title: Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.
abstract: Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.
authors.affiliation: Department of Biological Sciences, Virginia Polytechnic Institute and State University , 1981 Kraft Drive, Blacksburg, Virginia 24061, United States.
authors.forename: Wooram
authors.initials: W
authors.lastname: Lee
authors.forename: Iulia M
authors.initials: IM
authors.lastname: Lazar
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D011506:Proteins