Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry
|
|
- Melinda Newman
- 5 years ago
- Views:
Transcription
1 Available online at Analytical Biochemistry 376 (2008) ANALYTICAL BIOCHEMISTRY Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry David J. Harvey a, *, Louise Royle a,1, Catherine M. Radcliffe a,2, Pauline M. Rudd a,b, Raymond A. Dwek a a Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, U.K. b Dublin-Oxford Glycobiology Laboratory, National Institute for Bioprocessing Research and Training, Conway Institute, University College Dublin, Ireland Received 31 October 2007 Available online 31 January 2008 Abstract Negative ion electrospray (ESI) fragmentation spectra derived from anion-adducted glycans were evaluated for structural determination of N-linked glycans and found to be among the most useful mass spectrometric techniques yet developed for this purpose. In contrast to the more commonly used positive ion spectra that contain isobaric ions formed by losses from different regions of the molecules and often lead to ambiguous deductions, the negative ion spectra contain ions that directly reflect structural features such as the branching pattern, location of fucose, and the presence of bisecting GlcNAc. These structural features are sometimes difficult to determine by traditional methods. Furthermore, the spectra give structural information from mixtures of isomers and from single compounds. The method was evaluated with well-characterized glycans from IgG and used to explore structures of N-linked glycans released from serum glycoproteins with the aim of identifying biomarkers for cancer. Quantities of glycans were measured by ESI and by matrix-assisted laser desorption ionization mass spectrometry; each technique produced virtually identical results for the neutral desialylated glycans. Ó 2008 Elsevier Inc. All rights reserved. Keywords: N-glycans; MALDI; Electrospray; Mass spectrometry; Quantitation; Negative ions The current interest in oligosaccharides as markers of disease has highlighted the need for rapid glycan analysis. The structural analysis of oligosaccharides is generally regarded more difficult than that of other biopolymers because of their branched structures and the variable ring size and anomericity of the constituent monosaccharides. However, the problem simplifies dramatically for many carbohydrate types, such as the N-linked glycans, because of the specific enzymology involved in their biosynthesis. * Corresponding author. Fax: address: dh@glycob.ox.ac.uk (D.J. Harvey). 1 Current address: Ludger Ltd., Culham Science Centre, Abingdon, Oxfordshire OX14 3EB, U.K. 2 Current address: Lonza Biologics plc, 228 Bath Road, Slough, Berkshire SL1 4DX, U.K. The general structure of these N-linked carbohydrates is highly conserved across most species yet studied. N-linked glycans are attached to an asparagine residue in an Asn- Xaa-Ser(Thr or occasionally Cys) consensus sequence in glycoproteins where Xaa is any amino acid except proline. An outline of their biosynthesis is shown in Fig. 1 and it can be seen that all N-linked glycans contain a common trimannosyl-chitobiose core variously substituted, mainly on the two outer mannose residues with either additional mannose residues or antennae consisting predominantly of Gal-b-(1?4(or 3))-GlcNAc-b-(1?) groups. Among other modifications, additional substituents such as fucose can be attached to the core GlcNAc or to the antennae. The analysis of such compounds in common mammalian and plant systems where the biosynthetic enzymology is well defined, therefore, reduces to the determination of /$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi: /j.ab
2 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Fig. 1. Biosynthesis of N-linked glycans. Arrows are illustrative and do not necessarily depict intact biochemical pathways. The antennae of the complex glycans often contain an additional N-acetyl-neuraminic acid in either a2-3- or a2-6-linkage. The structures that are shown are only a few of those possible. Key to symbols used for the constituent monosaccharides in this scheme and subsequent figures: } = galactose, j = GlcNAc, s = mannose, h = glucose, = fucose, w = N-acetylneuraminic (sialic) acid. such factors as the type of glycan (high-mannose, hybrid, or complex; see Fig. 1 for definitions), the number and composition of the antennae, the number and location of other substituents (fucose, xylose, sulfate, etc.), the linkage of sialic acids, and the presence or absence of a