Yehia Mechref

High-throughput solid-phase permethylation of glycans prior to mass spectrometry.

Permethylation of glycans prior to their mass spectrometric determination has now become a time-honored methodology in glycoconjugate analysis due to the advantage of a simultaneous analysis of neutral and acidic glycans as well as enhanced sensitivity and easier tandem mass spectrometry interpretation. While the different solvent extraction-based versions of this method often suffice in different structural studies, they are generally less satisfactory in the quantitative determinations aiming at minor quantities of the analyzed materials. To overcome these difficulties, we recently introduced a solid-phase capillary permethylation technique (Kang et al., Rapid Commun. Mass Spectrom. 2005; 19: 3421) for microscale determination. Here, we describe a very useful high-throughput extension of the solid-phase methodology utilizing spin columns packed with sodium hydroxide beads. This procedure has been thoroughly optimized to match the analytical performance parameters of the previously used capillary technique. As demonstrated with a high-precision glycomic profiling analysis of human blood serum, this methodological improvement offers simplicity and reproducibility, allowing the complete permethylation of 12-18 samples in less than 20 min.

Characterization of glycopeptides by combining collision-induced dissociation and electron-transfer dissociation mass spectrometry data

Structural characterization of a glycopeptide is not easily attained through collision-induced dissociation (CID), due to the extensive fragmentation of glycan moieties and minimal fragmentation of peptide backbones. In this study, we have exploited the potential of electron-transfer dissociation (ETD) as a complementary approach for peptide fragmentation. Model glycoproteins, including ribonuclease B, fetuin, horseradish peroxidase, and haptoglobin, were used here. In ETD, radical anions transfer an electron to the peptide backbone and induce cleavage of the N-Calpha bond. The glycan moiety is retained on the peptide backbone, being largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is being achieved with a mass spectrometer capable of alternating between CID and ETD on-the-fly during an LC/MS/MS analysis. This is demonstrated here with several tryptic glycopeptides.

Profiling of Human Serum Glycans Associated with Liver Cancer and Cirrhosis by IMS-MS

Aberrant glycosylation of human glycoproteins is related to various physiological states, including the onset of diseases such as cancer. Consequently, the search for glycans that could be markers of diseases or targets of therapeutic drugs has been intensive. Here, we describe a high-throughput ion mobility spectrometry/mass spectrometry analysis of N-linked glycans from human serum. Distributions of glycans are assigned according to their m/z values, while ion mobility distributions provide information about glycan conformational and isomeric composition. Statistical analysis of data from 22 apparently healthy control patients and 39 individuals with known diseases (20 with cirrhosis of the liver and 19 with liver cancer) shows that ion mobility distributions for individual m/z ions appear to be sufficient to distinguish patients with liver cancer or cirrhosis. Measurements of glycan conformational and isomeric distributions by IMS-MS may provide insight that is valuable for detecting and characterizing disease states.

Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-MS.

Ion mobility-mass spectrometry (IMS-MS) and molecular modeling techniques have been used to characterize ovalbumin N-linked glycans. Some glycans from this glycoprotein exist as multiple isomeric forms. The gas-phase separation makes it possible to resolve some isomers before MS analysis. Comparisons of experimental cross sections for selected glycan isomers with values that are calculated for iterative structures generated by molecular modeling techniques allow the assignment of sharp features to specific isomers. We focus here on an example glycan set, each having a m/z value of 1046.52 with formula [H5N4+2Na]2+, where H corresponds to a hexose, and N to a N-acetylglucosamine. This glycan appears to exist as three different isomeric forms that are assignable based on comparisons of measured and calculated cross sections. We estimate the relative ratios of the abundances of the three isomers to be in the range of ∼1.0:1.35:0.85 to ∼1.0:1.5:0.80. In total, IMS-MS analysis of ovalbumin N-linked glycans provides evidence for 19 different glycan structures corresponding to high-mannose and hybrid type carbohydrates with a total of 42 distinct features related to isomers and/or conformers.

