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A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)* [S]

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for... Technological Innovation and Resources © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N- glycosylation Analysis, as Demonstrated for □ S Human Serum Immunoglobulin M (IgM)* Martin Pabst‡, Simon Karl Ku¨ ster‡, Fabian Wahl§, Jasmin Krismer‡, Petra S. Dittrich‡, and Renato Zenobi‡¶ We demonstrate a new approach for the site-specific plished on the adjacent, untreated spots with high mass identification and characterization of protein N-glyco- resolution and high mass accuracy using a matrix as- sylation. It is based on a nano-liquid chromatography mi- sisted laser desorption ionization-Fourier transform-MS. croarray-matrix assisted laser desorption/ionization-MS We present the first detailed and comprehensive mass platform, which employs droplet microfluidics for on- spectrometric analysis on the glycopeptide level for hu- plate nanoliter reactions. A chromatographic separation man polyclonal IgM with high mass accuracy. Besides of a proteolytic digest is deposited at a high frequency on complex type glycans on Asn 395, 332, 171, and on the J the microarray. In this way, a short separation run is chain, we observed oligomannosidic glycans on Asn 563, archived into thousands of nanoliter reaction cavities, and Asn 402 and minor amounts of oligomannosidic glycans chromatographic peaks are spread over multiple array on the glycosite Asn 171. Furthermore, hybrid type gly- spots. After fractionation, each other spot is treated with cans were found on Asn 402, Asn 171 and in traces Asn PNGaseF to generate two correlated traces within one 332. Molecular & Cellular Proteomics 14: 10.1074/mcp. run, one with treated spots where glycans are enzymati- O114.046748, 1645–1656, 2015. cally released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is per- formed by searching for the predicted deglycosylated Glycosylation is one of the most common post-translational peptides in the treated trace. An identified peptide then modifications and it is known to play an important role in leads directly to the position of the “intact” glycopeptide protein structure, protein function, cell signaling and recogni- clusters, which are located in the adjacent spots. Further- tion (1–4). Almost all proteins involved in the immune re- more, the deglycosylated peptide can be sequenced effi- sponse are glycoproteins and their attached glycans are ciently in a simple collision-induced dissociation-MS ex- thought to be components of the immune system effector periment. We applied the microarray approach to a mechanism (5). Whereas the glycosylation of IgG, IgE and IgA detailed site-specific glycosylation analysis of human se- is well studied, there are no detailed mass spectrometric data rum IgM. By scanning the treated spots with low-resolu- available describing the site-specific glycosylation profiles of tion matrix assisted laser desorption/ionization-time-of- human serum IgM (6–9). Human IgG has one conserved N- flight-MS, we observed all five deglycosylated peptides, glycosylation site on each heavy chain CH2 domain at Asn including the one originating from the secretory chain. A detailed glycopeptide characterization was then accom- 297, and 15–20% of normal polyclonal IgG bears additional Fab (fragment antigen binding) glycosylation (6, 10, 11). Other antibody classes such as IgM or IgA show a higher complexity From the ‡Department of Chemistry and Applied Biosciences, with respect to the number of glycosylation sites and variety ETH Zu¨ rich, Vladimir-Prelog-Weg 3, 8093 Zu¨ rich, Switzerland; of glycoforms (6, 9). Only recently, also monoclonal IgM an- §Sigma-Aldrich Chemie GmbH, Industriestrasse 25, 9471 Buchs tibodies came into the focus of pharmaceutical industry, be- (SG), Switzerland cause they show great potential for the treatment of diseases Received November 26, 2014, and in revised form, March 20, 2015 Published, MCP Papers in Press March 23, 2015, DOI 10.1074/ (12–14). mcp.O114.046748 Determination of the site-specific N-glycosylation pattern of Author contributions: M.P., S.K.K., F.W., P.S.D., and R.Z. designed complex proteins is a challenging task and therefore glyco- research; M.P., S.K.K., and J.K. performed research; M.P., S.K.K., sylation is often solely analyzed on released glycan pools F.W., P.S.D., and R.Z. contributed new reagents or analytic tools; (15–17). Whereas the analysis of released pools is necessary M.P. and J.K. analyzed data; M.P., J.K., P.S.D., and R.Z. wrote the paper. when linkage and positional isomers have to be investigated Molecular & Cellular Proteomics 14.6 1645 This is an Open Access article under the CC BY license. Glycoprotein Analysis by Microarray MALDI-MS (18, 19), the information obtained is here limited to the car- strategies are often required in order to obtain a reproducible bohydrate portion and does not allow any conclusion on a and sensitive signal (34, 50, 51). protein site specific heterogeneity. N-linked glycans in partic- The identification of a potential glycopeptide cluster is fur- ular are attached by an amide bond to an asparagine residue ther performed by tandem mass spectrometry, either by scan- of the protein, where this particular asparagine is necessarily ning for marker ions (52, 53) or with the assistance of peak part of a consensus sequence NX(S/T) or more rarely part of (-pattern) interpretation tools searching for a characteristic a NXC motif (where X can be any amino acid except proline) glycoprofile mass pattern (7, 54). (Lower energy) collision- (20, 21). Enzymes like PNGaseF or PNGaseA can be used to induced dissociation (CID) experiments in ESI-MS mostly de- specifically release the N-glycans form peptides. Thereby the liver fragments of the glycan backbone. Thus, more sophisti- asparagine residue undergoes a deamidation leading in an cated fragmentation techniques such as MS , SORI-CID, aspartic acid residue with a mass increase of 0.9848 Da IRMPD, ECD or ETD have been employed for a simultaneous (22–24). sequencing of peptides and glycans (30, 55). Unfortunately, Selective methods allowing a zoom onto a particular glyco- these techniques result in complex fragmentation spectra site of the protein are highly desirable. Unfortunately, state- requiring specific tools and databases for interpretation. of-the-art methods are not straightforward and a great deal of MALDI-TOF/TOF-MS because of its elevated collision energy, effort is required to perform a detailed analysis of a complex has also been reported to provide also significant fragmenta- protein sample. tion of the peptide backbone in parallel to the glycan (55–58). Site-specific analysis of single proteins has already been The glycopeptide is then usually identified by the appearance carried out some decades ago, by fractionation of glycopep- of three characteristic fragment ions: The first peak derives tides from proteolytic digested proteins and following analysis from a loss of the glycan (peptide fragment), the second peak by 1D and 2D NMR spectroscopy as well as later by fast atom derives from a cross-ring fragmentation of the inner core bombardment-MS (25–29). Nowadays, mass spectrometric GlcNAc (peptide  84Da), and the third peak results from the analysis of glycopeptides is carried out in several ways, for fragmentation of the two core GlcNAc residues (peptide example, by online electrospray ionization-MS (ESI) or offline 204Da) (30, 36, 58). Identification of a glycopeptide can be matrix-assisted laser desorption/ionisation mass spectrome- performed in ESI-MS and MALDI-MS by various different try-MS (MALDI) as well as by means of a combination of diagnostic fragment ions, very much depending on the frag- different techniques (30–36). “Bottom-up” approaches are mentation technique and the mass analyzer employed (30). the most promising strategies, where trypsin or a combination However, it has to be considered, that low parent ion inten- of proteases can be used to generate homogeneous medium- sity, large glycopeptides as well as multiple glycosylated/ sized peptides/glycopeptides, which provide sufficient infor- modified species might not always deliver sufficient fragment mation to assign the glycan to a specific site in the protein (9, ions to clearly identify the glycosylation site and the peptide. 37–43). In contrast, digestion using Pronase results in a “small Complex samples require in theory also the fragmentation of peptide footprint,” which might not always be sufficient for an hundreds of peaks in order to confidently assign potential unambiguous identification (4, 44). Glycosylation is usually glycopeptide peaks. Furthermore there is always the risk that heterogeneous and a single peptide peak splits into multiple the rather large and less intense glycopeptide peaks are over- signals, accompanied by reduced signal intensity and altered looked and not selected for automatic fragmentation. For this, retention on separation systems when compared with its un- LC connected offline to MS (e.g. LC-MALDI-MS) has some glycosylated counterpart (35). Furthermore, glycopeptide sig- advantages: the MS analysis is time independent from the LC nals can be suppressed in the presence of peptides as a separation and individual fractions can be reanalyzed for a result of competitive ionization (45–47). Therefore, reduction more specific investigation at any time (34, 36, 59). of the sample complexity by a selective enrichment of glyco- On the other hand, an additional enzymatic de-glycosyla- peptides is often beneficial. This can be realized by means of tion step is often included in the sample preparation. For this, chemical capturing techniques (48) or by a solid phase ex- the proteolytic digest is split into two parts, in whichby one traction prior, or after a proteolytic digestion (34, 46, 49). In the part is deglycosylated using PNGaseF and the other one is case in which single proteins or less complex protein mixtures analyzed untreated (32, 34, 35, 60). The appearance of new are investigated, an enrichment can be compensated by a peaks in the deglycosylated sample indicates then for the chromatographic separation as it is usually the case in LC- presence of glycopeptides. The deglycosylated peptides are ESI-MS (35). In MALDI-MS, suppression of glycopeptide sig- then also sequenced efficiently by a “standard CID” MS/MS nals is found to be a major issue and sample enrichment experiment. However, this combined approach presents some challenges, because pairs of peaks from different chro- matographic separation runs have to be correlated. The abbreviations used are: ESI, electrospray ionization; MALDI, Here we demonstrate a systematic and straightforward ap- matrix assisted laser desorption ionization; FTMS, Fourier transform proach for the site-specific identification and characterization MS; RP-ESI-MS, reverse phase-ESI-MS; CID, collision-induced dis- sociation; TOF, time-of-flight. of protein N-glycosylation using human serum IgM. The ap- 1646 Molecular & Cellular Proteomics 14.6 Glycoprotein Analysis by Microarray MALDI-MS proach is based on a recently developed nano-LC-microar- Microarray plates were cleaned in acetone using ultrasonic agita- tion, then rinsed with ethanol, then with water and finally dried and ray-MALDI-MS platform that uses droplet microfluidics to stored under nitrogen until use. store an analytical nano-LC run on a microarray chip (at 1 Proteolytic Digest of IgM—Human serum IgM (Sigma, Buchs, Swit- fraction per second) and which was recently also applied for zerland) was digested in solution as described in Grass et al. (67). 