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Pierre Massion
Faculty Member
Last active: 1/11/2018

Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry.

Zhang H, Liu Q, Zimmerman LJ, Ham AJ, Slebos RJ, Rahman J, Kikuchi T, Massion PP, Carbone DP, Billheimer D, Liebler DC
Mol Cell Proteomics. 2011 10 (6): M110.006593

PMID: 21357624 · PMCID: PMC3108838 · DOI:10.1074/mcp.M110.006593

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.

MeSH Terms (17)

Adenocarcinoma Alkaline Phosphatase Amino Acid Sequence Biomarkers, Tumor Carcinoma, Squamous Cell Cell Line, Tumor Chromatography, Liquid Humans Isotope Labeling Lung Neoplasms Mass Spectrometry Molecular Sequence Data Oligopeptides Peptide Fragments Peptides Proteins Reference Standards

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