bisecting GlcNAc residue. However, less common substituents such as galacturonic acid and N-acetylgalactosamine [1] may also be encountered, especially in glycans derived from less-well-studied species. The presence of these less common constituents often results in the failure of techniques that rely solely on traditional methods such as exoglycosidase sequencing. The classical method for analyzing these compounds involves successive incubation with exoglycosidases of known specificity that remove constituent monosaccharide residues from the nonreducing terminals, coupled with profiling by gel-filtration chromatography, high-performance liquid chromatography, or, more recently, mass spectrometry [2,3], both before and after digestion. The substituent and its linkage are revealed by knowledge of the enzyme specificity and the number of substituents is deduced by the change in the property being measured (retention time, mass, etc.). Although powerful, the technique requires fluorescent or related tagging of the glycan for detection by chromatography and is dependent on a readily available source of the necessary enzymes in a pure state. Furthermore, specific enzymes are not available for determination of all structural features, e.g., the presence of a bisecting GlcNAc. Consequently, there is increasing use of techniques such as mass spectrometry for complete structural
3 46 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Fig. 2. MS/MS spectra of the (a) [M + H] +, (b) [M + Na] +, and (c) [M + H 2 PO 4 ] ions from the high-mannose N-glycan Man 5 GlcNAc 2. The [M + H] + ions fragment mainly by losses of mannose residues from the molecular and B 4 (B R-1 ) ion (Y-type glycosidic cleavages). More cross-ring cleavage ions (indicated by a star) are present in the spectrum of the [M + Na] + ion (spectrum b) but the glycosidic cleavages still originate from losses from several sites. Fragmentation of the [M + H 2 PO 4 ] ion, on the other hand, (spectrum 2c) contains many more specific cross-ring cleavage ions formed by mechanisms similar to that shown in Fig. 3 (top). analysis. Mass spectrometry is essential for identifying any mass differences produced by unusual monosaccharide constituents and for providing a composition of the glycan in terms of its isobaric monosaccharide composition. Even then, some glycans are substituted with other noncarbohydrate groups necessitating the use of MS/MS or other analytical techniques for their identification. The ideal mass spectrometric approach is one in which the initial spectrum produces the glycan profile with no fragmentation and subsequent spectra give extensive fragmentation to provide the detailed structural information. Entire glycan profiles are conveniently obtained by matrix-assisted laser desorption/ionization (MALDI) 3 mass spectrometry [4 7], a technique that gives a quantitative response and negligible fragmentation for neutral N- glycans of varying structure [8,9]. Sialylated glycans, although less stable, can be neutralized and stabilized by methyl ester formation [10] but tend to fragment by loss of sialic acid if not derivatized. The somewhat milder technique of electrospray ionization (ESI) is being increasingly used but, again, sialic acid derivatization is needed if multiple charging is to be avoided in negative ion mode. Older, less sensitive techniques such as fast-atom bombardment mass spectrometry have also been widely used in this context [11,12] but produce extensive fragmentation, require 3 Abbreviations used: MALDI, matrix-assisted laser desorption ionization; ESI, electrospray ionization; TOF, time-of-flight. derivatization of the glycans, and are now mainly of historical interest. The mass of the glycan provides the constituent monosaccharide composition in terms of sugar type (hexose, deoxy-hexose, etc.) but the arrangement of these monosaccharides in the glycan chain must be deduced from fragmentation studies. Fragmentation from MALDIgenerated ions produced in time-of-flight (TOF) instruments can be obtained by analysis of spontaneously produced post-source decay ions [13] but resolution is generally poor. Greatly improved performance can be obtained by generating the ions by collision in a tandem instrument such as a quadrupole/time-of-flight (Q-Tof) mass spectrometer [14 18] and multiple successive fragmentations can be produced with a storage instrument such as an ion trap [19 21]. The type of ion that is formed has a major influence on the information that can be obtained. MALDI produces mainly [M + Na] + ions (Fig. 2b) that fragment predominantly by cleavage between the sugar rings (glycosidic cleavage) to provide sequence information but few linkage data. This information is provided by cross-ring cleavage fragments but these are present at only very low relative abundance in low-energy spectra of the type provided by ion trap and Q-Tof-type instruments. Cross-ring fragments of higher abundance are found in the spectra recorded with instruments providing higherenergy collisions [22 24] but all spectra of [M + Na] + ions contain ambiguous information because fragment ions of the same mass and composition can arise from different
