[Show abstract][Hide abstract] ABSTRACT: An improved separation of the human serum N-glycome using hydrophilic interaction chromatography (HILIC) technology with UPLC is described, where more than 140 N-glycans were assigned. Using this technique serum samples from 107 healthy controls and 62 newly diagnosed breast cancer patients were profiled. The most statistically significant alterations were observed in cancer patients compared to healthy controls: an increase in sialylation, branching and outer arm-fucosylation and a decrease in high mannosylated and biantennary core-fucosylated glycans. In the controls and cases combined systemic features were analysed; serum estradiol was associated with increase in digalactosylated glycans, higher mammographic density was associated with increase in biantennary digalactosylated glycans and with decrease in trisialylated and in outer arm-fucosylated glycans. Furthermore, particular glycans were altered in some features of the breast carcinomas - bisected biantennary non-fucosylated glycans were decreased in patients with progesterone receptor positive tumours and core-fucosylated biantennary bisected monogalactosylated glycans were decreased in patients with the TP53 mutation. Systemic features show more significant associations with the serum N-glycome than do the features of the breast carcinomas. In conclusion, the UPLC-based glycan analysis technique described here reveals highly significant differences between healthy women and breast cancer patients. Significant associations with breast carcinoma and systemic features are described.
Journal of Proteome Research 03/2014; 13(5). DOI:10.1021/pr401092y · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of Etanercept was carried out using LC/MS methods in a systematic approach in which we analysed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and the glycans separated using UPLC-HILIC. Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the glycan pool. The removal of the N-glycans from Etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the Core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α(2->3,6,8,9) sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC-MSE protocol to identify the O-glycopeptides. Electron transfer dissociation (ETD) was then used to pinpoint the 12 occupied glycosylation sites. The determination of glycans and glycosylation sites in Etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics.
[Show abstract][Hide abstract] ABSTRACT: The objective was to analyze the proteomic composition of uterine flushes collected from beef heifers on day 7 after insemination. Estrus was synchronized in crossbred beef heifers by using a protocol with a controlled intravaginal drug releasing device. Heifers detected in standing estrus (within 24-48 h after removal of controlled intravaginal drug releasing device) were inseminated (estrus = day 0) with frozen-thawed semen from a single ejaculate of a bull with proven fertility. Heifers from which an embryo was recovered (after slaughter on day 7) were classified as either having a viable embryo (morula/blastocyst stage) or a degenerate embryo (arrested at the 2- to 16-cell stage). The overall recovery rate (viable and degenerate combined) was 64%. Global liquid chromatography coupled to tandem mass spectrometry proteomic analysis of the histotroph collected identified 40 high-confidence proteins present on day 7; 26 proteins in the viable group, 10 in the degenerate group, and 4 shared between both groups. Five proteins (platelet-activating factor acetylhydrolase IB subunit γ [PAFAH1B3], tubulin α-1D chain, tubulin β-4A chain, cytochrome C, and dihydropyrimidinase-related protein-2) were unique or more abundant in the histotroph collected from animals with a viable embryo, and 1 protein (S100-A4) was more abundant in the histotroph collected from animals with a degenerate embryo. Of interest, PAFAH1B3, detected only in histotroph from the group yielding viable embryos, belongs to the group of platelet-activating factors that are known to be important for the development of the pre-implantation embryo in other species. To our knowledge this is the first report of PAFAH1B3 in relation to bovine early embryonic development.
[Show abstract][Hide abstract] ABSTRACT: Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.
Journal of Proteome Research 03/2012; 11(5):3004-18. DOI:10.1021/pr300144q · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Characterization of mono- and bis-mannose-6-phosphate (M6P) bearing oligosaccharides present on acid hydrolase enzymes poses a considerable analytical challenge. In the current paper, we investigated the use of UPLC profiling on a 1.7 μm HILIC phase and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) combined with exoglycosidase digestion and weak anion exchange fractionation for the characterization of M6P bearing glycans on recombinant β-glucuronidase expressed in Chinese Hamster Ovary (CHO) cells. Using this multidimensional approach a number of peaks were observed to resist digestion, suggesting the presence and blocking activity of the M6P tag. To investigate further, mixed oxide affinity purification on a combined TiO(2)/ZrO(2) resin facilitated the selective enrichment of oligosaccharides bearing mono- or diphospho-esters that corresponded to those peaks previously identified to resist exoglycosidase digestion. Alkaline phosphatase digestion identified Man(6)GlcNAc(2) and Man(7)GlcNAc(2) glycans as the primary carriers of the M6P tag. Site-specific glycoproteomic analysis revealed that Man(7)GlcNAc(2)-M6P oligosaccharides were present at asparagine 272 and 420, while asparagine 631 displayed Man(6)GlcNAc(2)-M6P. The analytical strategy applied herein represents a novel yet simple approach for the qualitative and semiquantitative structural characterization of M6P containing oligosaccharides on therapeutic enzymes.
[Show abstract][Hide abstract] ABSTRACT: Post-translational modifications, in particular glycosylation, represent critical structural attributes that govern both the pharmacodynamic and pharmacokinetic properties of therapeutic glycoproteins. To guarantee safety and efficacy of recombinant therapeutics, characterization of glycosylation present is a regulatory requirement. In the current paper, we applied a multidimensional strategy comprising a shallow anion exchange gradient in the first dimension, followed by analysis using the recently introduced 1.7 μm HILIC phase in the second dimension for the comprehensive separation of complex N-glycans present on the European Biological Reference Preparation (BRP) 3 erythropoietin standard. Tetra-antennary glycans with multiple sialic acids and poly-N-acetyl lactosamine extensions were the most abundant oligosaccharides present on the molecule. Site-specific glycan analysis was performed to examine microheterogeneity. Tetra-antennary glycans with up to four sialic acids and up to five poly-N-acetyl lactosamine extensions were observed at asparagine 24 and 83, while biantennary glycans were the major structures at asparagine 38. The combined AEC x UPLC HILIC allows for the rapid and comprehensive analysis of complex N-glycosylation present on therapeutic glycoproteins, such as BRP3 erythropoietin.