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.

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.

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Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a largescale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated 90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.