Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry

Abstract

Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two-dimensional liquid chromatography, TiO₂-mediated phosphopeptide enrichment, high-resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.

Keywords: Bioinformatics; Isobaric labeling; Liquid chromatography; Mass spectrometry; Phosphoproteome; Proteomics; Tandem mass tags.

Fig. 1

TMT10-plex reagents. (A) TMT reagents label amine-reactive group (e.g. Lys residues and the amino termini of peptides). (B) The 10 reagents in the TMT10-plex kit (asterisks indicate the positions of 13C and 15N heavy isotopes). (C) TMT reporter ions in an MS/MS spectrum. The intensities of these reporters are used for protein quantitation.

Fig. 2

Workflow of TMT-LC/LC-MS/MS for profiling whole proteome and phosphoproteome. (A) The analysis scheme. (B) Basic pH RPLC fractionation for whole proteome analysis. (C) Basic pH RPLC fractionation for phosphoproteome analysis.

Fig. 3

Quality control analysis of one batch of 10 samples by Western blotting analysis. Protein A is a positive control protein expressed at a higher level in samples 6–10. Protein B is a loading control showing no difference in the samples.

Fig. 4

Labeling efficiency analysis. (A) An unlabeled peptide of strong intensity at retention time of 7.4 min during LC-MS/MS. (B) Complete absence of the same peptide at 7.2 min and the presence of the labeled peptide at 8.2 min, indicating fully TMT labeling.

Fig. 5

Analysis of experimental variance and control-case comparison. Distribution of log2 ratios between biological replicates (null experiment) and the control-case comparison.