Introduction: Protein identification using peptide mass maps and database searching has become an important technique for proteomic studies. MALDI-TOF MS has been used extensively to identify samples extracted from gel spots by this approach. However, MALDI-TOF MS has the disadvantage that MS/MS is better performed on tandem mass spectrometers. The power to fragment selected ions further can be key to assigning the position of modifications as well as determining the structure of the modification. As an alternative, this report describes an atmosphere-pressure MALDI source coupled to an ion-trap MS. This new MALDI-Ion Trap is compatible with the current control and data-handling software, which allows samples to be run and data to be analyzed automatically.
Methods: Proteins from gel spots or solution (range from femtomole to picomole) were reduced and alkylated and then digested by trypsin (in-gel or solution digestion). An aliquot of 1 uL of the digested solution mixed with 0.5 uL of alpha-4-hydroxycinamic acid (10 ug/uL) was placed on a gold plate, and then inserted into the MALDI source with a nitrogen laser for analysis. The MALDI source parameters were similar to the nanospray source parameters (no sheath gas and a low spray voltage). Protein identifications were made with Bioworks (Sequest) using both MS and MS/MS spectra.
Preliminary Data: The heavy chain and light chain of a monoclonal antibody from SDS-PAGE gel were identified by MS (with 7 unique peptide related molecular ions) and MS/MS (with 8 unique peptide sequence assignment). Human glycoproteins from 2-D gel spots were also rapidly identified with the assignment of 2 or more peptides. The structure of modifications (e.g. disulfide linkage, glycosylation, or phosphorylation) was determined by further fragmentation in this device (e.g. up to MS to 3 in the ion trap).
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