. Here, we present the workflow and demonstrate its functionality by analyzing a phosphoproteomic data set from two lymphoma cell lines treated with kinase inhibitors. We also show that using a pervanadate treated cell line as a BOOST channel in a TMT experiment can aid in the identification of low abundance pTyr containing peptides. Over 5000 unique pTyr sites were quantified using RTS (real time search) on a Orbitrap Eclipse.

Who should attend:

• Director, PIs & Managers of Proteomics labs
• Director, PIs & Managers of Biopharma labs
• Current users of Q Exactive technology & QTOF MS analyzing peptides & proteins

What you will learn:

• Sensitivity and Speed of Orbitrap Eclipse
• Use of pervanadate treated cells as BOOST channel for phosphotyrosine data acquisition

Presenter: Ricky D. Edmondson, Ph.D. (Associate Professor of Medicine
Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences)

Dr. Edmonson’s area of expertise is in mass spectrometry-based proteomics and he has been involved in discovery proteomics for over 20 years. For the last 12 years, Dr. Edmonson has Co-Directed the UAMS Proteomics Core Facility where he is involved in project design and the daily operation and maintenance of the UPLC nanospray mass spectrometry systems. He has worked with hundreds of researchers to provide discovery proteomics services through the UAMS Core facility. Prior to his arrival at UAMS, he served for 5 years as the Director of the Center for Proteomics at the FDA’s National Center for Toxicological Research. In addition, Dr. Edmonson has 6 years of experience in leading Discovery Proteomics in a commercial setting within the pharmaceutical and biotechnology industry. He received his PhD in Analytical Chemistry from Texas A&M University.