4 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) regions of the molecule. The situation is even more difficult with the [M + H] + ions frequently encountered in ESI spectra (Fig. 2a); fragmentation of these ions rarely yield crossring cleavages and are prone to internal rearrangements [25 28] that again yield ambiguous information. The solution to these problems is to be found with negative ion spectra. Under these conditions, cross-ring cleavage reactions are frequently dominant [29 35] (Fig. 2c) and the redundancy seen in the positive ion spectra is largely absent. Consequently, the spectra contain ions that are diagnostic of specific structural features that cannot be obtained easily from the positive ion spectra or from the more classical analytical methods. This paper describes the fragmentation of negative ions derived from N-linked glycans with applications to glycans released from specific glycoproteins and glycoprotein mixtures. The samples consisted of N-glycans that had been released from glycoproteins from within SDS PAGE gels as described fully in the companion paper by Royle et al. [36]. Materials and methods Sample preparation from released glycans Human serum was obtained from normal individuals and human serum IgG was prepared as described in the companion paper [36]. An aqueous solution (1 ll, 20%) of glycans from about 1 lg of glycoprotein that had been released with protein N-glycosidase F (PNGase F; Roche Diagnostics GmbH, Mannheim, Germany or Prozyme, Leandro, CA, USA) from within a SDS PAGE gel by the method described by Küster et al. [37] as modified according to Rudd et al. [36,38] and as described in the accompanying paper [36], was placed on a small piece of Nafion 117 membrane (Aldrich Chemical Co., Poole, UK) [39] that was floating on water. After about 30 min, the solution was removed and examined by MALDI- TOF MS as described below. If sialylation was detected, a further 1-lL sample of glycans was treated as above, dried, and dissolved in methanol (20 ll). A trace of 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) catalyst, prepared by the method described by Kunishima et al. [40] was added and the solution was heated at 80 C for 2 h. The methanol was evaporated, the glycans were redissolved in water (1 ll), and the DMT-MM was removed with a Nafion membrane as above. Full details of this technique will be published in a further communication. Samples were reexamined by MALDI-TOF MS. Fragmentation of derivatized and nonderivatized glycans was performed on further cleaned samples by nanospray-ms/ms as described below. Quantification of neutral (desialylated) N-glycans derived from human serum glycoproteins N-glycans were released in-gel with PNGase F from six aliquots of a sample of pooled human serum from normal control subjects, desialylated with Arthrobacter ureafaciens sialidase (Prozyme), and cleaned with a Nafion 117 membrane. MALDI and ESI spectra were acquired as detailed below. Quantities of the major glycans were measured by both techniques using either peak height measurements on peaks that had been smoothed to remove isotopic resolution or summation of the peak heights of all isotopes. MALDI-TOF mass spectrometry The aqueous solution of glycans (1 ll) was placed on the stainless steel MALDI target together with 0.3 ll of matrix (saturated solution of 2,5-dihydroxybenzoic acid in acetonitrile) and allowed to dry under ambient conditions. The dried sample/matrix mixture was then redissolved in ethanol and again allowed to dry to produce a more homogeneous target surface. MALDI spectra were acquired with a Waters- Micromass (Manchester, UK) TofSpec 2E reflectron-tof mass spectrometry operated in reflectron mode with delayed extraction. Operating conditions were as follows: accelerating voltage, 20 kv; delayed extraction delay, 500 ms (setting of 39 on the control