title: Deep Coverage of Global Protein Expression and Phosphorylation in Breast Tumor Cell Lines Using TMT 10-plex Isobaric Labeling.
abstract: Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8,706 proteins and 28,186 phosphopeptides, including 7,394 proteins and 23,739 phosphosites common to all channels. The majority of technical replicates correlated with a R2≥ 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide data sets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta, and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2> 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics.
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York 10016, United States.
authors.forename: Fang-Ke
authors.initials: FK
authors.lastname: Huang
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York 10016, United States.
authors.forename: Guoan
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
authors.forename: Kevin
authors.initials: K
authors.lastname: Lawlor
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
authors.forename: Arpi
authors.initials: A
authors.lastname: Nazarian
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
authors.forename: John
authors.initials: J
authors.lastname: Philip
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Tempst
authors.affiliation: Sandra and Edward Meyer Cancer Center, Department of Biochemistry, Weill Cornell Medical College , New York, New York 10065, United States.
authors.forename: Noah
authors.initials: N
authors.lastname: Dephoure
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York 10016, United States.
authors.forename: Thomas A
authors.identifier: 0000-0001-7049-2088
authors.initials: TA
authors.lastname: Neubert
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D013194:Staining and Labeling; D014443:Tyrosine / Q000378:metabolism
chemical_list: D010748:Phosphopeptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D014443:Tyrosine; C508053:ERBB2 protein, human
keywords: hierarchical clustering; mass spectrometry; phosphoproteomics; quantitative proteomics; tandem mass tag; titanium dioxide; tyrosine phosphorylation