Determination of glycopeptide structures by multistage mass spectrometry with low‐energy collision‐induced dissociation: comparison of electrospray ionization quadrupole ion trap and matrix‐assisted laser desorption/ionization quadrupole ion trap reflectron time‐of‐flight approaches

Abstract

Multistage mass spectrometry, as implemented using low-energy collision-induced dissociation (CID) analysis in three-dimensional (3D) quadrupole ion traps (QITs), has become a powerful tool for the investigation of protein glycosylation. In addition to the well-known combination of QITs with electrospray ionization (ESI), also a matrix-assisted laser desorption/ionization--quadrupole ion trap--reflectron time-of-flight (MALDI-QIT-rTOF) mass spectrometer has recently become available. This study systematically investigates the differences between these types of instrument, as applied to characterization of glycopeptides from human antithrombin. The glycopeptides were obtained by tryptic digestion followed by lectin-affinity purification. Some significant differences between the ESI-QIT and MALDI-QIT-rTOF approaches appeared, most of them are causally related to the desorption/ionization process. The combination of a vacuum MALDI source with an ion-trap analyzer accentuates some characteristic differences between MALDI and ESI due the longer time frame needed for the trapping process. In contrast to ESI, MALDI generated ions that exhibited considerable metastable fragmentation during trapping. The long time span of the QIT process (ms range) compared with that for conventional rTOF experiments (micros range) significantly magnified the extent of this metastable fragmentation. With the investigated glycopeptides, a complete depletion of the terminal sialic acids of the glycopeptides as well as a variety of other fragment ions was already found in the MS1 spectra from the MALDI-QIT-rTOF instrument. The positive ion low-energy CID spectra (MS2) of the selected glycopeptides obtained using the two different QIT equipped instruments were found to be quite similar. In both approaches, fragmentation of the glycan and peptide structures occurred sequentially, allowing unambiguous sequence determination. In the case of ESI-QIT-MS, fragmentation of the glycan structure occurred at the MS2 stage and fragmentation of the peptide structure was obtained only at the MS3 stage, which indicates the necessity of multistage CID experiments for complete structure elucidation. The MALDI-QIT-rTOF instrument yielded both kinds of fragments at the MS2 stage but without mutual interference.

Characterization of moenomycin antibiotic complex by multistage MALDI-IT/RTOF-MS and ESI-IT-MS

“…Negative ion mode MALDI measurements using the matrix substance 2,4,6-THAP and diammonium hydrogen citrate as additive (to suppress alkali ad- A consequence of the coupling of vacuum MALDI with an ion trap device is the enhanced probability of post source decay in the larger time frame inherent to the trapping process, resulting in the occasionally observed fragmentation of the precursor ions already in MS 1 [33,41]. Indeed, the rather labile phosphoglycolipids exhibited prompt decay in MS 1 as well (most abundant fragment ion/deprotonated molecular ion ratio of approximately 2/1).…”

“…The applicability of the MALDI-IT/RTOF-MS technique to the structural analysis of oligosaccharides and glycopeptides has been described in previous work [33][34][35][36][37][38] and is extended to the class of medically relevant phosphoglycolipids.…”

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

“…In the case in which single proteins or less complex protein mixtures are investigated, an enrichment can be compensated by a chromatographic separation as it is usually the case in LC-ESI-MS (35). In MALDI-MS, suppression of glycopeptide signals is found to be a major issue and sample enrichment strategies are often required in order to obtain a reproducible and sensitive signal (34,50,51).…”

Tandem Mass Spectrometry and Glycoproteins

“…However, glycosylation sites of glycopeptides produced by trypsinization of complex glycoprotein mixtures are particularly difficult to identify because glycopeptides are usually present in relatively low abundance (2% to 5%) in peptide mixtures compared to the nonglycosylated peptides, and because the sugar fragments in the MS spectrum often dominate the peptide fragments due to glycosidic bonds being more labile than peptide bonds. Some approaches have been developed to overcome this problem, mainly based on multistage MS (Bateman et al 1998, Demelbauer et al 2004, electron capture dissociation (ECD) (Håkansson et al 2001(Håkansson et al , 2003 or electron transfer dissociation (ETD) (Hogan et al 2005).…”