screen); pulse voltage, 3 kv; laser repetition rate, 5 Hz. Instrument control, spectral acquisition, and processing were performed with MassLynx software (version 3.3). Quantitative data were obtained with a Waters-Micromass MALDI-Micro MX mass spectrometer under similar conditions. Nano-electrospray mass spectrometry Nano-electrospray mass spectrometry was performed with a Waters-Micromass Q-Tof Ultima Global instrument. Samples in 1:1 (v:v) methanol:water containing 0.5 mm ammonium phosphate were infused through Proxeon (Proxeon Biosystems, Odense, Denmark) nanospray capillaries. The ion source conditions were as follows: temperature, 120 C; nitrogen flow, 50 L/h; infusion needle potential, 1.2 kv; cone voltage, 100 V; RF-1 voltage, 180 V. Spectra (2-s scans) were acquired with a digitization rate of 4 GHz and accumulated until a satisfactory signal:noise ratio had been obtained. For MS/MS data acquisition, the parent ion was selected at low resolution (about 4 m/z mass window) to allow transmission of isotope peaks and fragmented with argon. The voltage on the collision cell was adjusted with mass and charge to give an even distribution of fragment ions across the mass scale. Typical values were V. Other voltages were as recommended by the manufacturer. Instrument control, data acquisition, and data processing were performed with MassLynx software version 4.0. Results Diagnostic fragments The specificity of the negative ion fragmentation reactions arises from the initial ionization of the molecules by
5 48 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) removal of a proton from one of several hydroxyl groups. Subsequent electron shifts stabilize the initially formed ion (Fig. 3, top). The [M-H] ions themselves are relatively unstable and tend to fragment within the ion source. However, more stable molecular ions can be obtained by adduction with various anions such as chloride, nitrate, phosphate, etc. [41 49] with concomitant increases in sensitivity and with only minimal in-source fragmentation. Among the univalent ions, nitrate gives good stability and sensitivity, producing glycan profiles that closely parallel those obtained by MALDI. The ions can be prepared conveniently by addition of a nitrate salt, preferably ammonium nitrate, to the ESI solvent. Chloride provides comparable stability but higher halogens, although forming stable products yield weak or nonexistent fragment ions. However, sensitivity of chloride adducts is compromised by the isotopic profile of chlorine. In our initial study [48], divalent anions were sometimes found to give anomalous results and were not studied further. However, later work involving studies with N-glycans released with PNGase F from within SDS PAGE gels revealed an almost quantitative conversion to phosphate adducts that, fortunately, fragmented in a manner analogous to that of the nitrate adducts. Consequently, subsequent work, and that described in this paper, uses phosphate addition. With a few minor exceptions, fragmentation of all of these adducts resembles that of the [M-H] ions because the first elimination is that of the corresponding adduct acid (e.g., H 3 PO 4 from the phosphate adduct) to give what is essentially the [M-H] ion. Following abstraction of the initial proton, very specific electron shifts lead to stable ions that are diagnostic of specific structural features. Significant diagnostic ions are listed in Table 1. For example, abstraction of the proton from the 3-hydroxy group of the reducing terminal Glc- NAc residue results in the formation of a prominent 2,4 A R ion (Fig. 3, top and m/z 1072 in Fig. 2c) (the ion nomenclature is that proposed by Domon and Costello [50] modified by the use of the subscript R rather than a variable subscript number to denote reducing terminus [20]). The carbon atom at position 6 of this GlcNAc residue, together with any substituent, is eliminated in this process, providing an unambiguous method for detecting fucose substitution at this position. Fucose substitution at carbon-3, the only other position available for substitution in this GlcNAc residue and a common substituent in plant N-glycans, would prevent formation of the 2,4 A R ion because of the absence of a removable proton. Simi- Fig. 3. (Top) Proposed mechanism for the production of the 2,4 A ion from the reducing terminus of N-glycans. (Bottom) Biantennary glycan showing the major fragment ions. The linking oxygen atoms have been shown on the glycosidic bonds so that B and C ions can be differentiated.
