Targeted Proteomic Analysis of Glycolysis in Cancer Cells

• Published in: Journal of proteome research Vol. 10; no. 2; pp. 604 - 613
• Main Authors: Murphy, J. Patrick, Pinto, Devanand M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.02.2011
• Subjects: Cell Line, Tumor; Computer Simulation; Female; Glycolysis; Humans; Insulin-Like Growth Factor I - metabolism; Isotope Labeling; Mass Spectrometry; Neoplasms - enzymology; Neoplasms - metabolism; Proteome - analysis; Proteome - metabolism; Proteomics; Sequence Analysis, Protein; lactic acid; invasiveness; multiple reaction monitoring; glycolysis; pyruvate kinase M2; targeted proteomics; Warburg effect
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/pr100774f

Altered 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-1)-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.