title: Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients.
abstract: The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 10 h of nanoLC-MS/MS measurement time.
authors.affiliation: Research Institute of Molecular Pathology, Vienna, Austria. thomas.koecher@imp.ac.at
authors.forename: Thomas
authors.initials: T
authors.lastname: Köcher
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.forename: Remco
authors.initials: R
authors.lastname: Swart
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D002457:Cell Extracts / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002457:Cell Extracts; D011506:Proteins