6 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Table 1 Ions defining structural features in the negative ion spectra of N-linked glycans Structural feature Ion Ionic composition Composition Molecular [M + X] Antenna sequence Antenna composition Fucose at 6-position of reducing terminus Absence of fucose at 6-position of reducing terminus Composition of 6-antenna Composition of 3-antenna Presence of bisect m/z C Gal, Man, Glc 179 GlcNAc, GalNAc 220 [Fuc]Gal 325 Gal-GlcNAc 382 Gal-[Fuc]GlcNAc 528 agal-gal, Man-Man 341 Man-[Man]Man 503 GalNAc-GlcNAc 423 F Man 221 GlcNAc 262 Gal-GlcNAc 424 Gal-[Fuc]GlcNAc 570 agal-gal 383 agal-gal-glcnac 586 GalNAc-GlcNAc 465 (Gal-GlcNAc) (Gal-GlcNAc) 2 Fuc 935 (Gal-GlcNAc) B 3 Neu5Ac-Gal-GlcNAc 655 Neu5Gc-Gal-GlcNAc 671 2,4 A R [M-Cl-307] [M-342] [M-NO 3-307] [M-369] [M-H 2 PO 4-307] [M-405] 2,4 A R [M-Cl-161] [M-196] [M-NO 3-161] [M-223] [M-H 2 PO 4-161] [M-259] D and [D-18] ([D-36] ) O,3 A R-2 and O,4 A R-2 GlcNAc 526, 508 Gal-GlcNAc 688, 670 Gal-[Fuc]GlcNAc 834, 816 (Gal-GlcNAc) , 1035 (1017) (Gal-GlcNAc) 2 Fuc 1199, 1181 (1163) Man 3 647, 629 Man 4 809, 791 Man 5 971, 953 GlcNAc 292, 262 a Gal-GlcNAc 454, 424 Gal-[Fuc]GlcNAc 600, 570 (Gal-GlcNAc) 2 819, 789 (Gal-GlcNAc) 2 Fuc 965, 935 Man 3 251, 221 Man 4 413, 383 Man 5 575, 545 O,4 A R-3 (E) ion Gal-GlcNAc 466 GlcNAc Gal-GlcNAc (Gal-GlcNAc) (Gal-GlcNAc) 2 Fuc 977 Abundant [D-221] ion. Ion at [M ] Missing Ion D GlcNAc 508 Gal-GlcNAc 670 Gal-[Fuc]GlcNAc 816 (Gal-GlcNAc) (Gal-GlcNAc) 2 Fuc 1181 Man Man Man Table 1 (continued) Structural feature Presence of sialic acid Presence of a2? 6-linked sialic acid Ion Ionic composition larly, the mass or absence of the abundant 2,4 A R-1 ion, resulting from abstraction of the proton from the other core GlcNAc residue (m/z 869; Fig. 2c), provides information about substitution around this residue. Cleavages of, or around, the branching mannose residue result in ions that are diagnostic of the individual antenna. Possibly the most significant of these ions is one, termed ion D (Fig. 3, bottom), that arises from formal loss of the chitobiose core and the substituents forming the 3-antenna and, thus, contains only the 6- antenna and the branching mannose residue. This ion can be seen at m/z 647 in Fig. 2c. It is accompanied by a second ion (m/z 629; Fig. 2c) formed by an additional loss of water (18 mass units) or, additionally, two such losses in the case of a tetra-antennary glycan (X, Fig. 1; see below). The initial water loss is probably from the 4- position of the branching mannose because in spectra of glycans containing a bisecting GlcNAc residue at this position (see structure XI; Fig. 1) the D ion is missing but the ion equivalent of D-18, in this case D-221 (D-Glc- NAc), is very prominent and diagnostic (as shown in Fig. 8e, below, at m/z 670) and is accompanied by another ion 18 mass units lower. Identification of bisecting Glc- NAc residues by traditional techniques is laborious because of the lack of a specific exoglycosidase but the negative ion fragmentation spectra easily highlight this structural feature. Other ions that are diagnostic for the composition of the 6-antenna are the cross-ring O,3 Aand O,4 A fragments from the branching mannose (m/z 575 and 545, respectively, in Fig. 2c). A problem that has often hindered the use of mass spectrometry for biomolecular analysis is the difficulty of differentiating isomers; frequently their presence in positive ion spectra is reflected only in relative ion abundance [51], a property that is more uncertain than differences in mass. The negative ion fragmentation spectra of isomeric N-glycans, however, usually yield fragments that differ in mass, allowing isomers to be detected and, in many cases, quantified within a single spectrum. An example of isomer differentiation of high-mannose glycans is given in an earlier paper [52]. Other examples of isomer detection are shown below in Figs. 7c and 8c. An O,4 A cleavage of the mannose residues attached to the core branching mannose results in the formation of m/z B 1 Neu5Ac 290 Neu5Gc 306 O,4 A 2 -CO 2 Neu5Ac 306 Neu5Gc 322 a These ions are normally of relatively low abundance. An abundant ion at the mass of the O,4 A ion is more likely to be an F-type ion.
