The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously-described reductive β-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.

Silica-based lectin microcolumns are described in this study together with the chemical procedures necessary for their preparation. The analytical merits of Canavalia ensiformis and Sambucus nigra lectins, [immobilized on activated macroporous silica], such as binding capacity, trapping reproducibility, and substrate selectivity, have been evaluated using model glycoproteins. The described microcolumns are applicable to high-pressure analytical schemes utilizing microvalving procedures, washing steps, and quantitative desorption for LC/MS analysis. The described analytical systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation.High-performance affinity chromatography (HPAC) offers a number of advantages 1-4 over the classical and commonly used chromatographic technique which utilizes various immobilized ligands to retain selectively an analyte or a group of analytes. These advantages primarily include speed, ease of automation, and the application versatility. During the past decade, HPAC has been employed in biomolecular purification and enrichment and chiral separations, as well as in the study of biological interactions. 5-7 The success of any affinity chromatography, including HPAC, is largely dependent on chemical procedures employed in the attachment of a small-molecule ligand or an interacting biopolymer to a respective chromatographic support. 8-10 Their immobilization should not interfere with the native state of a ligand, facilitating access of analyte molecules and proper interactions.Lectins comprise a group of proteins with unique affinities toward carbohydrate structures. They have long been used in biomolecular isolation, carbohydrate chemistry, and histochemistry, and more recently as the mimicking agents in intercellular interaction studies. For a number of years, lectins have been utilized in various glycoprotein isolation efforts, 11-14 albeit few addressed the quantitative aspects of this approach. 15,16 Lectins with different specificities toward oligosaccharides have been immobilized to agarose and other conventional biochemical matrixes. 11-14 Some of these column materials have been commercialized. In addition, lectins have been immobilized to magnetic beads, 17 gold foils, 18 silica materials, 19-21 and to affinity membranes. 22The uses of lectins in contemporary glycoproteomics and glycomics are likely to grow due to the frequent needs for fractionation and preconcentration for high-sensitivity mass spectrometry (MS). 23 Although glycoproteins and glycopeptides could be fractionated through lectins with different specificity, in most reported cases, lectin affinity procedures were employed to isolate the entire pools of glycoconjugates rather than specific structures. 24,25 Consequently, wheat germ agglutinin and concanavalin A have been widely used because of their broad specificity. Although it is assumed that different glycoproteins produced by a certain cell type possess a similar type...

In simplifying the evaluation and quantification of high-throughput label-free quantitative proteomic data, we introduce ProteinQuant Suite. It comprises three standalone complementary computer utilities, namely ProtParser, ProteinQuant, and Turbo RAW2MGF. ProtParser is a filtering utility designed to evaluate database search results. Filtering is performed according to different criteria that are defined by the end-user. ProteinQuant then utilizes this parsed list of peptides and proteins in conjunction with mzXML or mzData files generated from the raw files for quantification. This quantification is based on the automatic detection and integration of chromatographic peaks representative of the liquid chromatography/mass spectrometry (LC/MS) elution profiles of identified peptides. Turbo RAW2MGF was developed to extend the applicability of ProteinQuant Suite to data collected from different types of mass spectrometers. It directly processes raw data files generated by Xcalibur, a ThermoElectron data acquisition software, and generates a MASCOT generic file (MGF). This file format is needed since the protein identification results generated by the database search employing this file format include information required for the precise identification and quantification of chromatographic peaks. The performance of ProteinQuant Suite was initially validated using LC/MS/MS generated for a mixture of standard proteins as well as standard proteins spiked in a complex biological matrix such as blood serum. Automated quantification of the collected data resulted in calibration curves with R(2) values higher than 0.95 with linearity spanning over more than 2 orders of magnitude with peak quantification reproducibility better than 15% (RSD). ProteinQuant Suite was also applied to confirm the binding preference of standard glycoproteins to Con A lectin using a sample consisting of both standard glycoproteins and proteins.

We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.

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Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.

Posttranslational modifications (PTM) of proteins are among the key biological regulators of function, activity, localization, and interaction. The fact that no more than 30,000-50,000 proteins are encoded by the human genome underlines the importance of posttranslational modifications in modulating the activities and functions of proteins in health and disease. With approximately 50% of all proteins now considered to be glycosylated, its physiological importance in mammalian systems is imperative. Aberrant glycosylation has now been recognized as an attribute of many mammalian diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular conditions, and cancer. As many potential disease biomarkers may be glycoproteins present in only minute quantities in tissue extracts and physiological fluids, glycoprotein isolation and enrichment may be critical in a search for such biomarkers. For decades, efforts have been focused on the development of glycoprotein enrichment from complex biological samples. Logically, the great majority of these enrichment methodologies rely on the use of immobilized lectins, which permit selective enrichment of the pools of glycoproteins for proteomic/glycomic studies. In this chapter, lectin affinity chromatography in different formats are described, including tubes; packed columns, and microfluidic channels.

Hepatocellular cancer is a serious human disease with an unfortunately low survival rate. It further poses a significant epidemic threat to our society through its viral vectors associated with cirrhosis conditions preceding the cancer. A search for biomarkers of these diseases enlists analytical glycobiology, in general, and quantitative biomolecular mass spectrometry (MS), in particular, as valuable approaches to cancer research. The recent advances in quantitative glycan permethylation prior to MALDI-MS oligosaccharide profiling has enabled us to compare the glycan quantitative proportions in the small serum samples of cancer and cirrhotic patients against control individuals. We were further able to fractionate the major serum proteins from the minor components and compare statistically their differential glycosylation, elucidating some causes of quantitatively unusual glycosylation events. Numerous glycan structures were tentatively identified and connected with the origin proteins, with a particular emphasis on sialylated and fucosylated glycans.