Targeted proteomic analysis of glycolysis in cancer cells.
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.
National Research Council Institute for Marine Biosciences, Halifax, NS, Canada.
J Patrick
JP
Murphy
Devanand M
DM
Pinto
D045744:Cell Line, Tumor; D003198:Computer Simulation; D005260:Female; D006019:Glycolysis; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry; D009369:Neoplasms / Q000201:enzymology / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics; D020539:Sequence Analysis, Protein
D020543:Proteome; D007334:Insulin-Like Growth Factor I