Open AccessHigh-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry
Author(s) -
Zhen Wang,
Kanisha Kavdia,
Kaushik Dey,
Vishwajeeth Pagala,
Kiran Kodali,
Danting Liu,
Dong Geun Lee,
Huihui Sun,
Surendhar Reddy Chepyala,
Ji-Hoon Cho,
Mingming Niu,
Anthony A. High,
Junmin Peng
Publication year - 2020
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/61684
Subject(s) - isobaric labeling , tandem mass tag , tandem mass spectrometry , proteome , proteomics , mass spectrometry , chemistry , chromatography , fractionation , quantitative proteomics , computational biology , biology , protein mass spectrometry , biochemistry , gene
Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.