title: Identification of gel-separated proteins by liquid chromatography-electrospray tandem mass spectrometry: comparison of methods and their limitations.
abstract: We have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The efficiency of peptide recovery from trypsin-digested gel bands or electroblotted membrane slices was examined using 35S-labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC-ESI-MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit. We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow-split microspray HPLC-MS interface system. In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng. In general, protein identification was slightly better in samples prepared from gels rather than membranes. A dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydrase and 100 ng of BSA.
authors.affiliation: Department of Molecular Biotechnology, University of Washington, Seattle 98195-7730, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Haynes
authors.forename: N
authors.initials: N
authors.lastname: Fripp
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D011506:Proteins / Q000302:isolation & purification*; D012588:Scintillation Counting; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods; D013462:Sulfur Radioisotopes / Q000378:metabolism
chemical_list: D005782:Gels; D011506:Proteins; D013462:Sulfur Radioisotopes; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D002256:Carbonic Anhydrases