title: 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.
authors.affiliation: St. Jude Children's Research Hospital, Memphis, TN, United States.
authors.forename: B
authors.initials: B
authors.lastname: Bai
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, United States.
authors.forename: H
authors.initials: H
authors.lastname: Tan
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, United States.
authors.forename: V R
authors.initials: VR
authors.lastname: Pagala
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, United States.
authors.forename: A A
authors.initials: AA
authors.lastname: High
authors.affiliation: St. Jude Children's Research Hospital, Memphis, TN, United States.
authors.forename: V P
authors.initials: VP
authors.lastname: Ichhaporia
authors.affiliation: St. Jude Children's Research Hospital, Memphis, TN, United States.
authors.forename: L
authors.initials: L
authors.lastname: Hendershot
authors.affiliation: St. Jude Children's Research Hospital, Memphis, TN, United States; St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, United States. Electronic address: junmin.peng@stjude.org.
authors.forename: J
authors.initials: J
authors.lastname: Peng
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D010750:Phosphoproteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010750:Phosphoproteins; D020543:Proteome
keywords: Bioinformatics; Isobaric labeling; Liquid chromatography; Mass spectrometry; Phosphoproteome; Proteomics; Tandem mass tags