25 the application of nanoliter phosphatase digests for the iden- g of IgM was dissolved in 100 mM ammonium bicarbonate, reduced using dithiotreitol, and subsequently carbamido-methylated using tification of protein phosphorylation (59, 61). Here, droplet iodoacetamide. The protein was then purified by a precipitation step microfluidics furthermore allows a PNGaseF on-chip reaction using 80% ice cold acetone and a subsequent five-minute centrifu- in nanoliter volumes to selectively remove the glycan portion gation using a bench top centrifuge at maximum speed. The precip- from the peptide on each second spot. This generates two itated protein was then resuspended in 25 mM ammonium bicarbon- time-correlated traces: one PNGaseF-treated trace in which ate and digested overnight using trypsin (Sigma, Cat. No. T6567) and GluC (Sigma, Cat. No. P6181) at 37 °C. Sequence as well as potential glycans and peptides are separated and a second untreated N-glycosylation sites for human Ig mu chain and J-chain were ob- trace containing the intact glycopeptides exactly as they were tained from Kehry et al. (68) as well as from the UniprotKB database fractionated (Fig. 1). To minimize competitive ionization and to (IGHM_HUMAN (P01871), IGJ_HUMAN (P01591)). Deglyco- allow digests within a chromatographic peak, one-second sylated peptides were investigated manually, by considering up to intervals are fractionated and stored in a micro-spot. Identi- two missed cleavages and methionine oxidation. Nano-liquid Chromatography (nano-LC) and High Frequency Frac- fying or mining for distinct glycosites is then carried out by tionation—For nano-liquid flow chromatography, we used an Eksi- searching for the predicted deglycosylated peptides in the gent/Ekspert (Dublin/AC, USA) nano-LC400 and a reversed-phase treated trace; the intact glycopeptides are necessarily present separation column (0,075  150 mm, Eksigent RPC18-CL-120). A in the adjacent spots. Furthermore, the deglycosylated pep- flow rate of 300 nL/min was maintained throughout all experiments. Solvent A consisted of 0.1% unbuffered formic acid and solvent B of tides can be sequenced in CID fragmentation experiments 95% acetonitrile in H O. An aliquot of 5 l sample (prepared as and analyzed by a common shot-gun proteomics pipeline described above) containing 0.75 g of IgM digest was directly in- (31). Although the pioneering work on the analysis of IgM jected and analyzed by the following gradient: 6–26% B from 0 to 15 glycosyl- min, 26–67% B from 15 to 20 min and a washing step at 67% B from ation was already performed in 1979 (62, 63), there are yet no 20 to 30 min. Fractions were collected at a rate of 1 Hz (circa 5 nL) using a droplet-based spotting device as described in Ku¨ ster et al. detailed data available presenting a complete picture of the (59). After fractionating the nano-LC outflow in a serpentine-like pat- IgM site-specific N-glycosylation profiles of all sites including tern on the microarray chip, the chip was placed in a vacuum to dry the one from the J chain. Previous analysis are based on the for 20 min. After this, the slide was put under oil (perfluorodecalin, released glycan pool, carried out by Arnold et al. in 2005 (64) Fluka, Buchs, Switzerland) and enzyme solution was spotted to dis- and by Loos et al. on glycopeptides, (13) who demonstrated tinct spots as described below. Human serum IgM, LC solvents and 2,5-dihydroxy benzoic acid (DHB) MALDI matrix were obtained from the recombinant production of a hetero-multimeric IgM with a Sigma Aldrich (Buchs, Switzerland). The best signal for glycopeptides human-like glycosylation in plants. in positive mode was obtained by using a 10 mg/ml DHB matrix Here, starting with a trypsin/GluC in-solution digestion of solution in 50:50 acetonitrile/water containing 0.75% phosphoric human polyclonal IgM, we performed an in-depth analysis of acid. all predicted glycosylation sites with a single nano-LC run Reference Measurements Using nLC-ESI-MS—For LC-ESI-MS ref- erence experiments, the same nano-LC from the droplet-spotting using MALDI-MS without prior glycopeptide enrichment. We device was connected to a SYNAPT G2 mass spectrometer (Waters, observed oligomannosidic glycans (Asn 171, Asn 402 and Asn Manchester, UK) using a nano-flow interface with TaperTip emitters 563) on three sites, where the major glycoforms on site Asn (50  5 m, New Objective). The analysis was performed using the 171 are complex type structures (95%). Glycosite Asn 563 is same gradient as for the nanoLC-MALDI-MS experiment. Acquisition the only site, which carries solely oligomannosidic structures. was performed in “sensitivity mode” at a scan rate of 1 Hz over the mass range of m/z  500 to 2000. Glycopeptides were enriched using Glycosites Asn 402, Asn 332 and Asn 171 show small a ZIC-HILC solid phase extraction cartridge (SeQuant, Sweden), amounts of hybrid-type glycans. Asn 395 and the J chain only following the protocol from Parker et al. (49). Briefly, samples were present complex-type glycans with one or two sialic acid diluted in 80% acetonitrile containing 0.1% trifluoroacetic acid, residues and a bisecting GlcNAc. Unglycosylated portions loaded to a ZIC-HILIC SPE column (SeQuant, Sweden, 1 ml bed size), washed with 500 l 80% acetonitrile and eluted with 350 lofH O, were not noticed in the present study. 2 and were finally speed vac dried. An aliquot was additionally degly- cosylated using PNGaseF. In consecutive LC runs, the deglycosy- EXPERIMENTAL PROCEDURES lated sample, the control sample and a mixture of both was analyzed. Production of Microarray Chips—Microarrays for Mass Spectrom- For each run, a glycopeptide extract starting from 2.5 g IgM etry (MAMS) were fabricated as described in Urban et al., and Ku¨ ster digestion was injected to the LC system. Identification of the glyco- et al. (65, 66) In brief, a coated ITO-glass slide (12–18 Ohm/m , Sigma peptides was realized by searching for the expected glycopeptide Aldrich) was structured using a laser ablation system to generate a pattern, eluting slightly before the deglycosylated peptide counterpart checkerboard-like array of 2800 hydrophilic sample deposition areas as described in Pabst et al. 2012. (35) Enrichment of the glycopeptide of 300 m diameter and 35 m depth each (720 m center-to- fractions was found to be beneficial for an unambiguous assignment center distance within one row). of the peaks. Molecular & Cellular Proteomics 14.6 1647 Glycoprotein Analysis by Microarray MALDI-MS TABLE I N-glycosylation sites from IGHM_human (P01871) and IGJ_human (P01591). The glycopeptides from the glycosylation site Asn 171 was predominantly detected with one missed trypsin cleavage and glycopeptides from Asn 563 / Asn 332 were found by a majority oxidized, presumably on methionine. In-silico protein digestion and theoretical mass calculation was performed using PeptideMass tool on ExPASy (http://web.expasy.org/peptide_mass/). The observed masses from the initial screening were obtained on a low resolution MALDI-TOF-MS and identification was based on appearance in the treated spots. Mass errors for identified glycopeptides are shown in Table II Peptide Calculated Observed Asn (mu chain) (Trypsin/GluC) MC [MH] MSO (Delta Da) Glycoform 171 YKNNSDISSTR 1 1285.6 x 0.23 Complex (hybrid, oligomannosidic) 171 NNSDISSTR 0 994.44 x x Complex (hybrid, oligomannosidic) 332 GLTFQQNASSMCVPDQDTAIR 0 2340.05 Detected 0.39 Complex (hybrid) 395 THTNISE 0 802.35 x 0.12 Complex 402 SHPNATFSAVGE 0 1217.54 x 0.2 Oligomannosidic (hybrid) 563 STGKPTLYNVSLVMSDTAGTCY 0 2366.08 Detected 0.41 Oligomannosidic Asn (J chain) Peptide (Trypsin/GluC) MC [MH] MSO (Delta Da) Glycoform 71 ENISDPTSPLR 0 1229.6 x 0.21 Complex On-chip PNGaseF Digest—On-chip PNGaseF digest was carried Glycopeptide Mining and Analysis—The location of the deglyco- out with the assistance of droplet microfluidics directly on the mi- sylated peptides on the microarray slide was first identified by extract- croarray chip as described in Ku¨ ster et al. 2014 (59). The under-oil ing the ion traces of the masses for the deglycosylated peptides from reaction employs a perfluorinated oil bath in which aqueous liquids an initial MALDI-TOF-MS scan. The sequence as well as potential can be spotted under oil on the chip predefined spots using a droplet N-glycosylation sites for the human Ig mu chain were obtained from spotting device as described in Ku¨ ster et al. (59). In this work, Kehry et al. (68) as well as from the UniprotKB database (IGHM_ PNGaseF (Roche Diagnostics, Mannheim, Germany) was dissolved in HUMAN (P01871) and IGJ_HUMAN (P01591)). Spectra from the ad- 100 lH O and 8 l of it was further diluted in 200 lof10mM jacent untreated spots of the identified areas were then acquired as ammonia bicarbonate solution containing 10% acetonitrile and 0.5 control, to proof absence of the deglycosylated peptides. Identified M fluorescein. An aliquot of approx. 3 nanoliter was added to each regions were then further analyzed using MALDI-FTMS. Mass lists micro-spot with the under-oil spotting technique. Carry-over of en- were exported to Microsoft Excel and further evaluated using the zyme solution to adjacent spots was found to be minimal. The slide glycoMod tool (http://web.expasy.org/glycomod/)(69) by allowing a was incubated at room temperature for 60 min and the oil was finally maximum mass deviation of 0.025 Da for mainly present [MH] decanted and residual liquid was evaporated under vacuum (p  1 ions. Larger deviations were just accepted after manual investigation mbar) for 15 min. In a final step, a 2,5-DHB matrix solution was for peaks of lower intensity, which is also indicated in Table II. (Gly- applied to each spot using the same droplet spotter. Spotting for comod structural parameters  Hexose range 0–9, HexNAc range enzyme or for matrix application required 20–30 minutes. DHB with 2–6, deoxyhexose range 0–2, NeuAc range 0–4, NeuGc no, Pentose 0.75% phosphoric acid generated homogeneous fine crystals for a no, Sulfate/Phosphate no, KDN no, HexA no, UniCarbKB entries were reproducible and sensitive detection of the glycopeptides. This was listed separately). Furthermore, results from the previous work of true for both employed MALDI-MS instruments using positive ion Arnold et al. were used if a decision between potentially possible mode (AB5800, Bruker solariX). isobaric compositions had to be made. MS Analysis Using MALDI-TOF/TOF, MALDI-FTMS, and ESI-Q/ TOF-MS—MALDI-MS analysis was carried out by analyzing the treated spots first with an AB Sciex TOF/TOF 5800 mass spectrom- RESULTS eter (AB Sciex, Darmstadt, Germany; analyzing the whole treated The potential glycosites from IgM are listed in Table I. After trace required 20 min.) and subsequently by using a MALDI-FTMS an in-solution trypsin/GluC digest of human polyclonal IgM, instrument (SolariX Bruker, Bremen, Germany) for a detailed glyco- peptide characterization. Microarrays were mounted onto an AB an aliquot of 750 ng was injected to the LC system without Sciex sample target carrier using the mask delivered with the Laser- prior glycopeptide enrichment. Separation was carried out Bio Labs™ Mass Spectrometry Imaging Starter Kit. For the Bruker within a short 15-min gradient as described in the experimen- solariX FTMS instrument, we used the MTP slide adapter II. Spot set tal procedures section. As shown in the workflow graph (Fig. templates for the AB Sciex 5800 and geometry files for the SolariX were programmed in house. For the AB Sciex 5800 MALDI-TOF-MS 1 and Fig. 2), the nano-LC separation was fractionated at a instrument, the laser energy was set to 4950 (arbitrary units), and 23 rate of 1 Hz onto the microarray substrate. As described in the sub-spectra on 25 different positions were acquired per spot. Spectra experimental procedures section, a PNGaseF digestion of were acquired over a mass range from 500–3000 Da. MALDI-FTMS every other peak was carried out under a protecting oil analysis was performed over the mass range of 1000–5000 Da and a phase, with the aim to generate two traces on the chip: one defocused “smart beam” laser (100 m diameter) was used at 60% laser energy (arbitrary units). The total ion chromatogram and ex- PNGaseF-treated trace with deglycosylated peptides and free tracted ion chromatograms obtained from the AB Sciex MALDI-MS glycans and an untreated trace containing still the intact gly- measurements were analyzed using the DataAnalysis tool within the copeptides (Fig. 1). Peptides that are not N-glycosylated are TOF/TOF series Explorer software and with Microsoft Excel. Mass not affected by this treatment and are therefore found in both spectra from the MALDI-FTMS instrument were analyzed and anno- tated with the Compass Data Analysis 4.0 software (Bruker). traces. 1648 Molecular & Cellular Proteomics 14.6 Glycoprotein Analysis by Microarray MALDI-MS FIG.1. Panels A to D describe the on chip PNGaseF digest approach: A, On chip peak fractionation of a (glyco-)peptide eluting from the nano-LC column. B, The content of each other spot is digested with 3 nanoliter of PNGaseF solution. C, MALDI matrix addition. D, MALDI-MS analysis results in two traces: (1) trace of treated spots deglycosylated peptides present and (2) a trace of untreated spots still containing the intact glycopeptides. (Total 2800 microarray spots with a diameter of 300 m). FIG.2. Workflow for the site-specific glycosylation analysis as nd FIG.3. Upper trace: TIC of the treated spots (each 2 spot on used for IgM (a-e). After fractionation of the eluent from the nano-LC the chip) generated by the lower-resolution scan using a MALDI- separation (a), a specific portion of the collected fractions (every other TOF-MS instrument. The elution areas of the deglycosylated peptides spot) was digested using PNGaseF (b). Subsequently, a first low- from individual sites are indicated with boxes, which were identified by resolution MALDI-TOF-MS scan in the lower mass range of the the appearance of the deglycosylated peptide after PNGaseF treat- PNGaseF-treated spots was performed (c). After the localization of ment. The glycopeptide for Asn 563 provided a broad tailing peak the deglycosylated peptides in the initial data set (d), a high-resolution (broad box). Lower trace (red): Example for an extracted ion chromat- MALDI-FTMS scan (at a high mass range) was performed solely on ogram (XIC) from the treated spots, as shown for the deglycosylated the selected untreated spots. peptide from glycosite Asn 332 ([MH ]  2340 Da). Data Mining and Analysis of the Glycosylation Sites from In order to facilitate a detailed analysis of the glycopeptide Human Serum IgM—By extracting ion traces for the masses clusters, we finally analyzed the untreated spots of the iden- of the potential deglycosylated peptides, we were able to tified areas with a MALDI-FTMS instrument (Bruker solariX). This identify the position of the deglycosylated peptides within the straightforward and systematic approach allowed the analysis treated trace on the microarray slide. The adjacent spots were of IgM site-specific glycosylation within one single LC run. Data further investigated to proof the absence of the same peaks. evaluation was performed using glycoMod Tool (69), by allow- Molecular & Cellular Proteomics 14.6 1649 Glycoprotein Analysis by Microarray MALDI-MS FIG.4. The two panels show the MALDI-TOF-MS spectra from two adjacent spots (n and n1 s). In the treated spot at Nr 608 ( spot n), the deglycosylated peptide of Asn 332 can be detected. This peak is absent in the consecutive spot Nr 609 ( spot n1), which is untreated. The untreated spots contain the intact glycopeptides and were further analyzed with a high resolution MALDI-FTMS instrument (Fig. 5, and supplemental Fig. S3). (All peaks are labeled with monoisotopic masses). ing a maximum mass deviation of 0.025 Da in average for the RP-ESI-MS and RP-microarray-MALDI-FTMS, revealed very individual structures. The investigated and detected glycosites similar profiles for all glycosites (supplemental information are indicated in the BPI chromatogram in Fig. 3 and were also figures 2a-f). This was rather surprising, because a recent summarized in Table I. Adjacent untreated spots are supposed inter laboratory study by Leymarie et al., revealed for the to be absent of the deglycosylated peptides, as it is shown for common approaches used in glycoproteomics rather strong Asn 332 in Fig. 4 (more detailed also in supplemental Fig. S3). deviations in their quantitative as well as qualitative results A complete list of identified glycopeptides including the pro- between different laboratories (71). posed glycan composition can be found in Table II. A repre- Mining for Glycopeptides by Means of a Pairwise Peak-list sentative MALDI-FTMS spectrum of each glycosite is shown Comparison—The method furthermore offers the option of a in Fig. 5. Taking into account that MALDI-MS is more prone to pairwise peak lists comparison between treated and un- in source fragmentation of labile sugar residues like sialic treated spots, where the appearance of a peptide in the acids, the here presented method was not primary evaluated treated spot indicates a deglycosylated peptide. There is an for exact quantification of the glycoforms, but provides more intrinsic correlation between the two different traces, that is, a fast and complete identification of a site specific glycan the presence of a deglycosylated peptide in one spot n as- profile. Nevertheless, profiles were found very similar as ob- sures the presence of the intact glycopeptide cluster in spot tained by RP-ESI-MS, presumably a result of the low post n-1 or n1. If the peptide mass is obtained from the digested source fragmentation in the FTMS instrument. peak, the glycan composition can then be identified by using Glycopeptides for Asn 563 and Asn 332 show an almost a prediction software like the GlycoMod tool (69), as well as by equal portion of methionine oxidation. The glycopeptide for fragmentation experiments performed on the intact glycopep- the glycosite Asn 171 was mainly detected with one missed tides. The peptide sequence can then be also more easily trypsin cleavage. A glycan sub-group distribution graph was obtained by sequencing the deglycosylated peptide in the evaluated by considering the most intense spectra from each treated trace, instead of gathering sequence tags from the site, which is provided in supplemental Fig. S1. Furthermore, fragmentation of the intact glycopeptide. we compared the identified profiles, which were obtained by Human Serum IgM Site-specific Glycosylation Profile— employing reversed-phase (RP) ESI-MS (35, 70). Here, the Here, we provide the first complete and detailed site-specific data-mining process was assisted by knowledge gained from mass spectrometric analysis of the glycosylation of the hu- the MALDI-MS analysis and by the present literature (13, 62, man serum IgM using a high-resolution and high mass accu- 64). Representative (deconvoluted) spectra for both methods, racy MALDI-MS instrument. Initial work published by Chap- 1650 Molecular & Cellular Proteomics 14.6 Glycoprotein Analysis by Microarray MALDI-MS Molecular & Cellular Proteomics 14.6 1651 TABLE II Identified glycopeptide peaks and corresponding glycan compositions obtained by analyzing the peak lists from the FTMS spectra with GlycoMod tool (mass deviations were averaged over acquired spectra). An absolute mass deviation below or equal 0.025 Da (in average if the structure was identified in more than one spectrum) was set as limit. (Greater deviations were accepted for some lower intense peaks whose corresponding spectra were further investigated manually) Glycopep. Glycan Peptide Deviation Deviation Nr. Asn MH (mi) Composition Type (mi) Sequence ppm (av) Da (av) calculated calc. calc calc. 1 171 3838.5280 0.8 0.003 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 2 3 2 2 171 3635.4480 2.2 0.008 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 2 3 2 3 171 3547.4320 0.0 0.000 2262.81 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 1 3 2 4 171 3401.3740 6.8 0.023 2116.76 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 3 2 5 171 3344.3530 0.3 0.001 