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High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2228, pp. 205-224. - Publication Year :
- 2021
-
Abstract
- Mass spectrometry (MS)-based proteomic profiling of whole proteome and protein posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome with high throughput and deep coverage. The reproducibility of quantification benefits not only from the fascinating developments in high-performance liquid chromatography (LC) and high-resolution MS with enhanced scan rates but also from the invention of multiplexed isotopic labeling strategies, such as the tandem mass tags (TMT). In this chapter, we introduce a 16-plex TMT-LC/LC-MS/MS protocol for proteomic profiling of biological and clinical samples. The protocol includes protein extraction, enzymatic digestion, PTM peptide enrichment, TMT labeling, and two-dimensional reverse-phase liquid chromatography fractionation coupled with tandem mass spectrometry (MS/MS) analysis, followed by computational data processing. In general, more than 10,000 proteins and tens of thousands of PTM sites (e.g., phosphorylation and ubiquitination) can be confidently quantified. This protocol provides a general protein measurement tool, enabling the dissection of protein dysregulation in any biological samples and human diseases.
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 2228
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 33950493
- Full Text :
- https://doi.org/10.1007/978-1-0716-1024-4_15