Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers

Glycosylation is highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Glycan compositional profiling of human serum with mass spectrometry has already identified potential biomarkers for several types of cancer and diseases; however, composition alone does not fully describe glycan stereo- and regioisomeric diversity. The vast structural heterogeneity of glycans presents a formidable analytical challenge. We have developed a method to identify and quantify isomeric native glycans using nanoflow liquid chromatography (nano-LC)/mass spectrometry. A microfluidic chip packed with graphitized carbon was used to chromatographically separate the glycans. To determine the utility of this method for structure-specific biomarker discovery, we analyzed serum samples from two groups of prostate cancer patients with different prognoses. More than 300 N-glycan species (including isomeric structures) were identified, corresponding to over 100 N-glycan compositions. Statistical tests established significant differences in glycan abundances between patient groups. This method provides comprehensive, selective, and quantitative glycan profiling.     

Detection of milk oligosaccharides in plasma of infants

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are non-digestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.     

Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.     

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.     

A topologically segregated one-bead-one-compound combinatorial glycopeptide library for identification of lectin ligands

A glycopeptide library containing more than 500,000 compounds has been constructed from a combination of Asn-linked carbohydrates using one-bead-one-compound combinatorial methodologies. The library was encoded with peptide markers that were topologically segregated on the interior of the solid support to negate interference with carbohydrate/protein recognition during lectin screening. Both the peptide backbone and carbohydrate components were randomized, but the glycosamine was limited to position 3 at the center of the pentapeptide to evaluate the influence of the peptide backbone in lectin recognition. Of the four lectins that were evaluated, remarkable selectivity was observed with wheat germ agglutinin (WGA), which recognizes N-acetyl glucosamine (GlcNAc). From more than 80,000 possible combinations, only six ligands were identified, all possessing GlcNAc. These compounds were independently synthesized, characterized, and evaluated in solution. All six of the glycopeptides showed higher affinity for WGA than GlcNAc, with one having a 4-fold increase. Modeling studies indicate that the peptide backbone is capable of interacting with amino acids in the active site of WGA, but these interactions are not strongly correlated with activity, suggesting that the primary role of the peptide is to properly orient the sugar in the recognition process.     

Unusual covalent bond-breaking reactions of beta-cyclodextrin inclusion complexes of nucleobases/nucleosides and related guest molecules

Host-guest complexes between nucleobases or nucleosides and beta-cyclodextrin can be observed by electrospray ionization mass spectrometry (ESI-MS) and their relative abundances appear to correlate with the condensed-phase binding order. Using Fourier transform ion cyclotron resonance mass spectrometry, the extent of the interactions between the host oligosaccharide and guest species have also been examined for permethylated beta-cyclodextrin : adenine/deoxyadenosine and permethylated maltoheptaose : adenine/deoxyadenosine using gas-phase exchange reactions with the gaseous amines, n-propylamine and ethylenediamine. The ease of guest exchange in the gas-phase follows the order : deoxyadenosine > adenine > deoxycytidine > cytosine, which is in contrast to their relative binding order in solution. Collision-induced dissociation (CID) has been used to probe the fragmentation behavior of oligosaccharide : nucleobase/nucleoside complexes. Under these conditions the inclusion complexes either (a) dissociate, (b) result in cleavage of the host oligosaccharide or (c) result in cleavage of the guest molecule. This study has shown that the preferred dissociation pathway of these complexes depends on the structures of both the cyclodextrin and guest molecule.     

Nanoliquid chromatography-mass spectrometry of oligosaccharides employing graphitized carbon chromatography on microchip with a high-accuracy mass analyzer

The nanoLC separations of oligosaccharides using microchip-based columns are described. Mixtures of alditols from mucins and human milk are separated on graphitized carbon. The nanoLC-MS device showed high mass accuracy for the oligosaccharides ranging between 1 and 6 ppm on routine analyses. The high mass accuracy readily allowed identification of oligosaccharide peaks and the determination of their compositions. High retention time reproducibility was exhibited by the microchip LC. Little variation was observed for standard sample either alone or in a complex heterogeneous mixture. The nanoLC-MS exhibits excellent capabilities in profiling mixtures of oligosaccharides.     

Parameters contributing to efficient ion generation in aerosol MALDI mass spectrometry

The Bioaerosol Mass Spectrometry (BAMS) system was developed for the real-time detection and identification of biological aerosols using laser desorption ionization. Greater differentiation of particle types is desired; consequently MALDI techniques are being investigated. The small sample size ( approximately 1 microm3), lack of substrate, and ability to simultaneously monitor both positive and negative ions provide a unique opportunity to gain new insight into the MALDI process. Several parameters known to influence MALDI molecular ion yield and formation are investigated here in the single particle phase. A comparative study of five matrices (2,6-dihydroxyacetophenone, 2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid, ferulic acid, and sinapinic acid) with a single analyte (angiotensin I) is presented and reveals effects of matrix selection, matrix-to-analyte molar ratio, and aerosol particle diameter. The strongest analyte ion signal is found at a matrix-to-analyte molar ratio of 100:1. At this ratio, the matrices yielding the least and greatest analyte molecular ion formation are ferulic acid and alpha-cyano-4-hydroxycinnamic acid, respectively. Additionally, a significant positive correlation is found between aerodynamic particle diameter and analyte molecular ion yield for all matrices. SEM imaging of select aerosol particle types reveals interesting surface morphology and structure.