2059.74 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 1 3 2 6 171 3198.2950 1.3 0.004 1913.68 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 3 2 7 171 3256.3370 7.7 0.025 1971.72 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 3 2 8 171 3053.2580 4.9 0.015 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 3 2 9 171 2907.137 20.0 0.025 1913.68 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 3 2 10 171 3303.326 5.8 0.019 2018.71 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 3 1 1 1 3 2 11 171 3141.2740 2.2 0.007 1856.66 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 2 1 1 1 3 2 12 171 2979.2210 0.3 0.001 1694.60 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Hybrid/complex 1283.61 YKNNSDISSTR (1 missed cleavage) 1 1 1 1 3 2 13 171 3012.2310 3.3 0.010 1727.61 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 3 1 1 3 2 14 171 2850.1780 9.5 0.025 1565.56 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 2 1 1 3 2 15 171 2688.1250 1.1 0.003 1403.51 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Hybrid/complex 1283.61 YKNNSDISSTR (1 missed cleavage) 1 1 1 3 2 16 171 2501.0410 1.6 0.004 1216.42 (Hex)  (Man) (GlcNAc) Oligomannosidic 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 2 17 171 2663.0940 4.1 0.011 1378.48 (Hex)  (Man) (GlcNAc) Oligomannosidic 1283.61 YKNNSDISSTR (1 missed cleavage) 3 3 2 18 171 3547.3690 19.7 0.025 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 3 1 2 3 2 19 171 3344.2890 19.7 0.025 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 2 3 2 20 171 3256.3370 10.0 0.025 2262.81 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 3 1 1 3 2 21 171 3110.216 10.1 0.025 2262.81 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 3 1 1 3 2 22 171 3053.195 9.8 0.025 2059.74 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 1 3 2 23 171 2907.137 1.4 0.004 1913.68 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 3 2 24 171 2762.1 2.0 0.006 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 3 2 25 171 2616.042 5.0 0.013 1622.58 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 3 2 26 171 2371.936 6.3 0.015 1378.48 (Hex)  (Man) (GlcNAc) Oligomannosidic 992.45 NNSDISSTR 3 3 2 27 171 2209.883 9.5 0.021 1216.42 (Hex)  (Man) (GlcNAc) Oligomannosidic 992.45 NNSDISSTR 2 3 2 28 395 3355.283 1.4 0.005 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 1 2 3 2 29 395 3152.203 0.6 0.007 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 2 1 2 3 2 30 395 3064.188 1.3 0.005 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 1 1 3 2 31 395 3209.226 0.5 0.003 2407.852 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 2 3 2 32 395 2699.056 0.9 0.004 1897.682 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 1 2 1 1 3 2 33 395 2861.109 1.7 0.006 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 2 1 1 3 2 34 395 2918.13 2.5 0.009 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 1 3 2 35 395 2902.136 0.9 0.008 2100.762 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 1 3 1 1 3 2 36 402 2270.929 1.8 0.004 1054.37 (Hex) (HexNAc) Paucimannosidic 1215.55 SHPNATFSAVGE 4 2 37 402 2432.982 0.4 0.001 1216.423 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 2 3 2 38 402 2595.035 0.0 0.000 1378.476 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 3 3 2 39 402 2757.087 0.7 0.002 1540.528 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 4 3 2 40 402 2919.14 0.7 0.002 1702.581 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 5 3 2 41 402 3081.193 2.6 0.008 1864.634 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 6 3 2 42 402 2311.956 0.9 0.002 1095.397 (HexNAc)  (Man) (GlcNAc) Hybrid/complex 1215.55 SHPNATFSAVGE 1 3 2 43 402 2474.008 4.9 0.012 1257.449 (Hex) (HexNAc)  (Man) (GlcNAc) Hybrid/complex 1215.55 SHPNATFSAVGE 1 1 3 2 44 402 2636.061 2.3 0.006 1419.502 (Hex) (HexNAc)  (Man) (GlcNAc) Hybrid/complex 1215.55 SHPNATFSAVGE 2 1 3 2 Glycoprotein Analysis by Microarray MALDI-MS 1652 Molecular & Cellular Proteomics 14.6 TABLE II—continued Glycopep. Glycan Peptide Nr. Asn MH Deviation Deviation Composition Type Sequence (mi) (mi) calculated 45 402 2798.114 3.2 0.009 1581.555 (Hex) (HexNAc)  (Man) (GlcNAc) Hybrid 1215.55 SHPNATFSAVGE 3 1 3 2 46 332 4908.98 75 0.025 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 2 3 2 47 332 4705.9 8.0 0.025 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 2 3 2 48 332 4617.884 0.9 0.004 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 1 3 2 49 332 4471.826 0.0 0.000 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 3 2 50 332 4455.832 4.0 0.018 2100.762 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 1 3 1 1 3 2 51 332 4414.805 4.3 0.019 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 1 3 2 52 332 4326.789 3.7 0.016 1971.719 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 3 2 53 332 4268.747 3.7 0.016 1913.677 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 3 2 54 332 4123.71 2.2 0.009 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 3 2 55 332 4430.8 4.3 0.019 2075.73 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Hybrid 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 3 2 1 3 2 56 332 4164.736 3.8 0.016 1809.666 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 1 3 1 3 2 57 332 4892.982 50 0.025 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 2 3 2 58 332 4689.905 1.5 0.007 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 2 3 2 59 332 4601.889 3.5 0.016 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 1 3 2 60 332 4455.831 4.0 0.018 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 3 2 61 332 4439.837 4.1 0.018 2100.762 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 1 3 1 1 3 2 62 332 4398.81 4.1 0.018 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 1 3 2 63 332 4310.794 3.0 0.013 1971.719 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 3 2 64 332 4252.752 6.6 0.025 1913.677 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 3 2 65 332 4107.715 6.6 0.002 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 3 2 66 332 4414.805 4.3 0.019 2075.73 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Hybrid 2338.07 GLTFQQNASSMCVPDQDTAIR 3 2 1 3 2 67 332 4148.741 1.9 0.008 1809.666 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 1 3 1 3 2 68 563 3435.47 5.5 0.019 1054.37 (Hex) (HexNAc) Paucimannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 4 2 69 563 3597.523 3.3 0.012 1216.423 (Hex)  (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 2 3 2 70 563 3759.576 2.9 0.011 1378.476 (Hex) (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 3 3 2 71 563 3921.628 2.3 0.009 1540.528 (Hex)  (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 4 3 2 72 563 4083.681 2.2 0.009 1702.581 (Hex) (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 5 3 2 73 563 4245.734 5.9 0.025 1864.634 (Hex)  (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 6 3 2 74 71 (J-chain) 3142.294 0.8 0.003 1913.677 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 1 3 2 75 71 (J-chain) 3288.352 0.3 0.001 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 1 1 3 2 76 71 (J-chain) 3345.373 2.4 0.008 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 1 3 2 77 71 (J-chain) 2851.199 2.5 0.007 1622.582 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 3 2 78 71 (J-chain) 3491.431 0.7 0.002 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 1 1 3 2 79 71 (J-chain) 2997.257 1.6 0.005 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 1 3 2 80 71 (J-chain) 3054.278 0.5 0.002 1825.661 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 3 2 81 71 (J-chain) 3200.34 2.2 0.007 1826.661 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 1 3 2 Glycoprotein Analysis by Microarray MALDI-MS FIG.5. MALDI-FTMS spectra representative for each glycosite of the IgM mu chain as well as of the J-chain. Peaks were labeled with monoisotopic masses [MH ] . A direct comparison with the ESI-MS reference measurements is presented in the electronic supplemental information (supplemental Fig. S2A–S2F). man and Kornfeld in 1979 was based on the preparative Man6 on Asn 402 and Man6 and Man8 on Asn 563. A recent fractionation of glycopeptides, amino acid analysis by liquid study by Loos et al. using LC-ESI-MS assigned major glyco- chromatography, and monosaccharide analysis by gas-liquid forms of the mu chain, which also confirmed the pioneering chromatography (62, 63). The authors describe Man5 and work of Chapman et al. (62, 63). The major glycoforms found Molecular & Cellular Proteomics 14.6 1653 Glycoprotein Analysis by Microarray MALDI-MS Acknowledgments—We thank Alfredo J. Iba´n˜ ez, Robert Steinhoff, here by the microarray-MALDI-FTMS approach were in and Stephan Fagerer for helpful advices with our MALDI-MS mea- agreement with the results obtained by the previous studies. surements. Furthermore we thank Konstantins Jefimovs and Rolf In addition we could provide a comprehensive and a detailed Bro¨ nnimann from EMPA Du¨ bendorf, Jens Boertz from Sigma-Aldrich, analysis, assigning a total of 81 glycopeptide peaks to the IgM Rolf Ha¨ flinger and Dr. Xiangyang Zhang from the MS-Service for their assistance with the MALDI-FTMS system. mu chain and to the J chain with highest mass accuracy (Table II). Thereby we detected some minor portions of oligo- * This work was supported by the European Research Council (ERC mannosidic glycans as well as hybrid-type glycans, also on Starting Grant nLIPIDS, Grant No. 203428) and the Swiss KTI (Kom- Asn 171. Glycosite Asn 563 carried exclusively oligomanno- mission fu¨ r Technologie und Innovation; Grant No. 13123.1 PFNM- NM). Simon K. Ku¨ ster acknowledges financial support from the schol- sidic structures, but site Asn 402 exhibited 10% hybrid-type arship fund of the Swiss Chemical Industry (SSCI). glycans. Glycosites Asn 395 and the J chain carried only □ S This article contains supplemental Figs. S1 to S3. complex type glycans with one or two sialic acid residues, ¶ To whom correspondence should be addressed: Department of bisecting GlcNAc and one fucose residue. Arnold et al. (64), Chemistry and Applied Bioscience, ETH Zurich, HCI E 329, Zurich demonstrated by exoglycosidase digests that the fucose res- CH-8093 Switzerland. Tel.: 4144-6324376; E-mail: zenobi@org. chem.ethz.ch. idue present is a core fucosylation. 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A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)* [S]

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American Society for Biochemistry and Molecular Biology
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Copyright © 2015 Elsevier Inc.
