Targeted proteomic analysis of glycolysis in cancer cells

  1. Get@NRC: Targeted proteomic analysis of glycolysis in cancer cells (Opens in a new window)
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Journal titleJournal of Proteome Research
Pages604613; # of pages: 10
SubjectGlycolysis; Invasiveness; Lactic acid; Multiple reaction monitoring; Pyruvate kinase M2; Targeted proteomics; Warburg effect
AbstractAltered expression of glycolysis proteins is an important yet poorly understood characteristic of cancer. To better understand the glycolytic changes during tumorigenesis, we designed a liquid chromatography multiple reaction monitoring (LC-MRM) assay targeting the "glycolysis proteome" in MCF-7 breast cancer cells, using isotope-coded dimethylation of peptides for relative quantification. In silico, dimethyl labeled tryptic peptides [M + 2H] 2+ (of length n) and their y n-1 fragment ions were determined based on UniprotKB database sequence entries for glycolysis proteins, related branching pathways, and reference proteins. Using predicted transitions ([M + 2H] 2+ - y n-1), MRM-initiated detection and sequencing (MIDAS) was performed on a dimethyl-labeled, tryptic digest from MCF-7 cells, using two- dimensional liquid chromatography mass spectrometry analysis. Three transitions for each peptide were selected from identified spectra and assessed using 1D-LC-MRM-MS. Collision energy (CE) and dwell times were optimized and matching transitions for "heavy" isotope-coded dimethylated peptides were calculated. Resulting LC-MRM transitions were then used to measure changes in the glycolytic proteome in insulin-like growth factor-1 (IGF-l)-stimulated MCF-7 cells and other breast cell lines. Increases in the expression of glycolysis proteins leading to lactic acid production were observed common to IGF-1-stimulated MCF-7 cells and the invasive MDA-MB-231 cell line. Preliminary analysis of lung tumors with varied states of differentiation demonstrated the clinical applicability of LC-MRM and showed decreased levels of PGK1 in poorly differentiated tumors.
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AffiliationNRC Institute for Marine Biosciences; National Research Council Canada
Peer reviewedYes
NPARC number19709156
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Record identifierd2e4b9e5-67c8-4ac1-b4c3-de269410465b
Record created2012-04-02
Record modified2016-05-09
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