title: Preparation and analysis of proteins and peptides using MALDI TOF/TOF mass spectrometry.
abstract: Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) is a valuable tool for the analysis of peptides and proteins. Particularly useful features include high sensitivity, fast data acquisition, ease of use, and robust instrumentation. Although MALDI is relatively tolerant to buffers and other impurities, substantial sensitivity enhancement can be achieved through removal of non-analyte components of samples. Therefore, sample processing to remove buffers and impurities can greatly improve the quality of results obtained by MALDI experiments. This unit describes optimized procedures for enzymatic digestion, preparation of MALDI target plates, thin layer matrix preparation, on-target sample cleanup, and capillary HPLC-MALDI co-spotting of analyte and matrix. Procedures are also described for analysis of on-membrane proteins by MALDI-TOF/TOF-MS before tryptic digestion. Some of these procedures are also applicable to protein spots from two-dimensional (2-D) gels. Guidance is also provided for acquisition and interpretation of MS and MS/MS spectra.
authors.affiliation: PO Royal Brisbane Hospital, Herston, Queensland, Australia.
authors.forename: Keyur A
authors.initials: KA
authors.lastname: Dave
authors.affiliation: PO Royal Brisbane Hospital, Herston, Queensland, Australia.
authors.forename: Madeleine J
authors.initials: MJ
authors.lastname: Headlam
authors.affiliation: Mater Adult's Hospital, South Brisbane, Queensland, Australia.
authors.forename: Tristan P
authors.initials: TP
authors.lastname: Wallis
authors.affiliation: PO Royal Brisbane Hospital, Herston, Queensland, Australia.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gorman
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003373:Coumaric Acids / Q000737:chemistry; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D003373:Coumaric Acids; D010455:Peptides; D011506:Proteins; C007175:alpha-cyano-4-hydroxycinnamate; D014357:Trypsin