Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.
Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.
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DOI：
10.1016/j.jprot.2010.03.016
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发表时间：
2010-06-16
影响因子：
3.3
通讯作者：
Solaro RJ
中科院分区：
文献类型：
--
作者：
Warren CM;Geenen DL;Helseth DL Jr;Xu H;Solaro RJ
Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1-hour without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissue. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of <5%. Compared to no fractionation prior to LC-MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four fold. We found approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.
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DOI：
10.1124/jpet.104.067926
发表时间：
2004-08-01
影响因子：
3.5
作者：
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影响因子：
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影响因子：
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