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Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

Soluble amyloid precursor protein alpha (sAPPalpha) is a cleavage product of the amyloid precursor protein (APP), the etiologic agent in Alzheimer's disease (AD). Reduced expression of sAPPalpha was previously found in the brains of AD patients, and it was suggested that sAPPalpha might counteract neurotoxic effects of Abeta, another APP cleavage product with enhanced abundance in Alzheimer's diseased brains. However, little is known about the biological functions of sAPPalpha. Thus, efficient production of this protein is a prerequisite for further studies. The unicellular eukaryotic parasite Leishmania tarentolae has recently emerged as a promising expression system for eukaryotic proteins due to its ability to posttranslationally modify proteins combined with easy cultivation and high protein yield. Interestingly, sAPPalpha produced in L. tarentolae was biologically active and glycosylated. In contrast to nonglycosylated sAPPalpha expressed in Eschericha coli, it also featured higher stability against enzymatic degradation. Detailed analysis of the glycosylation pattern of sAPPalpha produced in L. tarentolae by PGC-LC-ESI-MS/MS N-glycan analysis identified among eukaryotic species the highly conserved core pentasaccharide (Man3GlcNAc2) as being attached to Asn467 of sAPPalpha. Using oxonium ion scanning of CID-MS/MS spectra in combination with ETD fragmentation, we also identified two peptides (peptides 269-288 and 575-587) modified with N-acetyl hexosamine (HexNAc) residues. One of these O-glycosylation sites could be unambiguously assigned to Thr576 of sAPPalpha. This is the first time that O-glycosylation of a recombinant protein expressed in L. tarentolae has been demonstrated. Together, human sAPPalpha produced in L. tarentolae was N- and O-glycosylated on similar sites as described for mammalian-expressed sAPPalpha and showed similar biological activity. This demonstrates that L. tarentolae is a very suitable and simple to handle expression system for mammalian glycoproteins.

O-Linked glycosylation often occurs in mucin-type domains that are heavily and heterogeneously glycosylated and are challenging to analyze. The analysis of these domains is often overlooked because of these difficulties, but changes in mucinlike domain glycosylation are implicated in many diseases. Here we have explored several strategies to determine the heterogeneity of mucinlike O-glycosylated domains. Four glucanases secreted in large quantities from Trichoderma reesei, all containing heavily O-glycosylated mucinlike linker regions, were used as a model system. The strategies involved monosaccharide compositional analysis and identification of the released glycans by HPAEC-PAD and carbon-LC ESI-MS/MS. Glycosylated peptides were generated by different protease digestions (trypsin, papain, Asp-N, PreTAQ) and enriched by HILIC microcolumns, to determine the glycopeptide heterogeneity and glycosylation sites. The complex O-glycan heterogeneity on the intact glycoproteins and the enriched mucin-type domains was determined by MALDI-MS and ESI-MS, but the dense O-glycosylation in the mucin-type domains conferred high resistance to protease cleavage. ETD-MS/MS of the glycopeptide-enriched protease digests was unsuccessful for the de novo assignment of O-glycosylation at individual sites within the mucin-type domains but allowed several previously unknown O-linked sites outside the defined linker region to be found on two of the four glucanases. The protease digests produced many glycopeptides as determined by CID-MS/MS, but ETD fragmentation of these resulted in only a few interpretable spectra, suggesting that the use of ETD for determining the heterogeneous O-glycosylation at specific sites in regions of multiple occupancy is still in its infancy.

With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked,or=5 O-linked sites), IgA2 (4 N-linked sites) and J-chain (1 N-linked site) is described. GlycoSpectrumScan is freely available at www.glycospectrumscan.org .

Past proteomic studies of membrane proteins have often been hampered by the low abundance and relatively high hydrophobicity of these proteins. Proteins are often glycosylated, particularly on the extracellular surface of the plasma membrane, and this characteristic was targeted as an enrichment strategy for identifying membrane proteins. Here, we report a strategy for identifying the tissue membrane glycoproteome, which involves (1) Triton X-114 phase partitioning, (2) isolation of glycosylphosphatidylinositol (GPI)-anchored proteins, and (3) glycoprotein capture using lectin affinity or hydrazine chemistry. Surprisingly, the capture of membrane proteins by lectin affinity and hydrazine chemistry resulted in mostly different populations of enriched glycoproteins. Lectins enriched high molecular weight functional membrane proteins with more potential glycosylation such as those involved in signal transduction and cell adhesion. Conversely, hydrazine chemistry isolated a higher proportion of smaller, enzymatic and peripheral membrane proteins such as solute carrier transporters and cytochrome p450s. We have applied our strategy to characterize the rat liver membrane glycoproteome and identified four new predicted GPI-anchored proteins and two that have not previously been seen in the liver. We also identified 424 nonredundant membrane proteins, of which 335 had potential N-linked glycosylation sites.