The benefit of combining nLC-MALDI-Orbitrap MS data with nLC-MALDI-TOF/TOF data for proteomic analyses employing elastase.
The recently established coupling of a MALDI-type ion source to a linear ion trap and an orbitrap mass analyzer offers high-accuracy mass measurements compared to common MALDI-TOF/TOF instruments. Contrary to MALDI-TOF/TOF, the fragmentation of peptides in the new hybrid mass spectrometer is less efficient due to the generation of predominantly singly charged ions by the MALDI process. Therefore, data from two MALDI instruments, TOF/TOF and Orbitrap, were combined into a single data set in order to obtain accurate precursor masses as well as superior MS/MS spectra. This study demonstrates that an accurate precursor mass is particularly important for the nLC-MS/MS analyses of less-specific proteolytic digests. A potential gain of approximately one-third additional peptides identifications was theoretically estimated from previously published MALDI-TOF/TOF data. These calculations were verified by the nLC-MS/MS analysis of two elastatically digested proteomes, one cytosolic (Corynebacterium glutamicum) and one membrane (Halobacterium salinarium). Thereby it was discovered that the error distribution of a MALDI-Orbitrap can be significantly improved by applying an easy recalibration strategy. In summary, this study represents an updated workflow for the analysis of less-specific digests using nLC-MALDI.
Rietschel
Benjamin
B
Cluster of Excellence Macromolecular Complexes, Institute for Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany.
Baeumlisberger
Dominic
D
Arrey
Tabiwang N
TN
Bornemann
Sandra
S
Rohmer
Marion
M
Schuerken
Malte
M
Karas
Michael
M
Meyer
Bjoern
B
D019843:Archaeal Proteins / Q000032:analysis / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000737:chemistry; D019617:Halobacterium salinarum / Q000737:chemistry; D010196:Pancreatic Elastase / Q000378:metabolism*; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D057188:Workflow
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D010455:Peptides; D010196:Pancreatic Elastase