Yu-ki Matsuno

Quantitative Derivatization of Sialic Acids for the Detection of Sialoglycans by MALDI MS

Recently, glycans have been recognized as valuable biomarkers for various disease states. In particular, sialoglycans, which have sialic acids at their terminal end, are likely to have relevance to diseases such as cancer and inflammation. Mass spectrometry (MS) has become an indispensable tool for biomarker discovery. However, matrix-assisted laser desorption ionization (MALDI) MS of sialoglycans normally causes loss of sialic acid. Methylesterification or amidation of carboxyl functionality in sialic acid has been reported to suppress the loss of sialic acids. We found that the modifications of alpha2,3-linked sialic acids proceed less efficiently than those at alpha2,6-linkages. Furthermore, the modifications of the alpha2,3-linked sialic acids are incomplete. This variability in the extent of derivatization presents a major problem in terms of glycan biomarker discovery using MALDI MS. In this study, we developed a novel amidation using acetohydrazide which can completely modify both types of linkages of sialoglycans. With the use of this method, we demonstrate MS profiling of N-linked glycans released from a bovine fetuin which is rich in alpha2,3-linked sialic acids.

Supported Molecular Matrix Electrophoresis: A New Tool for Characterization of Glycoproteins

A new concept of separation technology, supported molecular matrix electrophoresis (SMME), is described. In SMME, analytes migrate in a molecular matrix supported by backbone materials. Here we introduce a novel strategy for the separation and characterization of mucins using SMME. Mucin, a highly tumor-associated glycoprotein, has great potential as clinical biomarker for diagnosis of various malignant tumors. However, due to their large size, polymeric nature, and heterogeneous glycosylation, analysis of mucins has been left behind by modern techniques. For mucin analysis, we employed a poly(vinylidene difluoride) (PVDF) membrane and poly(vinyl alcohol) (PVA) as the backbone material and the matrix molecule, respectively. Combining SMME with mass spectrometry and capillary electrophoresis, we demonstrate that a crude porcine stomach mucin consists of a neutral and a sulfated mucin and is contaminated by chondroitin sulfate-containing proteoglycan and hyaluronic acid. Furthermore, to demonstrate the feasibility of the strategy for biomarker discovery, we analyzed mucins in human pancreatic juice, which is an important source for clinical biomarkers of pancreatic tumors. This work revealed the presence of three types of mucin with distinct glycan profiles in human pancreatic juice.

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure

Mucins are a family of heavily glycosylated high molecular mass proteins that have great potential as novel clinical biomarkers for the diagnosis of various malignant tumors. Supported molecular matrix electrophoresis (SMME) is a new type of membrane electrophoresis that can be used to characterize mucins. In SMME, mucins migrate in a molecular matrix supported by membrane materials. Here, we have developed an immunostaining method for the identification of SMME‐separated mucins. The novel method involves stably fixing the mucins onto the SMME membrane and optimizing the molecular matrix for the fixation process. We applied this technique for the detection of MUC1 produced from three cancer cell lines (T47D, HPAF‐II and BxPC3) and also analyzed their O‐linked glycans by mass spectrometry. Our results revealed that properties of the MUC1 molecules from the three cell lines are different in terms of migrating position in SMME and glycan profile. The present method allows simple and rapid characterization of mucins in terms of both glycans and core proteins. The method will be a useful tool for the exploration of mucin alterations associated with various diseases such as cancer.

Serum protein fractionation using supported molecular matrix electrophoresis

Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue‐71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1‐nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N‐linked and O‐linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.

Identification of mucins by using a method involving a combination of on-membrane chemical deglycosylation and immunostaining

The characterization of mucins is critically important for gaining insights into the molecular pathology of diseases, including cancers, as well as for the discovery of biomarkers for disease diagnosis. However, no practical method has yet been reported for identifying mucin proteins. Here, we report a technique for immunological identification of mucins separated by supported molecular matrix electrophoresis (SMME), a recently developed membrane electrophoresis method. The technique involves on-membrane deglycosylation of mucins by using mild periodate oxidation/base-catalyzed elimination, followed by immunostaining with an antibody that specifically recognizes the mucin tandem repeat (TR) peptide. We demonstrated the methods feasibility by using MUC1 derived from 2 cancer cell lines, T47D and HPAF-II. The present method shows potential as an alternative approach for the identification of mucins separated by SMME or blotted from conventional gel electrophoresis, on a PVDF membrane.

Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes

Alcian blue staining has been widely used to visualize acidic mucins and mucopolysaccharides in supported molecular matrix electrophoresis (SMME) and on membrane transferred from electrophoresis gels. Mucins with low acidic glycan content, however, cannot be stained with Alcian blue, which is one of the major drawbacks of this staining method. On the other hand, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, regardless of the acidic residue content; however, periodic acid-Schiff staining decomposes glycans. Here, we introduce succinylation-Alcian blue staining as an alternative staining method to visualize mucins, regardless of the acidic residue content, and without glycan decomposition.

Determination of sulfate ester content in sulfated oligo- and poly-saccharides by capillary electrophoresis with indirect UV detection

Carbohydrates having sulfate groups such as glycosaminoglycans and chemically synthesized sucrose sulfate show interesting and important biological activities. We adapted CE with indirect UV detection technique to the determination of sulfate ester in sulfated carbohydrates, which were previously hydrolyzed with HCl. The liberated sulfate ion was analyzed using a background electrolyte consisting of triethanolamine-buffered chromate with hexamethonium bromide. Sulfate contents of glucose 3-sulfate and sucrose octasulfate used as a model were in good agreement with theoretical values (accuracy, 95.9-96.7 and 97.4-101.9%, respectively), and relative standard deviation values run-to-run were 0.977 and 1.90%, respectively. We applied the method to the determination of the sulfate contents of some glycosaminoglycan samples and showed that the contents were in good agreement with those calculated from sulfur content.

Supported Molecular Matrix Electrophoresis: A New Membrane Electrophoresis for Characterizing Glycoproteins

Protein blotting is often used for identification and characterization of proteins on a membrane to which proteins separated by gel electrophoresis are transferred. The transferring process is sometimes problematic, in particular, for mucins and proteoglycans. Here, we describe a novel membrane electrophoresis technique, termed supported molecular matrix electrophoresis (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used as the separation support. Proteins separated by this method can be immunoblotted without any transferring procedures.

Identification of Core Proteins Carrying the Sialyl Lewis a Epitope in Pancreatic Cancers

Identification of core proteins carrying the CA19-9 (carbohydrate antigen, sialyl Lewis a) epitope from various tissues will improve the diagnosis of pancreatic cancer in terms of specificity and sensitivity. In this study, we attempted to identify sialyl Lewis a-carrier proteins specifically expressed in pancreatic cancer. Pancreatic cancer is difficult to detect in the early stages of the disease, resulting in a high level of mortality. Therefore, in order to determine the correct course of treatment, it is vital to distinguish cancer from obstruction of the bile duct or other diseases. Our strategy to identify the carrier proteins was as follows: glycoproteins carrying sialyl Lewis a antigen were enriched from pancreatic cancer cell lines using anti-sialyl Lewis a antibody and then subjected to Peptide Mass Fingerprinting analysis. Based on these studies we identified nine glycoproteins carrying the sialyl Lewis a epitope. We evaluated candidate molecules by biochemical analyses of culture supernatants and human sera. In particular, we focused on one candidate molecule carrying a sialyl Lewis a epitope, Galectin-3BP/MAC2BP; M2BP, which was analyzed in detail. These results verified that our candidate molecule is a core protein carrying the sialyl Lewis a epitope. Furthermore, we demonstrated sandwich ELISA, which showed that the glycoprotein was able to detect CA19-9 antigen in culture supernatants. Our approach facilitated the identification of the core protein carrying the sialyl Lewis a epitope. We believe our approach will enable future developments in cancer glycobiomarker identification.