Hyphenated Technique for Releasing and MALDI MS Analysis of O-Glycans in Mucin-Type Glycoprotein Samples

Abstract

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.

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

“…Our lectin selection can be further justified by the composition of the human serum O-glycan pool. The dominance of the monosialylated mucin core-1 O-glycan, NeuAcα2–3Galβ1–3GalNAcα in human serum O-glycoproteins has been demonstrated by two independent studies [36, 37]. This structure represents more than 60% of the glycans, followed by the diasialo and asialo variants, at approximately 20% and 10%, respectively [37].…”

“…We aimed at the isolation of glycopeptides modified with mucin-type core-1 structures, as this glycoform has been reported to be the most abundant in human serum [36]: NeuAcα2–3Galβ1–3GalNAcα (~60%), NeuAcα2–3Galβ1–3(NeuAcβ2–6)GalNAcα (~20%), and Galβ1–3GalNAcα (~10%) represent ~90% of the total O-glycan pool [37]. Considering the wide dynamic range of serum protein levels, and the expected heterogeneity of O-glycosylation, we followed multistep enrichment strategies.…”

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…More than column selection, sample preparation is a big issue for O ‐glycan analysis. For non‐reducing β‐elimination, LiOH , NH 3 , and H 2 NNH 2 were used. Comparisons between H 2 NNH 2 , NH 3 , and NaOH , or NaOH, NH 3 , and Me 2 NH were carried out.…”

Protein O-glycosylation analysis

“…Over the years, a number of different reagents for nonreductive release have been developed. Chai et al (1997) reported the use of 70 % (w/v) aqueous ethylamine and Yamada et al (2010) reported the use of lithium hydroxide in an automatic setup, but the overall reaction Possible isomeric structures are given in different colors, and key product ions derived thereof are denoted in the respective color. Nonspecifi c product ions are labeled in black.…”