ISSN
1535-9476
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1535-9484
DOI
10.1074/mcp.o114.046748
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Abstract

Technological Innovation and Resources © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N- glycosylation Analysis, as Demonstrated for □ S Human Serum Immunoglobulin M (IgM)* Martin Pabst‡, Simon Karl Ku¨ ster‡, Fabian Wahl§, Jasmin Krismer‡, Petra S. Dittrich‡, and Renato Zenobi‡¶ We demonstrate a new approach for the site-specific plished on the adjacent, untreated spots with high mass identification and characterization of protein N-glyco- resolution and high mass accuracy using a matrix as- sylation. It is based on a nano-liquid chromatography mi- sisted laser desorption ionization-Fourier transform-MS. croarray-matrix assisted laser desorption/ionization-MS We present the first detailed and comprehensive mass platform, which employs droplet microfluidics for on- spectrometric analysis on the glycopeptide level for hu- plate nanoliter reactions. A chromatographic separation man polyclonal IgM with high mass accuracy. Besides of a proteolytic digest is deposited at a high frequency on complex type glycans on Asn 395, 332, 171, and on the J the microarray. In this way, a short separation run is chain, we observed oligomannosidic glycans on Asn 563, archived into thousands of nanoliter reaction cavities, and Asn 402 and minor amounts of oligomannosidic glycans chromatographic peaks are spread over multiple array on the glycosite Asn 171. Furthermore, hybrid type gly- spots. After fractionation, each other spot is treated with cans were found on Asn 402, Asn 171 and in traces Asn PNGaseF to generate two correlated traces within one 332. Molecular & Cellular Proteomics 14: 10.1074/mcp. run, one with treated spots where glycans are enzymati- O114.046748, 1645–1656, 2015. cally released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is per- formed by searching for the predicted deglycosylated Glycosylation is one of the most common post-translational peptides in the treated trace. An identified peptide then modifications and it is known to play an important role in leads directly to the position of the “intact” glycopeptide protein structure, protein function, cell signaling and recogni- clusters, which are located in the adjacent spots. Further- tion (1–4). Almost all proteins involved in the immune re- more, the deglycosylated peptide can be sequenced effi- sponse are glycoproteins and their attached glycans are ciently in a simple collision-induced dissociation-MS ex- thought to be components of the immune system effector periment. We applied the microarray approach to a mechanism (5). Whereas the glycosylation of IgG, IgE and IgA detailed site-specific glycosylation analysis of human se- is well studied, there are no detailed mass spectrometric data rum IgM. By scanning the treated spots with low-resolu- available describing the site-specific glycosylation profiles of tion matrix assisted laser desorption/ionization-time-of- human serum IgM (6–9). Human IgG has one conserved N- flight-MS, we observed all five deglycosylated peptides, glycosylation site on each heavy chain CH2 domain at Asn including the one originating from the secretory chain. A detailed glycopeptide characterization was then accom- 297, and 15–20% of normal polyclonal IgG bears additional Fab (fragment antigen binding) glycosylation (6, 10, 11). Other antibody classes such as IgM or IgA show a higher complexity From the ‡Department of Chemistry and Applied Biosciences, with respect to the number of glycosylation sites and variety ETH Zu¨ rich, Vladimir-Prelog-Weg 3, 8093 Zu¨ rich, Switzerland; of glycoforms (6, 9). Only recently, also monoclonal IgM an- §Sigma-Aldrich Chemie GmbH, Industriestrasse 25, 9471 Buchs tibodies came into the focus of pharmaceutical industry, be- (SG), Switzerland cause they show great potential for the treatment of diseases Received November 26, 2014, and in revised form, March 20, 2015 Published, MCP Papers in Press March 23, 2015, DOI 10.1074/ (12–14). mcp.O114.046748 Determination of the site-specific N-glycosylation pattern of Author contributions: M.P., S.K.K., F.W., P.S.D., and R.Z. designed complex proteins is a challenging task and therefore glyco- research; M.P., S.K.K., and J.K. performed research; M.P., S.K.K., sylation is often solely analyzed on released glycan pools F.W., P.S.D., and R.Z. contributed new reagents or analytic tools; (15–17). Whereas the analysis of released pools is necessary M.P. and J.K. analyzed data; M.P., J.K., P.S.D., and R.Z. wrote the paper. when linkage and positional isomers have to be investigated Molecular & Cellular Proteomics 14.6 1645 This is an Open Access article under the CC BY license. Glycoprotein Analysis by Microarray MALDI-MS (18, 19), the information obtained is here limited to the car- strategies are often required in order to obtain a reproducible bohydrate portion and does not allow any conclusion on a and sensitive signal (34, 50, 51). protein site specific heterogeneity. N-linked glycans in partic- The identification of a potential glycopeptide cluster is fur- ular are attached by an amide bond to an asparagine residue ther performed by tandem mass spectrometry, either by scan- of the protein, where this particular asparagine is necessarily ning for marker ions (52, 53) or with the assistance of peak part of a consensus sequence NX(S/T) or more rarely part of (-pattern) interpretation tools searching for a characteristic a NXC motif (where X can be any amino acid except proline) glycoprofile mass pattern (7, 54). (Lower energy) collision- (20, 21). Enzymes like PNGaseF or PNGaseA can be used to induced dissociation (CID) experiments in ESI-MS mostly de- specifically release the N-glycans form peptides. Thereby the liver fragments of the glycan backbone. Thus, more sophisti- asparagine residue undergoes a deamidation leading in an cated fragmentation techniques such as MS , SORI-CID, aspartic acid residue with a mass increase of 0.9848 Da IRMPD, ECD or ETD have been employed for a simultaneous (22–24). sequencing of peptides and glycans (30, 55). Unfortunately, Selective methods allowing a zoom onto a particular glyco- these techniques result in complex fragmentation spectra site of the protein are highly desirable. Unfortunately, state- requiring specific tools and databases for interpretation. of-the-art methods are not straightforward and a great deal of MALDI-TOF/TOF-MS because of its elevated collision energy, effort is required to perform a detailed analysis of a complex has also been reported to provide also significant fragmenta- protein sample. tion of the peptide backbone in parallel to the glycan (55–58). Site-specific analysis of single proteins has already been The glycopeptide is then usually identified by the appearance carried out some decades ago, by fractionation of glycopep- of three characteristic fragment ions: The first peak derives tides from proteolytic digested proteins and following analysis from a loss of the glycan (peptide fragment), the second peak by 1D and 2D NMR spectroscopy as well as later by fast atom derives from a cross-ring fragmentation of the inner core bombardment-MS (25–29). Nowadays, mass spectrometric GlcNAc (peptide  84Da), and the third peak results from the analysis of glycopeptides is carried out in several ways, for fragmentation of the two core GlcNAc residues (peptide example, by online electrospray ionization-MS (ESI) or offline 204Da) (30, 36, 58). Identification of a glycopeptide can be matrix-assisted laser desorption/ionisation mass spectrome- performed in ESI-MS and MALDI-MS by various different try-MS (MALDI) as well as by means of a combination of diagnostic fragment ions, very much depending on the frag- different techniques (30–36). “Bottom-up” approaches are mentation technique and the mass analyzer employed (30). the most promising strategies, where trypsin or a combination However, it has to be considered, that low parent ion inten- of proteases can be used to generate homogeneous medium- sity, large glycopeptides as well as multiple glycosylated/ sized peptides/glycopeptides, which provide sufficient infor- modified species might not always deliver sufficient fragment mation to assign the glycan to a specific site in the protein (9, ions to clearly identify the glycosylation site and the peptide. 37–43). In contrast, digestion using Pronase results in a “small Complex samples require in theory also the fragmentation of peptide footprint,” which might not always be sufficient for an hundreds of peaks in order to confidently assign potential unambiguous identification (4, 44). Glycosylation is usually glycopeptide peaks. Furthermore there is always the risk that heterogeneous and a single peptide peak splits into multiple the rather large and less intense glycopeptide peaks are over- signals, accompanied by reduced signal intensity and altered looked and not selected for automatic fragmentation. For this, retention on separation systems when compared with its un- LC connected offline to MS (e.g. LC-MALDI-MS) has some glycosylated counterpart (35). Furthermore, glycopeptide sig- advantages: the MS analysis is time independent from the LC nals can be suppressed in the presence of peptides as a separation and individual fractions can be reanalyzed for a result of competitive ionization (45–47). Therefore, reduction more specific investigation at any time (34, 36, 59). of the sample complexity by a selective enrichment of glyco- On the other hand, an additional enzymatic de-glycosyla- peptides is often beneficial. This can be realized by means of tion step is often included in the sample preparation. For this, chemical capturing techniques (48) or by a solid phase ex- the proteolytic digest is split into two parts, in whichby one traction prior, or after a proteolytic digestion (34, 46, 49). In the part is deglycosylated using PNGaseF and the other one is case in which single proteins or less complex protein mixtures analyzed untreated (32, 34, 35, 60). The appearance of new are investigated, an enrichment can be compensated by a peaks in the deglycosylated sample indicates then for the chromatographic separation as it is usually the case in LC- presence of glycopeptides. The deglycosylated peptides are ESI-MS (35). In MALDI-MS, suppression of glycopeptide sig- then also sequenced efficiently by a “standard CID” MS/MS nals is found to be a major issue and sample enrichment experiment. However, this combined approach presents some challenges, because pairs of peaks from different chro- matographic separation runs have to be correlated. The abbreviations used are: ESI, electrospray ionization; MALDI, Here we demonstrate a systematic and straightforward ap- matrix assisted laser desorption ionization; FTMS, Fourier transform proach for the site-specific identification and characterization MS; RP-ESI-MS, reverse phase-ESI-MS; CID, collision-induced dis- sociation; TOF, time-of-flight. of protein N-glycosylation using human serum IgM. The ap- 1646 Molecular & Cellular Proteomics 14.6 Glycoprotein Analysis by Microarray MALDI-MS proach is based on a recently developed nano-LC-microar- Microarray plates were cleaned in acetone using ultrasonic agita- tion, then rinsed with ethanol, then with water and finally dried and ray-MALDI-MS platform that uses droplet microfluidics to stored under nitrogen until use. store an analytical nano-LC run on a microarray chip (at 1 Proteolytic Digest of IgM—Human serum IgM (Sigma, Buchs, Swit- fraction per second) and which was recently also applied for zerland) was digested in solution as described in Grass et al. (67). 