Glycomic Alterations in the Highly-abundant and Lesser-abundant Blood Serum Protein Fractions for Patients Diagnosed with Hepatocellular Carcinoma.

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.

Effects of lead and mercury on the blood proteome of children.

Heavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding of biochemical interactions responsible for the health problems related to lead and mercury exposure at concentrations well below CDC guidelines. Blood plasma and serum samples from 34 children were depleted of their most abundant proteins using antibody-based affinity columns and analyzed using two different methods, LC-MS/MS and 2-D electrophoresis coupled with MALDI-TOF/MS and tandem mass spectrometry.

Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans: a potential methodology for cancer-biomarker discovery.

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.

A quantitative investigation of fucosylated serum glycoproteins with application to esophageal adenocarcinoma.

Although glycoproteomic studies provide unique opportunities for cancer research, it has been necessary to develop specific methods for analysis of oncologically interesting glycoproteins. We describe a general, multimethodological approach for quantitative glycoproteomic analysis of fucosylated glycoproteins in human blood serum. A total of 136 putative fucosylated glycoproteins were identified with very high confidence in three clinically relevant sample pools (N=5 for each), with a mean CV of 3.

Assigning glycosylation sites and microheterogeneities in glycoproteins by liquid chromatography/tandem mass spectrometry.

Glycosylation of proteins is one of the most common posttranslational modifications which has its bearing on function and biological activity. Assigning the glycosylation sites and their inherent microheterogeneities are key structural issues addressing various glycoprotein functions. This chapter describes three different approaches all based on liquid chromatography/tandem mass spectrometry (LC/MS-MS), which are commonly employed for the assignment of protein glycosylation sites and their microheterogeneities.

ProteinQuant Suite: a bundle of automated software tools for label-free quantitative proteomics.

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.

Efficacy of glycoprotein enrichment by microscale lectin affinity chromatography.

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.

Glycoprotein enrichment through lectin affinity techniques.

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.

High-sensitivity profiling of glycoproteins from human blood serum through multiple-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry.

We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7-10 orders of magnitude.

Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry.

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.

Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins.

Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.

Combining lectin microcolumns with high-resolution separation techniques for enrichment of glycoproteins and glycopeptides.

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.