A microarray-matrix-assisted laser desorption/ionization-mass spectrometry approach for site-specific protein nglycosylation analysis, as demonstrated for human serum immunoglobulin M (IgM)

Martin Pabst, Simon Karl Küster, Fabian Wahl, Jasmin Krismer, Petra S. Dittrich, Renato Zenobi

Research output: Contribution to journalArticleScientificpeer-review

15 Citations (Scopus)

Abstract

We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray- matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for onplate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-offlight- MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332.

Original languageEnglish
Pages (from-to)1645-1656
JournalMolecular and Cellular Proteomics
Volume14
Issue number6
DOIs
Publication statusPublished - 2015
Externally publishedYes

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