25 the application of nanoliter phosphatase digests for the iden- g of IgM was dissolved in 100 mM ammonium bicarbonate, reduced using dithiotreitol, and subsequently carbamido-methylated using tification of protein phosphorylation (59, 61). Here, droplet iodoacetamide. The protein was then purified by a precipitation step microfluidics furthermore allows a PNGaseF on-chip reaction using 80% ice cold acetone and a subsequent five-minute centrifu- in nanoliter volumes to selectively remove the glycan portion gation using a bench top centrifuge at maximum speed. The precip- from the peptide on each second spot. This generates two itated protein was then resuspended in 25 mM ammonium bicarbon- time-correlated traces: one PNGaseF-treated trace in which ate and digested overnight using trypsin (Sigma, Cat. No. T6567) and GluC (Sigma, Cat. No. P6181) at 37 °C. Sequence as well as potential glycans and peptides are separated and a second untreated N-glycosylation sites for human Ig mu chain and J-chain were ob- trace containing the intact glycopeptides exactly as they were tained from Kehry et al. (68) as well as from the UniprotKB database fractionated (Fig. 1). To minimize competitive ionization and to (IGHM_HUMAN (P01871), IGJ_HUMAN (P01591)). Deglyco- allow digests within a chromatographic peak, one-second sylated peptides were investigated manually, by considering up to intervals are fractionated and stored in a micro-spot. Identi- two missed cleavages and methionine oxidation. Nano-liquid Chromatography (nano-LC) and High Frequency Frac- fying or mining for distinct glycosites is then carried out by tionation—For nano-liquid flow chromatography, we used an Eksi- searching for the predicted deglycosylated peptides in the gent/Ekspert (Dublin/AC, USA) nano-LC400 and a reversed-phase treated trace; the intact glycopeptides are necessarily present separation column (0,075  150 mm, Eksigent RPC18-CL-120). A in the adjacent spots. Furthermore, the deglycosylated pep- flow rate of 300 nL/min was maintained throughout all experiments. Solvent A consisted of 0.1% unbuffered formic acid and solvent B of tides can be sequenced in CID fragmentation experiments 95% acetonitrile in H O. An aliquot of 5 l sample (prepared as and analyzed by a common shot-gun proteomics pipeline described above) containing 0.75 g of IgM digest was directly in- (31). Although the pioneering work on the analysis of IgM jected and analyzed by the following gradient: 6–26% B from 0 to 15 glycosyl- min, 26–67% B from 15 to 20 min and a washing step at 67% B from ation was already performed in 1979 (62, 63), there are yet no 20 to 30 min. Fractions were collected at a rate of 1 Hz (circa 5 nL) using a droplet-based spotting device as described in Ku¨ ster et al. detailed data available presenting a complete picture of the (59). After fractionating the nano-LC outflow in a serpentine-like pat- IgM site-specific N-glycosylation profiles of all sites including tern on the microarray chip, the chip was placed in a vacuum to dry the one from the J chain. Previous analysis are based on the for 20 min. After this, the slide was put under oil (perfluorodecalin, released glycan pool, carried out by Arnold et al. in 2005 (64) Fluka, Buchs, Switzerland) and enzyme solution was spotted to dis- and by Loos et al. on glycopeptides, (13) who demonstrated tinct spots as described below. Human serum IgM, LC solvents and 2,5-dihydroxy benzoic acid (DHB) MALDI matrix were obtained from the recombinant production of a hetero-multimeric IgM with a Sigma Aldrich (Buchs, Switzerland). The best signal for glycopeptides human-like glycosylation in plants. in positive mode was obtained by using a 10 mg/ml DHB matrix Here, starting with a trypsin/GluC in-solution digestion of solution in 50:50 acetonitrile/water containing 0.75% phosphoric human polyclonal IgM, we performed an in-depth analysis of acid. all predicted glycosylation sites with a single nano-LC run Reference Measurements Using nLC-ESI-MS—For LC-ESI-MS ref- erence experiments, the same nano-LC from the droplet-spotting using MALDI-MS without prior glycopeptide enrichment. We device was connected to a SYNAPT G2 mass spectrometer (Waters, observed oligomannosidic glycans (Asn 171, Asn 402 and Asn Manchester, UK) using a nano-flow interface with TaperTip emitters 563) on three sites, where the major glycoforms on site Asn (50  5 m, New Objective). The analysis was performed using the 171 are complex type structures (95%). Glycosite Asn 563 is same gradient as for the nanoLC-MALDI-MS experiment. Acquisition the only site, which carries solely oligomannosidic structures. was performed in “sensitivity mode” at a scan rate of 1 Hz over the mass range of m/z  500 to 2000. Glycopeptides were enriched using Glycosites Asn 402, Asn 332 and Asn 171 show small a ZIC-HILC solid phase extraction cartridge (SeQuant, Sweden), amounts of hybrid-type glycans. Asn 395 and the J chain only following the protocol from Parker et al. (49). Briefly, samples were present complex-type glycans with one or two sialic acid diluted in 80% acetonitrile containing 0.1% trifluoroacetic acid, residues and a bisecting GlcNAc. Unglycosylated portions loaded to a ZIC-HILIC SPE column (SeQuant, Sweden, 1 ml bed size), washed with 500 l 80% acetonitrile and eluted with 350 lofH O, were not noticed in the present study. 2 and were finally speed vac dried. An aliquot was additionally degly- cosylated using PNGaseF. In consecutive LC runs, the deglycosy- EXPERIMENTAL PROCEDURES lated sample, the control sample and a mixture of both was analyzed. Production of Microarray Chips—Microarrays for Mass Spectrom- For each run, a glycopeptide extract starting from 2.5 g IgM etry (MAMS) were fabricated as described in Urban et al., and Ku¨ ster digestion was injected to the LC system. Identification of the glyco- et al. (65, 66) In brief, a coated ITO-glass slide (12–18 Ohm/m , Sigma peptides was realized by searching for the expected glycopeptide Aldrich) was structured using a laser ablation system to generate a pattern, eluting slightly before the deglycosylated peptide counterpart checkerboard-like array of 2800 hydrophilic sample deposition areas as described in Pabst et al. 2012. (35) Enrichment of the glycopeptide of 300 m diameter and 35 m depth each (720 m center-to- fractions was found to be beneficial for an unambiguous assignment center distance within one row). of the peaks. Molecular & Cellular Proteomics 14.6 1647 Glycoprotein Analysis by Microarray MALDI-MS TABLE I N-glycosylation sites from IGHM_human (P01871) and IGJ_human (P01591). The glycopeptides from the glycosylation site Asn 171 was predominantly detected with one missed trypsin cleavage and glycopeptides from Asn 563 / Asn 332 were found by a majority oxidized, presumably on methionine. In-silico protein digestion and theoretical mass calculation was performed using PeptideMass tool on ExPASy (http://web.expasy.org/peptide_mass/). The observed masses from the initial screening were obtained on a low resolution MALDI-TOF-MS and identification was based on appearance in the treated spots. Mass errors for identified glycopeptides are shown in Table II Peptide Calculated Observed Asn (mu chain) (Trypsin/GluC) MC [MH] MSO (Delta Da) Glycoform 171 YKNNSDISSTR 1 1285.6 x 0.23 Complex (hybrid, oligomannosidic) 171 NNSDISSTR 0 994.44 x x Complex (hybrid, oligomannosidic) 332 GLTFQQNASSMCVPDQDTAIR 0 2340.05 Detected 0.39 Complex (hybrid) 395 THTNISE 0 802.35 x 0.12 Complex 402 SHPNATFSAVGE 0 1217.54 x 0.2 Oligomannosidic (hybrid) 563 STGKPTLYNVSLVMSDTAGTCY 0 2366.08 Detected 0.41 Oligomannosidic Asn (J chain) Peptide (Trypsin/GluC) MC [MH] MSO (Delta Da) Glycoform 71 ENISDPTSPLR 0 1229.6 x 0.21 Complex On-chip PNGaseF Digest—On-chip PNGaseF digest was carried Glycopeptide Mining and Analysis—The location of the deglyco- out with the assistance of droplet microfluidics directly on the mi- sylated peptides on the microarray slide was first identified by extract- croarray chip as described in Ku¨ ster et al. 2014 (59). The under-oil ing the ion traces of the masses for the deglycosylated peptides from reaction employs a perfluorinated oil bath in which aqueous liquids an initial MALDI-TOF-MS scan. The sequence as well as potential can be spotted under oil on the chip predefined spots using a droplet N-glycosylation sites for the human Ig mu chain were obtained from spotting device as described in Ku¨ ster et al. (59). In this work, Kehry et al. (68) as well as from the UniprotKB database (IGHM_ PNGaseF (Roche Diagnostics, Mannheim, Germany) was dissolved in HUMAN (P01871) and IGJ_HUMAN (P01591)). Spectra from the ad- 100 lH O and 8 l of it was further diluted in 200 lof10mM jacent untreated spots of the identified areas were then acquired as ammonia bicarbonate solution containing 10% acetonitrile and 0.5 control, to proof absence of the deglycosylated peptides. Identified M fluorescein. An aliquot of approx. 