Lectin approaches for glycoproteomics in FDA-approved cancer biomarkers (original) (raw)
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Molecular & Cellular Proteomics, 2009
Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and highthroughput manner. Thus, the established system should provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular & Cellular Proteomics 8:99-108, 2009. Glycan synthesis in individual cells is regulated by harmonized expression of more than a hundred glycosyltransferases. Importantly, detectable dynamics occurring on each cell surface during diverse biological events, e.g. differentiation, proliferation, and signal induction, indicate drastic changes in the glyco-machinery (1). During the last few decades, there have been enormous advances in the findings of glycosylation alterations related to oncogenesis. Cell surface sialylation and 1-6 branching of N-linked glycans are strongly correlated with metastatic potential of cancer cells (2, 3). Metastatic ability is also reflected by dramatic alteration of core structures of O-glycans (4). These observations suggest that novel tumor-specific glycoproteins accompanying substantial structural changes in glycan moieties will become reliable biomarkers with higher specificity than those established previously, e.g. CA19-9 (5), carcino-embryonic antigen family (6), ␣-fetoprotein (AFP) 1 (7), and prostate-specific antigen (PSA) (8). Recent advances in technologies such as mass spectrometry (MS), microarray, and laser microdissection have strongly advanced the proteomics-based biomarker discovery phase targeting both cultured cells and tissue specimens (9-14). This has resulted in the emergence of an extensive range of biomarker "candidates", many of which are glycoproteins. However, these candidate molecules need to be subsequently subjected to a verification step prior to a much large scale validation phase (e.g. treating with Ͼ1,000 incidences). Antibody microarray has taken the place of ELISA as a more versatile and high-throughput technique, and has enabled multiplexed quantitative analysis for over a hundred proteins with sufficient sensitivity (15). As an application of MS technology, multiple-reaction monitoring-MS has also been developed (16-19). However, taking into consideration the critical glycosylation changes occurring on diverse glycoproteins, differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen et al. (20) have recently developed a lectin-overlay antibody microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated
Analytical Chemistry, 2011
Aberrant glycosylation is a fundamental characteristic of progression of diseases such as cancer. Therefore, characterization of glycosylation patterns of proteins from disease tissues may identify changes specific to the disease development and improve diagnostic performance. Thus, analysis strategies with sufficient sensitivity for evaluation of glycosylation patterns in clinical specimens are needed. Here, we describe an analytical strategy for detection and verification of glycosylation patterns. It is based on a two-phase platform including a pattern discovery phase to identify the glycosylation changes using high-density lectin microarrays and a verification phase by developing lectin-based immunosorbent assays using the identified lectins. We evaluated the analytical performance of the platform using the glycoprotein standard, and found that the lectin microarray could detect specific bindings of glycoprotein to lectins at the nanogram level and the lectin-based immunosorbent assay could be used for verification of protein glycosylation. We then applied the approach to the analysis of glycosylation patterns of two glycoproteins, which are highly expressed in prostate cancer in our prior studies, PSA and membrane metalloendopeptidase (MME), from aggressive (AC) and non-aggressive prostate cancer (NAC) tissues. The observed differences in glycosylation patterns of PSA and MME may represent significant clinical importance, and could used to develop multiplex assays for diagnosis of aggressive prostate cancer.
Journal of Proteome Research, 2006
A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of R(2,3) and R(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to eludicate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using µLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using µLC-ESI-TOF-MS where the N83 glycosylation of R1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.
Targeted glycoproteomic identification of cancer cell glycosylation
Glycobiology, 2009
GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactosecontaining ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewis x and Lewis y structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewis x antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.
Advances in Cancer Research, 2015
Recent research is uncovering unexpected ways that glycans contribute to biology, as well as new strategies for combatting disease using approaches involving glycans. To make full use of glycans for clinical applications, we need more detailed information on the location, nature, and dynamics of glycan expression in vivo. Such studies require the use of specimens acquired directly from patients. Effective studies of clinical specimens require low-volume assays, high precision measurements, and the ability to process many samples. Assays using affinity reagents-lectins and glycan-binding antibodies-can meet these requirements, but further developments are needed to make the methods routine and effective. Recent advances in the use of glycan-binding proteins could meet that need. The advances involve improved determination of specificity using glycan arrays; the availability of databases for mining and analyzing glycan array data; lectin engineering methods; and the ability to quantitatively interpret lectin measurements. Here we describe many of the challenges and opportunities involved in the application of these new approaches to the study of biological samples. The new tools hold promise for developing methods to improve the outcomes of patients afflicted with diseases characterized by aberrant glycan expression.
Lectins: an effective tool for screening of potential cancer biomarkers
PeerJ
In recent years, the use of lectins for screening of potential biomarkers has gained increased importance in cancer research, given the development in glycobiology that highlights altered structural changes of glycans in cancer associated processes. Lectins, having the properties of recognizing specific carbohydrate moieties of glycoconjugates, have become an effective tool for detection of new cancer biomarkers in complex bodily fluids and tissues. The specificity of lectins provides an added advantage of selecting peptides that are differently glycosylated and aberrantly expressed in cancer patients, many of which are not possibly detected using conventional methods because of their low abundance in bodily fluids. When coupled with mass spectrometry, research utilizing lectins, which are mainly from plants and fungi, has led to identification of numerous potential cancer biomarkers that may be used in the future. This article reviews lectin-based methods that are commonly adopted ...
A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples
Journal of Visualized Experiments, 2009
Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls-fucosylated and sialylated human lactoferrin glycopeptides-and negative controls-high mannose glycopeptides from Saccharomyces cerevisiae-that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.
A lectin array-based methodology for the analysis of protein glycosylation
Journal of Biochemical and Biophysical Methods, 2007
Glycosylation is the most versatile and one of the most abundant protein modifications. It has a structural role as well as diverse functional roles in many specific biological functions, including cancer development, viral and bacterial infections, and autoimmunity. The diverse roles of glycosylation in biological processes are rapidly growing areas of research, however, Glycobiology research is limited by the lack of a technology for rapid analysis of glycan composition of glycoproteins. Currently used methods for glycoanalysis are complex, typically requiring high levels of expertise and days to provide answers, and are not readily available to all researcher.
Journal of Proteome Research, 2008
Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N-linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reverse-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal components analysis, hierarchical clustering, and Z-statistic analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential