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Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC–MS/MS approaches to cardiac proteomics by Chad M. Warren; David L. Geenen; Donald L. Helseth; Jr.; Hua Xu; R. John Solaro is a Chemistry article available to read on EtoBox.

What is Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC–MS/MS approaches to cardiac proteomics about?

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 h 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 tissues. 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 that approximately 22 u

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Author

Chad M. Warren; David L. Geenen; Donald L. Helseth; Jr.; Hua Xu; R. John Solaro

Publisher

Elsevier ; Elsevier BV (ISSN 1874-3919)

Published

2010

Language

EN

Field

Chemistry (Physical Sciences)