3 nanoliter was added to each regions were then further analyzed using MALDI-FTMS. Mass lists micro-spot with the under-oil spotting technique. Carry-over of en- were exported to Microsoft Excel and further evaluated using the zyme solution to adjacent spots was found to be minimal. The slide glycoMod tool (http://web.expasy.org/glycomod/)(69) by allowing a was incubated at room temperature for 60 min and the oil was finally maximum mass deviation of 0.025 Da for mainly present [MH] decanted and residual liquid was evaporated under vacuum (p  1 ions. Larger deviations were just accepted after manual investigation mbar) for 15 min. In a final step, a 2,5-DHB matrix solution was for peaks of lower intensity, which is also indicated in Table II. (Gly- applied to each spot using the same droplet spotter. Spotting for comod structural parameters  Hexose range 0–9, HexNAc range enzyme or for matrix application required 20–30 minutes. DHB with 2–6, deoxyhexose range 0–2, NeuAc range 0–4, NeuGc no, Pentose 0.75% phosphoric acid generated homogeneous fine crystals for a no, Sulfate/Phosphate no, KDN no, HexA no, UniCarbKB entries were reproducible and sensitive detection of the glycopeptides. This was listed separately). Furthermore, results from the previous work of true for both employed MALDI-MS instruments using positive ion Arnold et al. were used if a decision between potentially possible mode (AB5800, Bruker solariX). isobaric compositions had to be made. MS Analysis Using MALDI-TOF/TOF, MALDI-FTMS, and ESI-Q/ TOF-MS—MALDI-MS analysis was carried out by analyzing the treated spots first with an AB Sciex TOF/TOF 5800 mass spectrom- RESULTS eter (AB Sciex, Darmstadt, Germany; analyzing the whole treated The potential glycosites from IgM are listed in Table I. After trace required 20 min.) and subsequently by using a MALDI-FTMS an in-solution trypsin/GluC digest of human polyclonal IgM, instrument (SolariX Bruker, Bremen, Germany) for a detailed glyco- peptide characterization. Microarrays were mounted onto an AB an aliquot of 750 ng was injected to the LC system without Sciex sample target carrier using the mask delivered with the Laser- prior glycopeptide enrichment. Separation was carried out Bio Labs™ Mass Spectrometry Imaging Starter Kit. For the Bruker within a short 15-min gradient as described in the experimen- solariX FTMS instrument, we used the MTP slide adapter II. Spot set tal procedures section. As shown in the workflow graph (Fig. templates for the AB Sciex 5800 and geometry files for the SolariX were programmed in house. For the AB Sciex 5800 MALDI-TOF-MS 1 and Fig. 2), the nano-LC separation was fractionated at a instrument, the laser energy was set to 4950 (arbitrary units), and 23 rate of 1 Hz onto the microarray substrate. As described in the sub-spectra on 25 different positions were acquired per spot. Spectra experimental procedures section, a PNGaseF digestion of were acquired over a mass range from 500–3000 Da. MALDI-FTMS every other peak was carried out under a protecting oil analysis was performed over the mass range of 1000–5000 Da and a phase, with the aim to generate two traces on the chip: one defocused “smart beam” laser (100 m diameter) was used at 60% laser energy (arbitrary units). The total ion chromatogram and ex- PNGaseF-treated trace with deglycosylated peptides and free tracted ion chromatograms obtained from the AB Sciex MALDI-MS glycans and an untreated trace containing still the intact gly- measurements were analyzed using the DataAnalysis tool within the copeptides (Fig. 1). Peptides that are not N-glycosylated are TOF/TOF series Explorer software and with Microsoft Excel. Mass not affected by this treatment and are therefore found in both spectra from the MALDI-FTMS instrument were analyzed and anno- tated with the Compass Data Analysis 4.0 software (Bruker). traces. 1648 Molecular & Cellular Proteomics 14.6 Glycoprotein Analysis by Microarray MALDI-MS FIG.1. Panels A to D describe the on chip PNGaseF digest approach: A, On chip peak fractionation of a (glyco-)peptide eluting from the nano-LC column. B, The content of each other spot is digested with 3 nanoliter of PNGaseF solution. C, MALDI matrix addition. D, MALDI-MS analysis results in two traces: (1) trace of treated spots deglycosylated peptides present and (2) a trace of untreated spots still containing the intact glycopeptides. (Total 2800 microarray spots with a diameter of 300 m). FIG.2. Workflow for the site-specific glycosylation analysis as nd FIG.3. Upper trace: TIC of the treated spots (each 2 spot on used for IgM (a-e). After fractionation of the eluent from the nano-LC the chip) generated by the lower-resolution scan using a MALDI- separation (a), a specific portion of the collected fractions (every other TOF-MS instrument. The elution areas of the deglycosylated peptides spot) was digested using PNGaseF (b). Subsequently, a first low- from individual sites are indicated with boxes, which were identified by resolution MALDI-TOF-MS scan in the lower mass range of the the appearance of the deglycosylated peptide after PNGaseF treat- PNGaseF-treated spots was performed (c). After the localization of ment. The glycopeptide for Asn 563 provided a broad tailing peak the deglycosylated peptides in the initial data set (d), a high-resolution (broad box). Lower trace (red): Example for an extracted ion chromat- MALDI-FTMS scan (at a high mass range) was performed solely on ogram (XIC) from the treated spots, as shown for the deglycosylated the selected untreated spots. peptide from glycosite Asn 332 ([MH ]  2340 Da). Data Mining and Analysis of the Glycosylation Sites from In order to facilitate a detailed analysis of the glycopeptide Human Serum IgM—By extracting ion traces for the masses clusters, we finally analyzed the untreated spots of the iden- of the potential deglycosylated peptides, we were able to tified areas with a MALDI-FTMS instrument (Bruker solariX). This identify the position of the deglycosylated peptides within the straightforward and systematic approach allowed the analysis treated trace on the microarray slide. The adjacent spots were of IgM site-specific glycosylation within one single LC run. Data further investigated to proof the absence of the same peaks. evaluation was performed using glycoMod Tool (69), by allow- Molecular & Cellular Proteomics 14.6 1649 Glycoprotein Analysis by Microarray MALDI-MS FIG.4. The two panels show the MALDI-TOF-MS spectra from two adjacent spots (n and n1 s). In the treated spot at Nr 608 ( spot n), the deglycosylated peptide of Asn 332 can be detected. This peak is absent in the consecutive spot Nr 609 ( spot n1), which is untreated. The untreated spots contain the intact glycopeptides and were further analyzed with a high resolution MALDI-FTMS instrument (Fig. 5, and supplemental Fig. S3). (All peaks are labeled with monoisotopic masses). ing a maximum mass deviation of 0.025 Da in average for the RP-ESI-MS and RP-microarray-MALDI-FTMS, revealed very individual structures. The investigated and detected glycosites similar profiles for all glycosites (supplemental information are indicated in the BPI chromatogram in Fig. 3 and were also figures 2a-f). This was rather surprising, because a recent summarized in Table I. Adjacent untreated spots are supposed inter laboratory study by Leymarie et al., revealed for the to be absent of the deglycosylated peptides, as it is shown for common approaches used in glycoproteomics rather strong Asn 332 in Fig. 4 (more detailed also in supplemental Fig. S3). deviations in their quantitative as well as qualitative results A complete list of identified glycopeptides including the pro- between different laboratories (71). posed glycan composition can be found in Table II. A repre- Mining for Glycopeptides by Means of a Pairwise Peak-list sentative MALDI-FTMS spectrum of each glycosite is shown Comparison—The method furthermore offers the option of a in Fig. 5. Taking into account that MALDI-MS is more prone to pairwise peak lists comparison between treated and un- in source fragmentation of labile sugar residues like sialic treated spots, where the appearance of a peptide in the acids, the here presented method was not primary evaluated treated spot indicates a deglycosylated peptide. There is an for exact quantification of the glycoforms, but provides more intrinsic correlation between the two different traces, that is, a fast and complete identification of a site specific glycan the presence of a deglycosylated peptide in one spot n as- profile. Nevertheless, profiles were found very similar as ob- sures the presence of the intact glycopeptide cluster in spot tained by RP-ESI-MS, presumably a result of the low post n-1 or n1. If the peptide mass is obtained from the digested source fragmentation in the FTMS instrument. peak, the glycan composition can then be identified by using Glycopeptides for Asn 563 and Asn 332 show an almost a prediction software like the GlycoMod tool (69), as well as by equal portion of methionine oxidation. The glycopeptide for fragmentation experiments performed on the intact glycopep- the glycosite Asn 171 was mainly detected with one missed tides. The peptide sequence can then be also more easily trypsin cleavage. A glycan sub-group distribution graph was obtained by sequencing the deglycosylated peptide in the evaluated by considering the most intense spectra from each treated trace, instead of gathering sequence tags from the site, which is provided in supplemental Fig. S1. Furthermore, fragmentation of the intact glycopeptide. we compared the identified profiles, which were obtained by Human Serum IgM Site-specific Glycosylation Profile— employing reversed-phase (RP) ESI-MS (35, 70). Here, the Here, we provide the first complete and detailed site-specific data-mining process was assisted by knowledge gained from mass spectrometric analysis of the glycosylation of the hu- the MALDI-MS analysis and by the present literature (13, 62, man serum IgM using a high-resolution and high mass accu- 64). Representative (deconvoluted) spectra for both methods, racy MALDI-MS instrument. Initial work published by Chap- 1650 Molecular & Cellular Proteomics 14.6 Glycoprotein Analysis by Microarray MALDI-MS Molecular & Cellular Proteomics 14.6 1651 TABLE II Identified glycopeptide peaks and corresponding glycan compositions obtained by analyzing the peak lists from the FTMS spectra with GlycoMod tool (mass deviations were averaged over acquired spectra). An absolute mass deviation below or equal 0.025 Da (in average if the structure was identified in more than one spectrum) was set as limit. (Greater deviations were accepted for some lower intense peaks whose corresponding spectra were further investigated manually) Glycopep. Glycan Peptide Deviation Deviation Nr. Asn MH (mi) Composition Type (mi) Sequence ppm (av) Da (av) calculated calc. calc calc. 1 171 3838.5280 0.8 0.003 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 2 3 2 2 171 3635.4480 2.2 0.008 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 2 3 2 3 171 3547.4320 0.0 0.000 2262.81 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 1 3 2 4 171 3401.3740 6.8 0.023 2116.76 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 3 2 5 171 3344.3530 0.3 0.001 2059.74 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 1 3 2 6 171 3198.2950 1.3 0.004 1913.68 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 