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Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aberrant protein glycosylation has been shown to be associated with disease processes and identification of disease-specific glycoproteins and glycosylation changes may serve as potential diagnostic and therapeutic biomarkers. However despite recent advances in proteomic-based biomarker discovery, this knowledge has not yet translated into an extensive mining of the glycoproteome for potential biomarkers. The major challenge for a comprehensive glycoproteomics analysis arises primarily from the enormous complexity and the large dynamic range in protein constituent in biological samples. Methods that specifically target glycoproteins are therefore necessary to facilitate their selective enrichment prior to their identification by MS-based analysis. The use of lectins, with selective affinities for specific carbohydrate epitopes, to enrich glycoprotein fractions coupled with modern MS, have greatly enhanced the identification of the glycoproteome. On account of their ability to specifically bind cell surface carbohydrates lectins have, during the recent past, found extensive applications in elucidation of the architecture and dynamics of cell surface carbohydrates, glycoconjugate purification, and structural characterization. Combined with complementary depletion and MS technologies, lectin affinity chromatography is becoming the most widely employed method of choice for biomarker discovery in cancer and other diseases., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.

Plasma is an important biological material for biomarker discovery. However, the wide dynamic range in protein concentration remains a major challenge. In this paper, we introduce the development of a proteomic platform for analysis of plasma samples. The method utilizes a double fractionation approach which combines the MARS immunodepletion column with multi-lectin affinity chromatography, M-LAC, to deplete the most abundant proteins in plasma, the majority of which are glycosylated. To determine the suitability of this methodology, we applied the workflow described in this study to a sample set composed of four groups: a control pool and three different disease pools: obesity, diabetes, and hypertension. We were able to identify changes in the level of several proteins; for example, a protein such as angiotensinogen was found to be present at high levels in patients with obesity plus diabetes and hypertension. On the other hand, apolipoprotein CI was shown to be elevated in all disease groups. A review of the literature supported our observation. The methodology presented in this report was shown to be effective for profiling changes in the plasma proteome of subjects with obesity and its associated complications such as diabetes and hypertension.

An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.