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Isotopic N,N-Dimethyl Leucine (iDiLeu) for Absolute Quantification of Peptides Using a Standard Curve Approach

  • Tyler Greer
  • Lingjun Li
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)

Abstract

Quantitative proteomics studies require an absolute quantification step to accurately measure changes in protein concentration. Absolute quantification using liquid chromatography–mass spectrometry (LC–MS) traditionally combines triple quadrupole instrumentation with stable isotope-labeled standards to measure protein concentrations via their enzymatically produced peptides. Chemical modification of peptides using labels like mass differential tags for relative and absolute quantification (mTRAQ) provides another route to determine protein quantities. This chapter describes a cost-effective and high-throughput chemical labeling method that utilizes five amine-reactive, isotopic N,N-dimethyl leucine (iDiLeu) reagents. These tags enable generation of four-point calibration curves in one LC–MS run to determine protein concentrations from labeled peptides. In particular, we provide a detailed workflow for protein quantification using the iDiLeu reagent that includes important considerations like labeling conditions and isotopic interference correction.

Key words

Quantification iDiLeu DiLeu Mass difference labeling Proteomics Calibration curve 

Notes

Acknowledgments

The authors acknowledge support for this work by the National Institutes of Health grant (1R01DK071801). The Q-Exactive Orbitrap was purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531). L.L acknowledges an H.I. Romnes Faculty Research Fellowship.

References

  1. 1.
    Makawita S, Diamandis EP (2010) The bottleneck in the cancer biomarker pipeline and protein quantification through mass spectrometry-based approaches: current strategies for candidate verification. Clin Chem 56:212–222. doi: 10.1373/clinchem.2009.127019 CrossRefPubMedGoogle Scholar
  2. 2.
    Addona TA, Shi X, Keshishian H et al (2011) A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease. Nat Biotechnol 29:635–643. doi: 10.1038/nbt.1899 PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Barr JR, Maggio VL, Patterson DG et al (1996) Isotope dilution – mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin Chem 42:1676–1682PubMedGoogle Scholar
  4. 4.
    Gerber SA, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940–6945. doi: 10.1073/pnas.0832254100 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Zhao Y, Brasier AR (2013) Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways. Methods 61:313–322. doi: 10.1016/j.ymeth.2013.02.001 PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Warnken U, Schleich K, Schnölzer M, Lavrik I (2013) Quantification of high-molecular weight protein platforms by AQUA mass spectrometry as exemplified for the CD95 death-inducing signaling complex (DISC). Cells 2:476–495. doi: 10.3390/cells2030476 PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Sturm R, Sheynkman G, Booth C et al (2012) Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow. J Am Soc Mass Spectrom 23:1522–1533. doi: 10.1007/s13361-012-0411-1 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    DeSouza L, Taylor A, Li W (2008) Multiple reaction monitoring of mTRAQ-labeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal. J Proteome Res 7:3525–3534CrossRefPubMedGoogle Scholar
  9. 9.
    Zhou L, Wei R, Zhao P et al (2013) Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjögren’s syndrome-associated dry eye. Proteomics 13:2469–2481. doi: 10.1002/pmic.201200230 PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Zhang S, Wen B, Zhou B et al (2013) Quantitative analysis of the human AKR family members in cancer cell lines using the mTRAQ/MRM approach. J Proteome Res 12:2022–2033. doi: 10.1021/pr301153z CrossRefPubMedGoogle Scholar
  11. 11.
    Xiang F, Ye H, Chen R et al (2010) N,N-dimethyl leucines as novel isobaric tandem mass tags for quantitative proteomics and peptidomics. Anal Chem 82:2817–2825. doi: 10.1021/ac902778d PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Hui L, Xiang F, Zhang Y, Li L (2012) Mass spectrometric elucidation of the neuropeptidome of a crustacean neuroendocrine organ. Peptides 36:230–239. doi: 10.1016/j.peptides.2012.05.007 PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Sturm RM, Lietz CB, Li L (2014) Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry. Rapid Commun Mass Spectrom 28:1051–1060. doi: 10.1002/rcm.6875 PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Hao L, Zhong X, Greer T et al (2015) Relative quantification of amine-containing metabolites using isobaric N,N-dimethyl leucine (DiLeu) reagents via LC-ESI-MS/MS and CE-ESI-MS/MS. Analyst 140:467–475. doi: 10.1039/C4AN01582G PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Frost DC, Greer T, Li L (2015) High-resolution enabled 12-plex DiLeu isobaric tags for quantitative proteomics. Anal Chem 87:1646–1654. doi: 10.1021/ac503276z PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Greer T, Lietz CB, Xiang F, Li L (2015) Novel isotopic N,N-dimethyl leucine (iDiLeu) reagents enable absolute quantification of peptides and proteins using a standard curve approach. J Am Soc Mass Spectrom 26:107–119. doi: 10.1007/s13361-014-1012-y PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Mertins P, Udeshi ND, Clauser KR et al (2012) iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics. Mol Cell Proteomics. doi: 10.1074/mcp.M111.014423 Google Scholar
  18. 18.
    Shadforth IP, Dunkley TPJ, Lilley KS, Bessant C (2005) i-Tracker: for quantitative proteomics using iTRAQ. BMC Genomics 6:145. doi: 10.1186/1471-2164-6-145 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.School of PharmacyUniversity of Wisconsin-MadisonMadisonUSA

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