Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS.
The utility of matrix-assisted laser desorption time-of-flight (MALD-TOF) mass spectrometry for the analysis of recombinant glycopeptides is discussed and compared to information which may be obtained by fast atom bombardment mass spectrometry (FABMS) and tandem mass spectrometry (MS/MS). MALD-TOF appears to be 10-100 times more sensitive than FAB MS for the analysis of underivatized glycopeptides, providing qualitative site-specific information regarding the carbohydrate microheterogeneity without the extensive isolation and derivatization procedures required to obtain similar information by FAB MS. Analysis of a digest mixture in the positive and negative ion mode of MALD-TOF indicated that, in mixtures, sialylated glycopeptides are preferentially detected in the negative ion mode. The determination of the molecular masses of a glycopeptide with MALD-TOF prior to and after treatment with a variety of specific glycosidases, often without removal of the buffers, coupled to a comparison of molecular mass information available from a carbohydrate database facilitates the assignment of a carbohydrate composition. The vast majority of the molecular ion signal observed in the linear mode for sialylated glycopeptides are metastable ions. Reflector mass spectra reveal a shift to lower mass consistent with the loss of most of the neuraminic acid residues. The loss of Hex and HexNAc residues is also observed. Sequential lowering of the reflector potential reveals structurally significant fragment ions representing the carbohydrate and peptide portions of the molecule.
Huberty
M C
MC
Structural Biochemistry Department, Genetics Institute, Inc., Andover, Massachusetts 01810.
Vath
J E
JE
Yu
W
W
Martin
S A
SA
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry*
D002241:Carbohydrates; D006023:Glycoproteins; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D006026:Glycoside Hydrolases