Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests.
Mass spectrometric methods of glycopeptide-specific detection in liquid chromatography/electrospray mass spectrometry (LC/ESMS) of glycoprotein digests are explored using a variety of glycopeptide models and then applied to soluble complement receptor type I, a 240-kDa glycoprotein containing 25 potential sites of N-glycosylation. The most specific method, requiring a triple quadrupole, involves monitoring of sugar oxonium fragment ions during precursor-ion scan ESMS/MS. Signals derived from nonglycosylated peptides are virtually eliminated, resulting in a total-ion current chromatographic trace of only the glycopeptides present in the digest. The corresponding mass spectra yield molecular weight and glycopeptide microheterogeneity information. An alternative and complementary approach that we term collisional-excitation scanning also involves fragmentation of glycopeptides to sugar oxonium ion fragments but does not involve any mass-selection process, permitting the experiment to be performed on a single quadrupole instrument. The resulting total ion chromatogram is similar to the UV chromatogram (215 nm), but a selected-ion chromatogram for carbohydrate-specific ions such as the N-acetylhexosamine oxonium ion (m/z 204) produces a glycopeptide-specific trace. Although there can sometimes be peptide interferences in the spectra of the indicated glycopeptide-containing chromatographic peaks, this latter approach permits peptide mapping to be performed on the same data set that also indicates the location of glycopeptides in the chromatogram. Both methods are suitable for detection of glycopeptides with all common classes of oligosaccharides in either N- or O-linkage to the peptide.
Huddleston
M J
MJ
SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Bean
M F
MF
Carr
S A
SA
D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D017022:Flow Injection Analysis; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry
D006020:Glycopeptides; D009844:Oligosaccharides