title: Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes.
abstract: The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.
authors.affiliation: Institute of Pharmaceutical Chemistry, Cluster of Excellence Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt a. M., Germany.
authors.forename: Dominic
authors.initials: D
authors.lastname: Baeumlisberger
authors.forename: Marion
authors.initials: M
authors.lastname: Rohmer
authors.forename: Tabiwang N
authors.initials: TN
authors.lastname: Arrey
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Mueller
authors.forename: Tobias
authors.initials: T
authors.lastname: Beckhaus
authors.forename: Ute
authors.initials: U
authors.lastname: Bahr
authors.forename: Guenes
authors.initials: G
authors.lastname: Barka
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D048230:Corynebacterium glutamicum / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D010447:Peptide Hydrolases