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

Haixia Zhang, Qinfeng Liu, Lisa J. Zimmerman, Amy Joan L Ham, Robbert J C Slebos, Jamshedur Rahman, Takefume Kikuchi, Pierre P. Massion, David P. Carbone, David D Billheimer, Daniel C. Liebler

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
JournalMolecular and Cellular Proteomics
Volume10
Issue number6
DOIs
StatePublished - Jun 2011

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Liquid chromatography
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Peptides
Monitoring
Isotopes
Dilution
Proteins
Labels
Tumors
Galactosidases
Tissue
Plasma (human)
Costs and Cost Analysis
Biomedical Technology Assessment
Neoplasms
Lung
National Cancer Institute (U.S.)
Escherichia coli Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry
  • Medicine(all)

Cite this

Methods for peptide and protein quantitation by liquid chromatography- multiple reaction monitoring mass spectrometry. / Zhang, Haixia; Liu, Qinfeng; Zimmerman, Lisa J.; Ham, Amy Joan L; Slebos, Robbert J C; Rahman, Jamshedur; Kikuchi, Takefume; Massion, Pierre P.; Carbone, David P.; Billheimer, David D; Liebler, Daniel C.

In: Molecular and Cellular Proteomics, Vol. 10, No. 6, 06.2011.

Research output: Contribution to journalArticle

Zhang, H, Liu, Q, Zimmerman, LJ, Ham, AJL, Slebos, RJC, Rahman, J, Kikuchi, T, Massion, PP, Carbone, DP, Billheimer, DD & Liebler, DC 2011, 'Methods for peptide and protein quantitation by liquid chromatography- multiple reaction monitoring mass spectrometry', Molecular and Cellular Proteomics, vol. 10, no. 6. https://doi.org/10.1074/mcp.M110.006593
Zhang, Haixia ; Liu, Qinfeng ; Zimmerman, Lisa J. ; Ham, Amy Joan L ; Slebos, Robbert J C ; Rahman, Jamshedur ; Kikuchi, Takefume ; Massion, Pierre P. ; Carbone, David P. ; Billheimer, David D ; Liebler, Daniel C. / Methods for peptide and protein quantitation by liquid chromatography- multiple reaction monitoring mass spectrometry. In: Molecular and Cellular Proteomics. 2011 ; Vol. 10, No. 6.
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abstract = "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.",
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