Search

Background: Glycoproteins are often associated with cancer and are important in serum studies, for which glycosylation is a common posttranslational modification., Methods: We used multilectin affinity chromatography (M-LAC) to isolate glycoproteins from the sera of breast cancer patients and controls. The proteins were identified by HPLC-tandem mass spectrometry (MS/MS) analysis of the corresponding tryptic digests. We used the FuncAssociate Gene Ontology program for association analysis of the identified proteins. Biomarker candidates in these groups were comparatively quantitated by use of peak area measurements, with inclusion of an internal standard. We analyzed data for concordance within the ontology association groups for vector of change with the development of breast cancer., Results: Detection of the known low-concentration biomarker HER-2 (8-24 mug/L) enabled us to establish a dynamic range of 10(6), relative to the amount of albumin, for the depletion step. We then used ELISA to confirm this range. Proteins associated with lipid transport and metabolism, cell growth and maintenance, ion homeostasis, and protease inhibition were found to be differentially regulated in serum from women with breast cancer compared with serum from women without breast cancer., Conclusions: M-LAC for isolation of the serum glycoproteome, coupled with liquid chromatography-MS/MS and the use of gene ontology associations, can be used to characterize large panels of candidate markers, which can then be evaluated in a particular patient population.

The glycoproteome is a major subproteome present in human plasma. In this study, we isolated and characterized approximately 150 glycoproteins from the human plasma and serum samples provided by HUPO using a multilectin affinity column. The corresponding tryptic digest was separated by RP-HPLC coupled to an IT mass spectrometer (3-D LCQ). Also in this study, a new system, namely an Ettan MDLC system coupled to a linear ITLTQ, was compared with the previous LCQ platform and gave a greater number of protein identifications, as well as better quality. When we compared the composition of the glycoproteomes for the plasma and serum samples there was a close correlation between the samples, except for the absence of fibrinogen from the identified-protein list in the latter sample, which was presumably as a result of the clotting process. In addition, the analysis of the samples from three ethnic specimens, Caucasian American, Asian American, and African American, were very similar but showed a higher angiotensinogen plasma level and a lower histidine-rich glycoprotein level in Caucasian American samples, and a lower vitronectin level in African American blood samples.

Previously, we reported that the distribution of glycoproteins into the lectin displacement fractions of a multi-lectin affinity column was determined by the glycosylation patterns of the proteins. This distribution was observed by the sequential use of displacers specific to the lectins in the column. In this study we have evaluated the multi-lectin column (containing Concanavalin A, Wheat germ agglutinin and Jacalin lectin) to screen glycoproteins with known glycosylation pattern changes. The presence of a glycoprotein in a given displacer fraction was determined by LC-MS/MS analysis of a tryptic digest. We have used the enzyme neuraminidase to modify the oligosaccharide chains present in human transferrin, and used the enzymes, neuraminidase and fucosidase, to modify glycoproteins present in human serum. Then, by comparison with the untreated samples, we demonstrated a distribution shift of the enzyme-treated serum glycoproteins in the displacement fractions isolated from the multi-lectin column. The fractions were analyzed by a protein assay, Sequest rank comparison and peak area measurement from the extracted ion chromatogram. The results indicated that the multi-lectin affinity column (M-LAC) is sensitive to changes in the content of sialic acid and fucosyl residues present in serum glycoproteins, and has the potential to be used to screen serum proteins for glycosylation changes due to disease. In addition, the use of a glycosidase to induce specific structural changes in glycoproteins can support the development of multi-lectin column formats specific for detecting changes in the glycoproteome of certain diagnostic fluids and types of disease.

Glycosylation is one the most common post-translational modifications (PTM) and glycoproteins play fundamental roles in a diversity of biological processes. The development of an analytical approach to the study of variation of glycosylation patterns in serum samples has been hindered by the structural heterogeneity of this post-translational modification and the complexity of serum proteome. We have used the ability of different lectins to recognize specific glycosylation motifs to develop a specific affinity system that can achieve a comprehensive capture of serum glycoproteins. In a preliminary investigation, we evaluated the ability of five commonly used immobilized lectins to capture glycoproteins from human serum. SDS-PAGE analysis showed each lectin was able to enrich a subset of the serum glycoproteome and overlaps in lectin specificity were indeed observed. Based on these results and with the goal of studying the extent of the human serum glycoproteome, we then developed a multi-lectin affinity column containing Concanavalin A (Con A), Wheat germ and Jacalin lectin. The selection of lectins was also based on the known N-linked and O-linked glycan structures that are considered representative of the serum proteome. We then demonstrated that the capture of glycoproteins was specific, efficient and reproducible with this multi-lectin column. The results obtained with this affinity step indicated that about 10% of human serum proteins are glycosylated (weight/weight) and, after removal of six high abundance proteins, including albumin, at least 50% of the remaining proteins were glycosylated. We then evaluated the use of this affinity column to monitor changes in the pattern of glycosylation in serum samples by a controlled, stepwise release of the captured proteins from the multi-lectin affinity column with specific displacers.