7 50 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) an ion, termed Ion E, containing the 2-substituent (the antenna) plus 101 mass units from the mannose residue (C 4 H 5 O 3 ). In the spectra of neutral biantennary complex glycans in which each chain consists of Gal-GlcNAc units (VIII, Fig. 1), ions of this composition (e.g., m/z 466, Gal- GlcNAc+101) can arise from either antenna (Fig. 4a). However, in the spectra of triantennary glycans containing a branched 3-antenna (IX, Fig. 1), the presence of carbon-4 in Ion E results in both the branches remaining in this ion to give a prominent peak at, e.g., m/z 831 (Fig. 4b) where both antennae contain Gal-GlcNAc. Ions D and [D-18] remain at m/z 688 and 670, respectively, as in the spectra of biantennary glycans. If branching in a triantennary glycan occurs at the 6-position of the 6-antenna (Fig. 4c), no E ion is present at m/z 831 because carbon-6 (the substitution position) of the mannose residue is not retained by Ion E which again appears at m/z 466. However, the D and [D-18] ions shift by 365 mass units to m/z 1053 and 1035, respectively, and are accompanied by a third ion at m/z 1017 that has lost two molecules of water from the mass of Ion D. Thus, the masses of Ions D and E can be used to define the isomeric configuration of triantennary glycans; again, this differentiation is laborious by traditional methods. Consistent with these cleavages, the spectra of tetra-antennary glycans (X, Figs. 1 and 4d) show abundant E ions at m/z 831 and D, [D-18], and [D- (18) 2 ] ions at m/z 1053, 1035, and 1017, respectively. For complex glycans, the composition of the antennae can be ascertained from the mass of an abundant cross-ring cleavage ion of the mannose residue that leaves two carbon atoms attached to the ion (Ion F, Fig. 3, bottom, for 2- linked antennae). Ions of type F can be formed from antennae attached to other positions of the core mannose residues; i.e., a 4-linked antenna would give Ion F by a 2,4 A cleavage and a 6-linked antenna would produce it by an O,4 A cleavage. Sagi et al. [35] report that this latter cleavage Fig. 4. Negative ion MS/MS spectra of complex N-linked glycans. (a) Biantennary glycan. The absence of core fucose is reflected by the masses of the 2,4 A R and 2,4 A R-1 ions and the composition of the 6-antenna is shown by the D and [D-18] ions at m/z 688 and 670, respectively. The ion at m/z 424 defines the Hex-HexNAc (Gal-GlcNAc) composition of the antennae. (b) Triantennary glycan branched on the 3-antenna. The D and [D-18] ions appear at m/z 688 and 670 because there is no change in the composition of the 6-antenna from that of the biantennary glycan shown in (a). However, the presence of a branch on the 3-antenna causes a prominent E ion (identified by a subscript 3 preceding the E) to appear at m/z 831 (the E ion contains carbons 1, 2, 3, and 4 from the mannose residue). (c) Triantennary glycan branched on the 6-antenna. The D and [D-18] ions shift by 365 mass units to m/z 1053 and 1035, respectively, and another ion at [D-(18) 2 ] appears at m/z No E ion is present at m/z 831 because there is no branch on the 3- antenna and the 6-antenna is branched at the 2- and 6-carbons. (d) Tetra-antennary glycan. The D [D-18] and [D-(18) 2 ] ions appear at m/z 1053, 1035, and 1017 as in (c) and the branched 3-antenna produces an E ion at m/z 831. (e) Tetra-antennary glycan with a N-acetyl-lactosamine extension to one of the antennae. The extra Gal-GlcNAc residue causes a shift in the D [D-18] and [D-(18) 2 ] ions to m/z 1418, 1400, and 1382, respectively, indicating substitution in the 6-antenna and produces F-type ions at m/z 424 and 789. The high relative abundance of this latter ion is consistent with substitution at the 6-position of the 6-antenna [35].
