title: Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver.
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 urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches.
authors.affiliation: Proteomics R&D Facility, Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Arnold
authors.forename: Petra
authors.initials: P
authors.lastname: Hrncirova
authors.forename: Kiran
authors.initials: K
authors.lastname: Annaiah
authors.forename: Milos V
authors.initials: MV
authors.lastname: Novotny
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D002853:Chromatography, Liquid; D003600:Cytosol / Q000737:chemistry; D008110:Liver Extracts / Q000032:analysis / Q000737:chemistry*; D013058:Mass Spectrometry; D008861:Microsomes / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry*; D020543:Proteome; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D008110:Liver Extracts; D020543:Proteome; D013501:Surface-Active Agents; D014508:Urea; D014357:Trypsin