Nobuaki Takemori Naoka Komori Hiroyuki Matsumoto Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Received April 27, 2005 Revised August 9, 2005 Accepted September 29, 2005 Research Article Highly sensitive multistage mass spectrometry enables small-scale analysis of protein glycosylation from two-dimensional polyacrylamide gels Structural characterization of glycoproteins remains among the most challenging areas of glycomics due to the requirement of large quantities of samples and laborious bio- chemical steps involved in the analytical procedure. Here we report the structural characterization of glycoproteins separated on a 2-D gel by using a MALDI-QIT-TOF MS where QIT is quadrupole IT. The combination of MALDI-ion source and QITappears to generate a unique tendency to cause fragmentation of glycopeptides without colli- sion-induced dissociation. The majority of such fragmentations observed in our study result from the cleavage of sugar linkages, but not of peptide-peptide or peptide-sugar linkages. This unique feature allows us to perform pseudo-MS 3 analysis of a frag- mented glycopeptide. A small gel spot of a glycoprotein in the abundance range of low picomoles was enough for the mass spectrometer to analyze fragmentation pathway of the sugar linkage and peptide backbone. In this study, we demonstrate direct determination of glycosylation sites and N-linked glycan-sequences of the tryptic gly- copeptides of Drosophila glycoproteins. Glycopeptides with various MWs up to ,4000 Da were suitable for structural analysis, including its attachment site and the amino acid sequence, of the glycopeptide through multistage mass spectrometric analysis. Keywords: Glycomics / MALDI-QIT-TOF MS / N-Linked glycosylation / Structural characterization of glycoprotein / 2-DE DOI 10.1002/elps.200500324 1 Introduction Based on the genome information of an organism, prote- omics analysis reveals identities of proteins expressed in a cell. The genome information, however, seldom reveals post-translational modifications of proteins per se, and this makes interpretation of proteomics data compli- cated, since many proteins undergo multiple post-trans- lational modifications in vivo. In eukaryotic systems, gly- cosylation is one of the most commonly observed protein modifications resulting in a large number of proteins con- jugated with sugars [1, 2], and the process of glycosyla- tion mediates crucial cellular mechanisms such as protein folding and trafficking [3, 4]. The analysis of protein gly- cosylation generally requires multistep processes includ- ing separation, identification, and quantification of a gly- coprotein, characterization of the glycan structure, and determination of the glycosylation site. Following the separation of glycoproteins, mass spectrometer has been used as a highly sensitive tool in the analysis of glycan structures [5–7]. In conventional analysis, a MALDI or ESI MS is used to determine the molecular weight of a released glycan or endpeptidase-digests of a glycopro- tein [5–7], which would provide a putative structure of the glycan based on its molecular weight. To determine the actual glycan structure and glycosylation site, a glyco- protein is further subjected to fragmentation, or MS/MS, analysis in a tandem mass analyzer [8–16]. Because a Correspondence: Dr. Hiroyuki Matsumoto, Department of Biochem- istry and Molecular Biology, University of Oklahoma Health Sciences Center, P.O. Box 26901,Oklahoma City, OK 73190, USA E-mail: hiro-matsumoto@ouhsc.edu Fax: 11-405-271-3139 Abbreviations: FTICR, fourier transform ion cyclotron resonance; GlcNAc, N-acetylglucosamine; HexNAc, N-acetylhexosamine; MS n , multistage MS/MS; NCBI, National Center for Biotechnology Information; QIT , quadrupole IT 1394 Electrophoresis 2006, 27, 1394–1406  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com