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High-Throughput Quantitative Proteomics Enabled by Mass Defect-Based 12-Plex DiLeu Isobaric Tags

  • Dustin C. Frost
  • Lingjun Li
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)

Abstract

Isobaric labeling has become a popular technique for high-throughput, mass spectrometry (MS)-based relative quantification of peptides and proteins. However, widespread use of the approach for large-scale proteomics applications has been limited by the high cost of commercial isobaric tags. To address this, we have developed our own N,N-dimethyl leucine (DiLeu) multiplex isobaric tags as a cost-effective alternative that can be synthesized with ease using readily available isotopic reagents. When paired with high-resolution tandem mass (MS n ) acquisition, mass defect-based DiLeu isobaric tags allow relative quantification of up to twelve samples in a single liquid chromatography (LC)–MS2 experiment. Herein, we present detailed methods for synthesis of 12-plex DiLeu isobaric tags, labeling of complex protein digest samples, analysis by high-resolution nanoLC–MS n , and processing of acquired data.

Key words

Quantitative proteomics Isobaric labeling DiLeu High-resolution mass spectrometry Mass defect Isobaric tag synthesis Multiplexed quantitation 

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.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

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

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