title: One-hour proteome analysis in yeast.
abstract: Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-μm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350-375 nl/min) that increases the number of proteins identified across a single liquid chromatography-tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes ∼24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.
authors: 1] The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA. [2] Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
authors: Alicia L
authors: AL
authors: Richards
authors: 1] The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA. [2] Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
authors: Alexander S
authors: AS
authors: Hebert
authors: 1] The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA. [2] Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
authors: Arne
authors: A
authors: Ulbrich
authors: 1] The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA. [2] Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
authors: Derek J
authors: DJ
authors: Bailey
authors: The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA.
authors: Emma E
authors: EE
authors: Coughlin
authors: The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA.
authors: Michael S
authors: MS
authors: Westphall
authors: 1] The Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin, USA. [2] Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA. [3] Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
authors: Joshua J
authors: JJ
authors: Coon
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins