Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver

Journal of Proteome Research
Randy J ArnoldMilos V Novotny

Abstract

The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with u...Continue Reading

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Citations

Oct 15, 2010·Free Radical Research·Janos KernerCharles L Hoppel
Jun 22, 2005·Expert Review of Proteomics·Mamoun Ahram, David L Springer
Mar 21, 2007·Biochimica Et Biophysica Acta·Silke HenrichRichard I Christopherson
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Jan 18, 2006·Molecular & Cellular Proteomics : MCP·Francesca FornerMatthias Mann
Aug 12, 2009·Analytical Chemistry·Tyler CarlageWilliam S Hancock
Jun 15, 2005·Analytical Chemistry·Jean-Philippe LambertDaniel Figeys

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