419 ISSN: 1469-0667 © IM Publications LLP 2008 doi: 10.1255/ejms.694 All rights reserved EUROPEAN JOURNAL OF MASS SPECTROMETRY Glycosylation is an often observed protein modiication. 1,2 This changes physicochemical properties of proteins, but even more important, it is closely related to cell–cell communica- tion. 3 Although signiicance of glycosylation is becoming well recognized, our understanding is still far from being complete. Oligosaccharides are among the most complex biopolymers due to branching, varied stereochemistry and often a high degree of heterogeneity. Their analysis is very challenging, often requiring a variety of different approaches. 4–8 A commonly used approach is to cleave off the oligosaccharides from the protein and analyzing the resulting oligosaccharide mixture by mass spectrometry. 9–11 MALDI is used most often, 9,12 which is very eficient to determine oligosaccharide composition (i.e. based on molecular mass). A signiicant drawback is, however, that it lacks information on detecting/separating possible isomeric forms. Chromatography, in particular using porous graphitized carbon (PGC) columns (also called GCC, graph- itized carbon chromatography), was shown to be effective separating highly polar oligosaccharides. 13 Combining PGC chromatography with mass spectrometry has been shown to produce excellent results. 14,15 One option is off-line fractiona- tion using PGC chromatography followed by matrix-assisted laser desorption/ionization (MALDI)-MS analysis. 11 In the present Letter we have used another option, on-line combina- tion of PGC with MS using electrospray ionization (ESI). As a model, we study glycosylation of human a-1 acid glycoprotein (AGP). AGP is an acute-phase protein, having transporting and immunomodulatory functions. 16 There is a surging interest in the glycosylation of AGP, as it depends on patho-physiology and may have strong diagnostic potential. 16–18 AGP contains ive different N-glycosylation sites, each with a characteristic Letter Analysis of complex oligosaccharides using graphitized carbon liquid chromatography/mass spectrometry Lívia Budai, a Ferenc Pollreisz, a Olivér Ozohanics, a Krisztina Ludányi, b László Drahos a and Károly Vékey a,* a Hungarian Academy of Sciences, Chemical Research Center, H-1025, Pusztaszeri 59–67, Budapest, Hungary. E-mail: vekey@chemres.hu b Semmelweis University, Department of Pharmaceutics, H-1092. Ho˝gyes Endre 7, Budapest, Hungary The sugar fraction of α-1 acid glycoprotein (AGP) was studied using porous graphitized carbon (PGC) chromatography coupled to mass spectrometry. Electrospray ionization provides efficient control over fragmentation; at low collision energy only molecular species were observed, allowing accurate oligosaccharide profiling. PGC chromatography was useful separating 18 sugars differing in mono- saccharide composition. Most of these were separated into several isomeric forms; altogether 49 different oligosaccharides were found in AGP. Keywords: oligosaccharide, glycoprotein, glycoform, isoform, HPLC, UPLC, mass spectrometry, graphitized carbon chromatography (GPC) Introduction L. Budai et al., Eur. J. Mass Spectrom. 14, 419–422 (2008) Received: 15 September 2008 n Revised: 30 October 2008 n Accepted: 22 November 2008 n Publication: 8 December 2008 Special Issue: Honouring Miroslav Ryska