Post Translational Modification: Antibody Enrichment for Mass Spectrometry-based Proteomics
Continuing our theme of simplifying proteomics we present a webinar featuring Matthew Stokes, Ph.D., principal scientist of the proteomics group here at Cell Signaling Technology and Christopher Rose, Ph.D., a postdoctoral research fellow in the Gygi Lab at Harvard Medical School.
Dr. Stokes describes PTMScan® technology, a method that uses antibodies to enrich specific post-translationally modified (PTM) peptides (e.g., phosphorylated, methylated, ubiquitinated etc) from a complex mixture prior to LC-MS/MS analysis. Dr. Rose demonstrates how, by combining PTMScan technology with isobaric labeling, specifically with tandem mass tags (TMTs), his lab quantified over 15,000 ubiquitination events in Bortezomib treated cells.
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Abstract for PTMScan® Proteomics: Identification and Quantification of Post-Translationally Modified Peptides.
Protein post-translational modifications (PTMs) regulate all aspects of cellular growth, development, and disease biology. Proteomic analysis of these PTMs has become central to gaining a better understanding of cellular signaling. Current proteomic methods and instrumentation allow for identification of thousands of peptides in a single experiment. PTMScan proteomicsfrom Cell Signaling Technology combine antibody-based enrichment with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for identification and quantitation of post-translationally modified peptides. Products and services are available for analysis of protein phosphorylation, acetylation, ubiquitination, methylation, succinylation, and caspase cleavage from cell lines, tissues, xenografts, or other biological materials (PTMScan Discovery). Services have also been designed for targeted, in-depth coverage of critical signaling pathways (PTMScan Direct). PTMScan methods have been successfully applied to biomarker discovery, drug development, and studies to gain a better understanding of cellular signaling.
Multiplexed Quantitative Ubiquitylome Analysis utilizing Ubiquitin Remnant Immunoprecipitation, nLC-MS/MS, and Isobaric Labels. Large-scale, multiplexed quantitative proteomics experiments have recently become more accessible due to advances in both mass spectrometry instrumentation and reagents. Today, by utilizing isobaric labels (e.g., tandem mass tags), >8,000 proteins can be routinely quantified across 10 experimental samples in 1.5 days of analysis. However, to date, the analysis of the cellular ubiquitylome has been limited in the number of samples that can be multiplexed in one experiment. In this webinar I will highlight sample preparation methods including immunoprecipitation with the PTMScan ubiquitin remnant motif antibody (Cell Signaling Technology) and labeling of resulting peptides with isobaric tags. I will also discuss instrument methods (e.g., synchronous precursor selection MS3, SPS-MS3) that allow for accurate analysis of cellular ubiquitylomes. I will highlight the application of this method in which we quantified the change in >15,000 sites of ubiquitylation simultaneously across 10 quantitative channels upon treatment with Bortezomib.