Site-specific glycosylation of secretory immunoglobulin A from human colostrum.

Secretory immunoglobulin A (sIgA) is a major glycoprotein in milk and plays a key role in mediating immune protection of the gut mucosa. Although it is a highly glycosylated protein, its site-specific glycosylation and associated glycan micro-heterogeneity have still not been fully elucidated. In this study, the site-specific glycosylation of sIgA isolated from human colostrum (n = 3) was analyzed using a combination of LC-MS and LC-MS/MS and in-house software (Glycopeptide Finder). The majority of the glycans found are biantennary structures with one or more acidic Neu5Ac residues; however, a large fraction belonged to truncated complex structures with terminal GlcNAc. Multiple glycosites were identified with nearly 30 glycan compositions located at seven sites on the secretory component, six compositions at a single site on the J chain, and 16 compositions at five sites on the IgA heavy (H) chain. Site-specific heterogeneity and relative quantitation of each composition and the extent of occupation at each site were determined using nonspecific proteases. Additionally, 54 O-linked glycan compositions located at the IgA1 hinge region (HR) were identified by comparison against a theoretical O-glycopeptide library. This represents the most comprehensive report to date detailing the complexity of glycan micro-heterogeneity with relative quantitation of glycoforms for each glycosylation site on milk sIgA. This strategy further provides a general method for determining site-specific glycosylation in large protein complexes.

Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota: a proof-of-concept study.

Human milk oligosaccharides (HMOs) play a key role in shaping and maintaining a healthy infant gut microbiota. This article demonstrates the potential of combining recent advances in glycomics and genomics to correlate abundances of fecal microbes and fecal HMOs. Serial fecal specimens from two healthy breast-fed infants were analyzed by bacterial DNA sequencing to characterize the microbiota and by mass spectrometry to determine abundances of specific HMOs that passed through the intestinal tract without being consumed by the luminal bacteria. In both infants, the fecal bacterial population shifted from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life. An initial rise in fecal HMOs corresponded with bacterial populations composed primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation revealed that HMO consumption is highly structure-specific, with unique isomers being consumed and others passing through the gut unaltered. These results represent a proof-of-concept and are consistent with the highly selective, prebiotic effect of HMOs in shaping the gut microbiota in the first weeks of life. The analysis of selective fecal bacterial substrates as a measure of alterations in the gut microbiota may be a potential marker of dysbiosis.

Automated assignments of N- and O-site specific glycosylation with extensive glycan heterogeneity of glycoprotein mixtures.

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and complements experimental techniques that are optimal for glycopeptides, including nonspecific proteolysis and high mass resolution liquid chromatography/tandem mass spectrometry (LC/MS/MS). SSG for multiple N- and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of nonrandom glycopeptide matches and demonstrating the proof-of-concept for a self-consistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

In-gel nonspecific proteolysis for elucidating glycoproteins: a method for targeted protein-specific glycosylation analysis in complex protein mixtures.

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem mass spectrometry (MS) analysis of the resulting N- and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as Pronase, proteinase K, pepsin, papain, and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data, and predetermined protein identification (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (κ casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid determination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants.

Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother-infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children, and adults. This method may therefore provide diagnostic and therapeutic opportunities.

The glycolyzer: automated glycan annotation software for high performance mass spectrometry and its application to ovarian cancer glycan biomarker discovery.

Human serum glycomics is a promising method for finding cancer biomarkers but often lacks the tools for streamlined data analysis. The Glycolyzer software incorporates a suite of analytic tools capable of identifying informative glycan peaks out of raw mass spectrometry data. As a demonstration of its utility, the program was used to identify putative biomarkers for epithelial ovarian cancer from a human serum sample set. A randomized, blocked, and blinded experimental design was used on a discovery set consisting of 46 cases and 48 controls. Retrosynthetic glycan libraries were used for data analysis and several significant candidate glycan biomarkers were discovered via hypothesis testing. The significant glycans were attributed to a glycan family based on glycan composition relationships and incorporated into a linear classifier motif test. The motif test was then applied to the discovery set to evaluate the disease state discrimination performance. The test provided strongly predictive results based on receiver operator characteristic curve analysis. The area under the receiver operator characteristic curve was 0.93. Using the Glycolyzer software, we were able to identify a set of glycan biomarkers that highly discriminate between cases and controls, and are ready to be formally validated in subsequent studies.

Lacto-N-tetraose, fucosylation, and secretor status are highly variable in human milk oligosaccharides from women delivering preterm.

Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from 15 women delivering preterm and 7 women delivering at term using nanohigh performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene's test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2'-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments.

Identification and accurate quantitation of biological oligosaccharide mixtures.

Structure-specific characterization and quantitation is often required for effective functional studies of oligosaccharides. Inside the gut, HMOs are preferentially bound and catabolized by the beneficial bacteria. HMO utility by these bacteria employs structure-specific catabolism based on a number of glycosidases. Determining the activity of these enzymes requires accurate quantitation of a large number of structures. In this study, we describe a method for the quantitation of human milk oligosaccharide (HMO) structures employing LC/MS and isotopically labeled internal standards. Data analysis was accomplished with a newly developed software tool, LC/MS Searcher, that employs a reference structure library to process LC/MS data yielding structural identification with accurate quantitation. The method was used to obtain a meta-enzyme analysis of bacteria, the simultaneous characterization of all glycosidases employed by bacteria for the catabolism of milk oligosaccharides. Analysis of consumed HMO structures confirmed the utility of a ß-1,3-galactosidase in Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis). In comparison, Bifidobacterium breve ATCC 15700 showed significantly less HMO catabolic activity compared to B. infantis.

Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies.

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.

Site-specific protein glycosylation analysis with glycan isomer differentiation.

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining nonspecific protease digestion, nanoflow liquid chromatography, and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-Pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein, and bovine fetuin using nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC-Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan microheterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.

Enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry.

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.

N-linked glycan profiling of mature human milk by high-performance microfluidic chip liquid chromatography time-of-flight tandem mass spectrometry.

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.