Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry.
We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.
D002236:Carbohydrate Conformation; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007075:Immunoglobulin M / Q000032:analysis; D013058:Mass Spectrometry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis*; D008258:Waldenstrom Macroglobulinemia / Q000378:metabolism
D006023:Glycoproteins; D007075:Immunoglobulin M; D009844:Oligosaccharides