Hyphenated technique for releasing and MALDI MS analysis of O-glycans in mucin-type glycoprotein samples.

We developed an automatic apparatus for the release of O-glycans from mucin-type glycoproteins and proteoglycans (Matsuno, Y.-k.; Yamada, K.; Tanabe, A.; Kinoshita, M.; Maruyama, S.-z.; Osaka, Y.-s.; Masuko, T.; Kakehi, K. Anal. Biochem. 2007, 363, 245-257. Yamada, K.; Hyodo, S.; Matsuno, Y. K.; Kinoshita, M.; Maruyama, S. Z.; Osaka, Y. S.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52-61). The method allows rapid release of O-glycans as the reducing form within 10 min. In the present study, we connected the device to a MALDI-TOF MS spotter and achieved routine analysis of O-glycans in biological samples for clinical use after in situ derivatization of the released O-glycans with phenylhydrazine. We applied the method to the analysis of O-glycans expressed on MKN45 cells derived from human stomach cancer cells and found that MKN45 cells expressed characteristic trisialo-polylactosamine-type glycans as reported previously (Yamada, K.; Kinoshita, M.; Hayakawa, T.; Nakaya, S.; Kakehi, K. J. Proteome Res. 2009, 8, 521-537). We also applied the method to the analysis of O-glycans in serum samples. The present technique is the first attempt to use MS measurement for routine clinical diagnostic works.

Rapid and sensitive analysis of mucin-type glycans using an in-line flow glycan-releasing apparatus.

Rapid and sensitive analysis of glycans is essential for glycomics. We previously reported an apparatus, the AutoGlycoCutter (AGC), for rapid release of O-linked glycans under alkaline conditions and its application to rapid analysis of glycans in proteoglycans. We now report an application of the AGC to obtain mucin-type glycans with reducing end (i.e., hemiacetal group) within only 3 min. The released oligosaccharides could be labeled with fluorescent 2-aminobenzoic acid for analysis by normal-phase high-performance liquid chromatography (NP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We could detect O-glycans from as low as 5 pmol of bovine caseino glycomacropeptide (CGMP) by the proposed procedures. The validity of the current method was shown by the analyses of the released O-glycans from some standard glycoproteins: bovine submaxillary mucin, bovine fetuin, porcine stomach mucin, and human colostrum immunoglobulin A. The advantage of the current method was also demonstrated in comparative analysis of mucin-type glycans in CGMP derived from three different animal species.