3 2 7 171 3256.3370 7.7 0.025 1971.72 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 1 3 2 8 171 3053.2580 4.9 0.015 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 1 3 2 9 171 2907.137 20.0 0.025 1913.68 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 1283.61 YKNNSDISSTR (1 missed cleavage) 2 2 3 2 10 171 3303.326 5.8 0.019 2018.71 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 3 1 1 1 3 2 11 171 3141.2740 2.2 0.007 1856.66 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 2 1 1 1 3 2 12 171 2979.2210 0.3 0.001 1694.60 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Hybrid/complex 1283.61 YKNNSDISSTR (1 missed cleavage) 1 1 1 1 3 2 13 171 3012.2310 3.3 0.010 1727.61 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 3 1 1 3 2 14 171 2850.1780 9.5 0.025 1565.56 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Hybrid 1283.61 YKNNSDISSTR (1 missed cleavage) 2 1 1 3 2 15 171 2688.1250 1.1 0.003 1403.51 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Hybrid/complex 1283.61 YKNNSDISSTR (1 missed cleavage) 1 1 1 3 2 16 171 2501.0410 1.6 0.004 1216.42 (Hex)  (Man) (GlcNAc) Oligomannosidic 1283.61 YKNNSDISSTR (1 missed cleavage) 2 3 2 17 171 2663.0940 4.1 0.011 1378.48 (Hex)  (Man) (GlcNAc) Oligomannosidic 1283.61 YKNNSDISSTR (1 missed cleavage) 3 3 2 18 171 3547.3690 19.7 0.025 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 3 1 2 3 2 19 171 3344.2890 19.7 0.025 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 2 3 2 20 171 3256.3370 10.0 0.025 2262.81 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 3 1 1 3 2 21 171 3110.216 10.1 0.025 2262.81 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 3 1 1 3 2 22 171 3053.195 9.8 0.025 2059.74 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 1 3 2 23 171 2907.137 1.4 0.004 1913.68 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 3 2 24 171 2762.1 2.0 0.006 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 1 3 2 25 171 2616.042 5.0 0.013 1622.58 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 992.45 NNSDISSTR 2 2 3 2 26 171 2371.936 6.3 0.015 1378.48 (Hex)  (Man) (GlcNAc) Oligomannosidic 992.45 NNSDISSTR 3 3 2 27 171 2209.883 9.5 0.021 1216.42 (Hex)  (Man) (GlcNAc) Oligomannosidic 992.45 NNSDISSTR 2 3 2 28 395 3355.283 1.4 0.005 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 1 2 3 2 29 395 3152.203 0.6 0.007 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 2 1 2 3 2 30 395 3064.188 1.3 0.005 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 1 1 3 2 31 395 3209.226 0.5 0.003 2407.852 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 2 3 2 32 395 2699.056 0.9 0.004 1897.682 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 1 2 1 1 3 2 33 395 2861.109 1.7 0.006 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 2 1 1 3 2 34 395 2918.13 2.5 0.009 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 2 3 1 3 2 35 395 2902.136 0.9 0.008 2100.762 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 800.37 THTNISE 1 3 1 1 3 2 36 402 2270.929 1.8 0.004 1054.37 (Hex) (HexNAc) Paucimannosidic 1215.55 SHPNATFSAVGE 4 2 37 402 2432.982 0.4 0.001 1216.423 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 2 3 2 38 402 2595.035 0.0 0.000 1378.476 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 3 3 2 39 402 2757.087 0.7 0.002 1540.528 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 4 3 2 40 402 2919.14 0.7 0.002 1702.581 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 5 3 2 41 402 3081.193 2.6 0.008 1864.634 (Hex)  (Man) (GlcNAc) Oligomannosidic 1215.55 SHPNATFSAVGE 6 3 2 42 402 2311.956 0.9 0.002 1095.397 (HexNAc)  (Man) (GlcNAc) Hybrid/complex 1215.55 SHPNATFSAVGE 1 3 2 43 402 2474.008 4.9 0.012 1257.449 (Hex) (HexNAc)  (Man) (GlcNAc) Hybrid/complex 1215.55 SHPNATFSAVGE 1 1 3 2 44 402 2636.061 2.3 0.006 1419.502 (Hex) (HexNAc)  (Man) (GlcNAc) Hybrid/complex 1215.55 SHPNATFSAVGE 2 1 3 2 Glycoprotein Analysis by Microarray MALDI-MS 1652 Molecular & Cellular Proteomics 14.6 TABLE II—continued Glycopep. Glycan Peptide Nr. Asn MH Deviation Deviation Composition Type Sequence (mi) (mi) calculated 45 402 2798.114 3.2 0.009 1581.555 (Hex) (HexNAc)  (Man) (GlcNAc) Hybrid 1215.55 SHPNATFSAVGE 3 1 3 2 46 332 4908.98 75 0.025 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 2 3 2 47 332 4705.9 8.0 0.025 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 2 3 2 48 332 4617.884 0.9 0.004 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 1 3 2 49 332 4471.826 0.0 0.000 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 3 2 50 332 4455.832 4.0 0.018 2100.762 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 1 3 1 1 3 2 51 332 4414.805 4.3 0.019 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 1 3 2 52 332 4326.789 3.7 0.016 1971.719 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 3 1 3 2 53 332 4268.747 3.7 0.016 1913.677 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 3 2 54 332 4123.71 2.2 0.009 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 2 2 1 3 2 55 332 4430.8 4.3 0.019 2075.73 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Hybrid 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 3 2 1 3 2 56 332 4164.736 3.8 0.016 1809.666 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2354.06 GLTFQQNASSM(SO)CVPDQDTAIR 1 3 1 3 2 57 332 4892.982 50 0.025 2553.91 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 2 3 2 58 332 4689.905 1.5 0.007 2350.83 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 2 3 2 59 332 4601.889 3.5 0.016 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 1 3 2 60 332 4455.831 4.0 0.018 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 3 2 61 332 4439.837 4.1 0.018 2100.762 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 1 3 1 1 3 2 62 332 4398.81 4.1 0.018 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 1 3 2 63 332 4310.794 3.0 0.013 1971.719 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 3 1 3 2 64 332 4252.752 6.6 0.025 1913.677 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 3 2 65 332 4107.715 6.6 0.002 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 2 2 1 3 2 66 332 4414.805 4.3 0.019 2075.73 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Hybrid 2338.07 GLTFQQNASSMCVPDQDTAIR 3 2 1 3 2 67 332 4148.741 1.9 0.008 1809.666 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 2338.07 GLTFQQNASSMCVPDQDTAIR 1 3 1 3 2 68 563 3435.47 5.5 0.019 1054.37 (Hex) (HexNAc) Paucimannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 4 2 69 563 3597.523 3.3 0.012 1216.423 (Hex)  (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 2 3 2 70 563 3759.576 2.9 0.011 1378.476 (Hex) (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 3 3 2 71 563 3921.628 2.3 0.009 1540.528 (Hex)  (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 4 3 2 72 563 4083.681 2.2 0.009 1702.581 (Hex) (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 5 3 2 73 563 4245.734 5.9 0.025 1864.634 (Hex)  (Man) (GlcNAc) Oligomannosidic 2380.09 STGKPTLYNVSLV(MSO)SDTAGTCY 6 3 2 74 71 (J-chain) 3142.294 0.8 0.003 1913.677 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 1 3 2 75 71 (J-chain) 3288.352 0.3 0.001 2059.735 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 1 1 3 2 76 71 (J-chain) 3345.373 2.4 0.008 2116.756 (Hex) (HexNAc) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 1 3 2 77 71 (J-chain) 2851.199 2.5 0.007 1622.582 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 3 2 78 71 (J-chain) 3491.431 0.7 0.002 2262.814 (Hex) (HexNAc) (Deoxyhexose) (NeuAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 1 1 3 2 79 71 (J-chain) 2997.257 1.6 0.005 1768.64 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 2 1 3 2 80 71 (J-chain) 3054.278 0.5 0.002 1825.661 (Hex) (HexNAc)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 3 2 81 71 (J-chain) 3200.34 2.2 0.007 1826.661 (Hex) (HexNAc) (Deoxyhexose)  (Man) (GlcNAc) Complex 1227.61 ENISDPTSPLR 2 3 1 3 2 Glycoprotein Analysis by Microarray MALDI-MS FIG.5. MALDI-FTMS spectra representative for each glycosite of the IgM mu chain as well as of the J-chain. Peaks were labeled with monoisotopic masses [MH ] . A direct comparison with the ESI-MS reference measurements is presented in the electronic supplemental information (supplemental Fig. S2A–S2F). man and Kornfeld in 1979 was based on the preparative Man6 on Asn 402 and Man6 and Man8 on Asn 563. A recent fractionation of glycopeptides, amino acid analysis by liquid study by Loos et al. using LC-ESI-MS assigned major glyco- chromatography, and monosaccharide analysis by gas-liquid forms of the mu chain, which also confirmed the pioneering chromatography (62, 63). The authors describe Man5 and work of Chapman et al. (62, 63). The major glycoforms found Molecular & Cellular Proteomics 14.6 1653 Glycoprotein Analysis by Microarray MALDI-MS Acknowledgments—We thank Alfredo J. Iba´n˜ ez, Robert Steinhoff, here by the microarray-MALDI-FTMS approach were in and Stephan Fagerer for helpful advices with our MALDI-MS mea- agreement with the results obtained by the previous studies. surements. Furthermore we thank Konstantins Jefimovs and Rolf In addition we could provide a comprehensive and a detailed Bro¨ nnimann from EMPA Du¨ bendorf, Jens Boertz from Sigma-Aldrich, analysis, assigning a total of 81 glycopeptide peaks to the IgM Rolf Ha¨ flinger and Dr. Xiangyang Zhang from the MS-Service for their assistance with the MALDI-FTMS system. mu chain and to the J chain with highest mass accuracy (Table II). Thereby we detected some minor portions of oligo- * This work was supported by the European Research Council (ERC mannosidic glycans as well as hybrid-type glycans, also on Starting Grant nLIPIDS, Grant No. 203428) and the Swiss KTI (Kom- Asn 171. Glycosite Asn 563 carried exclusively oligomanno- mission fu¨ r Technologie und Innovation; Grant No. 13123.1 PFNM- NM). Simon K. Ku¨ ster acknowledges financial support from the schol- sidic structures, but site Asn 402 exhibited 10% hybrid-type arship fund of the Swiss Chemical Industry (SSCI). glycans. Glycosites Asn 395 and the J chain carried only □ S This article contains supplemental Figs. S1 to S3. complex type glycans with one or two sialic acid residues, ¶ To whom correspondence should be addressed: Department of bisecting GlcNAc and one fucose residue. Arnold et al. (64), Chemistry and Applied Bioscience, ETH Zurich, HCI E 329, Zurich demonstrated by exoglycosidase digests that the fucose res- CH-8093 Switzerland. Tel.: 4144-6324376; E-mail: zenobi@org. chem.ethz.ch. idue present is a core fucosylation. 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Published: Jun 1, 2015

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