title: Relative protein quantification and accessible biology in lung tumor proteomes from four LC-MS/MS discovery platforms.
abstract: Discovery proteomics experiments include many options for sample preparation and MS data acquisition, which are capable of creating datasets for quantifying thousands of proteins. To define a strategy that would produce a dataset with sufficient content while optimizing required resources, we compared (1) single-sample LC-MS/MS with data-dependent acquisition to single-sample LC-MS/MS with data-independent acquisition and (2) peptide fractionation with label-free (LF) quantification to peptide fractionation with relative quantification of chemically labeled peptides (sixplex tandem mass tags (TMT)). These strategies were applied to the same set of four frozen lung squamous cell carcinomas and four adjacent tissues, and the overall outcomes of each experiment were assessed. We identified 6656 unique protein groups with LF, 5535 using TMT, 3409 proteins from single-sample analysis with data-independent acquisition, and 2219 proteins from single-sample analysis with data-dependent acquisition. Pathway analysis indicated the number of proteins per pathway was proportional to the total protein identifications from each method, suggesting limited biological bias between experiments. The results suggest the use of single-sample experiments as a rapid tissue assessment tool and digestion quality control or as a technique to maximize output from limited samples and use of TMT or LF quantification as methods for larger amounts of tumor tissue with the selection being driven mainly by instrument time limitations. Data are available via ProteomeXchange with identifiers PXD004682, PXD004683, PXD004684, and PXD005733.
authors.affiliation: Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Stewart
authors.affiliation: Proteomics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Fang
authors.affiliation: Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Robbert J C
authors.initials: RJ
authors.lastname: Slebos
authors.affiliation: Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Guolin
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Borne
authors.affiliation: Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Fellows
authors.affiliation: Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Jamie K
authors.initials: JK
authors.lastname: Teer
authors.affiliation: Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Y Ann
authors.initials: YA
authors.lastname: Chen
authors.affiliation: Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Welsh
authors.affiliation: Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Eschrich
authors.affiliation: Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
authors.affiliation: Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Koomen
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D010455:Peptides; D020543:Proteome
keywords: Data-independent acquisition; Discovery proteomics; Label-free quantification; Lung squamous cell carcinoma; TMT