Dataset Information

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

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.

SUBMITTER: Blonder J

PROVIDER: S-EPMC3251957 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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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.

Blonder Josip J Yu Li-Rong LR Radeva Galina G Chan King C KC Lucas David A DA Waybright Timothy J TJ Issaq Haleem J HJ Sharom Frances J FJ Veenstra Timothy D TD

Journal of proteome research 20060201 2

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 ...[more]

PMID: 16457601

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Project description:Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane.

Dataset Information

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Publications

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Similar Datasets

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