Conference Program - ABRF 2011 - Association of Biomolecular ...

pattern of N-linked glycans in form of the respective N-glycopeptides.Targeted analysis of potential glycopeptides significantly increased theinformation content. Integrated glycoprotein analysis software tools(ProteinScape 2.2) allowed identification of glycan modifications andinteractive result validation. In this process software facilitated theinitial characterization of the antibody as well as the subsequent qualitycontrol tasks.204 Magnetic ZIC-HILIC BeadsEnrichment for Neutral and AcidicGlycopeptidesA. Resemann 1 , J. Wohlgemuth 2 , S. Andrecht 2 ,A. Schneider 1 , U. Schweiger-Hufnagel 1 , D. Suckau 11Bruker Daltonics, Bremen, Germany; 2 Merck KGaA,Darmastadt, GermanyGlycosylation is the most abundant protein posttranslational modificationand is involved in many relevant biological processes and crucial to theunderstanding of many diseases. In depth analysis of glycosylationsites is difficult, however, as glycopeptides exhibit a significant microheterogeneity at glycosylation sites. In addition, ion suppressioneffects require selective methods for glycopeptide enrichment. Massspectrometric analysis of glycopeptides is challenging because boththe peptide as well as the glycan moiety have to be elucidated for a fullstructural understanding. We used Fetuin, Asialo-Fetuin and Alpha-1-Acidglycoprotein to equally representing sialylated and non-sialylatedglycosylic structures. In addition, monoclonal antibodies were analyzedas a dedicated example for pharmaceutical QC. Proteins were digestedwith trypsin and glycopeptides were enriched using a dedicated ZIC-HILIC glycocapture beads in combination with an optimized buffersystem (EMD Chemicals Inc.). The glycopeptides were analyzed usingESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for indepth characterization of the glycopeptides. For database searches,an integrated software approach was used: protein searches of theglycopeptide MS/MS spectra were performed for obtaining the aminoacid sequence of the glycopeptide, and searches in glycan databasesbased on the same glycopeptide MS/MS spectra were carried outto complete the characterization of N-linked glycopeptides. In thecurrent study, two important features of glycoprotein analysis areshown: (1) The employed integrated software approach allowed theglycan identification in a similar way as peptide identification. Theimportant step of interactive result validation was facilitated by a suiteof dedicated data and result viewers. (2) Compared to MS analysis ofnative glycoprotein digests, the enriched samples allowed to detectmore glycopeptides and permitted the acquisition of higher qualityMS/MS spectra. For MALDI-TOF/TOF-MS analysis, linear positive ionmode detection of precursor ions proved to be highly suitable for theanalysis of even multi-sialylated glycopeptides.205 Fragment Analysis of CarbohydratesFollowing Capillary ElectrophoresisT. Snyder-Leiby, D. Hulce, F. Li, X. Li, C.S. LiuSoftGenetics, LLC, State College, PA, United StatesDepending on the macromolecule size and configuration, migrationrates through capillary electrophoresis vary greatly. Internal sizestandards for capillary electrophoresis of the same macromolecule maynot be readily available. The Macromolecule Tool in GeneMarker®aids with analysis of macromolecule fragments without an internallane size standard. Methods included importing raw data files to thesoftware and physically identifying reference peaks in the samplesknown to have the same size. The program uses this information tocalibrate from one capillary to another. Characteristics of the aligneddata (such as relative size, peak height, peak area, peak ratios) wereexported in an excel sheet. Ninety six raw data files from 4 dye capillaryelectrophoresis were analyzed. Peak height, height ratio, area, arearatio, and relative sizes were determined for all samples. These valuescan be used to determine characteristics such as number and relativesize of degradation products or other macromolecules, such as DNAbinding carbohydrates commonly functioning in gene regulation.206 Analysis of Complex Oligosaccharidesfrom Glycopeptides andGlycoproteins Using MSn Spectra andOligosaccharides Spectral LibraryF. XiangShimadzu Biotech, Pleasanton, CA, United StatesGlycosylation is a common post-translational modification to cellsurface and extra cellular matrix proteins as well as to lipids. Unlikeproteins and nucleic acids that are linear polymers of amino acidsand nucleotides respectively, with linkages at only one position,carbohydrates can adopt complex branched structures with individualmonomeric units linked at one of several sites. A detailed analysisof complex carbohydrate structures has been explored with massspectrometric techniques, and still presents challenge for the analyst.This presentation will describe a systematic approach of carbohydratesand glycoconjugates analysis with MSn techniques. Oligosaccharides,cleaved from glycoproteins (by hydrazinolysis or enzymatically), werecharacterized using a hybrid MALDI Ion Trap / TOF mass spectrometer.High mannose, biantennary and triantennary oligosaccharides wereanalyzed using MS, NS2, MS3 and MS4 modes. Intact oligosaccharideswere analyzed in MS mode using a cooling gas to prevent fragmentation.Individual precursor ions were isolated in the trap, subjected tofragmentation with Argon, to provide MS2 data. Product ions wereselected for further fragmentation, which was achieved by increasingthe energy for collisionally induced dissociation. In MS mode, the singlycharged sodium adduct form of these molecules was detected, whichis typical of the analysis on conventional MALDI mass spectrometers.In MS2 mode, high mannose oligosaccharides readily lost the coreN-acetylglucosamine residues, whilst MS3 and MS4 modes were usedto sequentially fragment the product ion corresponding to the residualbranched mannose oligomer. In MS2 mode analysis of biantennaryand triantennary structures also fragmented losing disaccharide units,such as the galactose - N - acetylglucosamine units that define eachantennary branch, or core fucosylated - N - acetylglucosamine units.MS3 of selected MS2 products ions could be used to differentiatefragments generated from either the reducing or non-reducing ends.Cross-ring cleavages were also observed during fragmentation in MS2,and the relevant product ions could be used to differentiate branchedstructures by further fragmentation in MS3 mode. Many tandem massspectrometric experiments have been revealing that oligosaccharidesmight have characteristic signal intensity profiles, depending on theglycosidic linkage and branching structures. In addition to the MSncapability of the platform, there is a library of observational mass spectraacquired from structurally defined oligosaccharides. The presentationwill show the enhancement of carbohydrate identification by utilizingcomparison procedure of the signal intensity profiles of MSn spectrabetween the analyte and structurally defined oligosaccharides in thelibrary.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 89