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

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journal contribution
posted on 2006-02-03, 00:00 authored by Josip Blonder, Li-Rong Yu, Galina Radeva, King C. Chan, David A. Lucas, Timothy J. Waybright, Haleem J. Issaq, Frances J. Sharom, Timothy D. Veenstra
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: quantitative proteomics • combined 16O/18O and ICAT stable isotopic labeling • Triton X-100 and Brij-96 detergent-insoluble membrane proteins