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.

Project description:Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [?-¹?O?]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min?¹ to 0.001 min?¹. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680.

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.

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