Pharmaceutical Sciences Faculty Publications

Title

Combined Chemical and Enzymatic Stable Isotope Labeling for Quantitative Profiling of Detergent-Insoluble Membrane Proteins Isolated Using Triton X-100 and Brij-96

Document Type

Article

Publication Date

2-1-2006

Journal Title

Journal of Proteome Research

ISSN

1535-3893

Volume

5

Issue

2

First Page

349

Last Page

360

DOI

10.1021/pr050355n

PubMed ID

16457601

PubMed Central® ID

PMC3251957

Abstract

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Keywords

Amino acid sequence, biotin, carbon radioisotopes, cell line, tumor, chromatography, affinity, detergents, deuterium, isotope labeling, microdomains, proteins, octoxynol, oxygen radioisotopes, plant oils, polyethylene glycols, proteomics, spectrometry, mass, electrospray ionization, trypsin

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