Automated interpretation of MS/MS spectra of oligosaccharides

MOTIVATIONnThe emerging glycomics and glycoproteomics projects aim to characterize all forms of glycoproteins in different tissues and organisms. Tandem mass spectrometry (MS/MS) is the key experimental methodology for high-throughput glycan identification and characterization. Fragmentation of glycans from high energy collision-induced dissociation generates ions from glycosidic as well as internal cleavages. The cross-ring ions resulting from internal cleavages provide additional information that is important to reveal the type of linkage between monosaccharides. This information, however, is not incorporated into the current programs for analyzing glycan mass spectra. As a result, they can rarely distinguish from the mass spectra isomeric oligosaccharides, which have the same saccharide composition but different types of sequences, branches or linkages.nnnRESULTSnIn this paper, we describe a novel algorithm for glycan characterization using MS/MS. This algorithm consists of three steps. First, we develop a scoring scheme to identify potential bond linkages between monosaccharides, based on the appearance pattern of cross-ring ions. Next, we use a dynamic programming algorithm to determine the most probable oligosaccharide structures from the mass spectrum. Finally, we re-evaluate these oligosaccharide structures, taking into account the double fragmentation ions. We also show the preliminary results of testing our algorithm on several MS/MS spectra of oligosaccharides.nnnAVAILABILITYnThe program GLYCH is available upon request from the authors.

Characterizing protein glycosylation sites through higher-energy C-trap dissociation.

Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher-energy C-trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N-glycosylation sites of glycoproteins. The combination of the very informative collision-induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple-stage tandem mass spectrometry. The resulting MS(3) spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut-off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites.

Glycomic analysis by capillary electrophoresis–mass spectrometry

The occurrence of multiple glycosylation sites on a protein, together with the number of glycan structures which could potentially be associated with each site (microheterogeneity) often leads to a large number of structural combinations. These structural variations increase with the molecular size of a protein, thus contributing to the complexity of glycosylation patterns. Resolving such fine structural differences has been instrumentally difficult. The degree of glycoprotein microheterogeneity has been analytically challenging in the identification of unique glycan structures that can be crucial to a distinct biological function. Despite the wealth of information provided by the most powerful mass spectrometric (MS) and tandem MS techniques, they are not able to readily identify isomeric structures. Although various separation methods provide alternatives for the analysis of glycan pools containing isomeric structures, capillary electrophoresis (CE) is often the method of choice for resolving closely related glycan structures because of its unmatched separation efficiency. It is thus natural to consider combining CE with the MS-based technologies. This review describes the utility of different CE approaches in the structural characterization of glycoproteins, and discusses the feasibility of their interface to mass spectrometry.

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker

Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase δ. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer.

Glycomic profiling of invasive and non-invasive breast cancer cells

Quantitative profiling of glycans with different structures appears essential for a better understanding of the cellular adhesion phenomena associated with malignant transformation and the underlying aberrant glycosylation of cancer cells. Using the recently developed glycomic techniques and mass-spectrometric measurements, we compare the N-linked and O-linked oligosaccharide profiles for different breast cancer cell lines with those of normal epithelial cells. Statistically significant differences in certain neutral, sialylated and fucosylated structures are readily discerned through quantitative measurements, indicating a potential of distinguishing invasive and non-invasive cancer attributes. The glycomic profile data cluster accordingly using Principal Component Analysis, verifying further glycobiological differences due to the differences between normal and cancer cell lines.

Quantitative serum glycomics of esophageal adenocarcinoma and other esophageal disease onsets.

Aberrant glycosylation has been implicated in various types of cancers and changes in glycosylation may be associated with signaling pathways during malignant transformation. Glycomic profiling of blood serum, in which cancer cell proteins or their fragments with altered glycosylation patterns are shed, could reveal the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease (N = 18), patients with high grade dysplasia (HGD, N = 11) and Barretts esophagus (N = 5), and patients with esophageal adenocarcinoma (EAC, N = 50) in an attempt to delineate distinct differences in glycosylation between these groups. The relative intensities of 98 features were significantly different among the disease onsets; 26 of these correspond to known glycan structures. The changes in the relative intensities of three of the known glycan structures predicted esophageal adenocarcinoma with 94% sensitivity and better than 60% specificity as determined by receiver operating characteristic (ROC) analysis. We have demonstrated that comparative glycomic profiling of EAC reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate disease-free from HGD, disease-free from EAC, and HGD from EAC. The clinical utility of these glycan biomarkers requires further validation.