High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry.

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.