8 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) is very favorable such that a very abundant ion of type F strongly suggests the presence of a 6-linked antenna. A Gal-GlcNAc chain produces an F-type ion at m/z 424 and substitution by other groups such as fucose or a-galactose can be detected by the appropriate mass shifts (to m/z 570 and 586 for these substituents, respectively; Table 1). The positions of these substituents can often be determined by the mass of a series of C-type ions (from the nonreducing terminus). Thus, antennae terminating in galactose or mannose produce a C 1 ion at m/z 179 whereas those terminating in GlcNAc or GalNAc show an ion at m/z 220. Again, mass shifts in these ions can be used to locate the substituents on the antennae. Thus, for example, a Gal-GlcNAc antenna containing fucose attached to the GlcNAc residue would fragment to give the C 1 ion at m/z 179 but a C 2 ion at m/z 528 rather than at m/z 382 (m/z ). Fucose substitution on the galactose residue would still produce m/z 528 but the C 1 ion would have shifted from m/z 179 to m/z 325 (see example in [53]). Structures of sialylated glycans (Fig. 5) are less easy to obtain by negative ion MS/MS because most of the charge following the initial loss of HX resides primarily on the acid groups rather than a hydroxyl group, effectively suppressing ions formed by hydroxyl proton abstraction. The ions of highest mass correspond to [M-nH] n-. Spectra of monosialylated glycans ([M-H] ions; Figs. 5a and b) generally give more informative spectra than those of the multiply charged ions (Fig. 5c). The presence of sialic acids is revealed by a prominent B ion at m/z 290 for N-acetylneuraminic acid and m/z 306 for N-glycoylneuraminic acid, allowing ready identification. A B-type cleavage adjacent to the GlcNAc residue in the spectra of sialylated complex glycans with Neu5Ac-Gal-GlcNAc chains gives an ion at m/z 655 or 671 if glycoylsialic acid is present. If these acids are a2-6-linked, a second ion of low abundance is present at m/z 306 or 322 for the two types of sialic acid, respectively [54]. Assignment of m/z 306 either to the presence of glycoylsialic acid or to an a2? 6-linked acetylsialic acid can usually be made by the presence or absence of the ion at m/z 671 indicating a glycoylsialic acid. Other fragments have been discussed by Sagi et al. [35]. Although many of the other diagnostic ions found in the spectra of the neutral glycans are usually missing from, or are of low abundance in, the spectra of multiply charged ions derived from glycans with more than one sialic acid, some specificity can be restored by blocking the acid group of the sialic acids by derivatization (methyl ester or lactone formation). If derivatization is performed in methanol with DMT-MM as catalyst, a2-6-linked sialic acids form methyl esters whereas a2-3-linked acids are converted into lactones. Their mass difference of 32 u thus provides a simple method for isomer determination (unpublished paper and Fig. 8a). However, although fragmentation of the lactones parallels that of the neutral glycans described above, the spectra of the methyl esters are complicated by losses of methanol from many of the ions. Nevertheless, it has been found possible to assign a2-3- or a2-6-linked sialic acids to specific antenna in many cases. Fig. 5. Negative ion MS/MS spectra of sialylated glycans released from human serum glycoproteins. (a) [M-H] ion from the biantennary glycan Hex 5 GlcNAc 4 Neu5Ac 1. The position of the sialic acid is shown on the 6-antenna for clarity although the spectrum is of a mixture of isomers. (b) [M-H] ion from the triantennary glycan Hex 6 GlcNAc 5 Neu5Ac 1. Branching of the 3-antenna can be deduced by the presence of the E-type ion at m/z 831 but the relative abundance of this ion is much lower than that of the corresponding ion in the spectrum of the corresponding neutral glycan (Fig. 4b). (c) [M-2H] 2 ion (m/z ) from the biantennary glycan Hex 5 GlcNAc 4 Neu5Ac 2. The singly charged ion at m/z is the result of elimination of one of the sialic acids with its charge.
9 52 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Quantitation of neutral glycans Quantitation by MALDI is often regarded as unreliable despite many studies showing the opposite. Although sialylated glycans fragment under most MALDI conditions, they may be stabilized by methylation, either permethylation or methyl ester formation [10], as above. Permethylation has been reported to provide good quantitation [55,56]. Neutral N-glycans have been quantified by MALDI [8] and complex N-glycans shown to give signals of the same strength for equivalent amounts deposited on the MALDI target [9]. Current interest in the use of glycans as biomarkers for various diseases such as rheumatoid arthritis and cancer has necessitated high-throughput quantitative measurements and to this end we have investigated the use of both MALDI-TOF and negative ion ESI- Q-Tof mass spectra to quantify neutral glycans. Both techniques have similar sensitivity. The decision to concentrate on the neutral glycans initially was based on several factors. As mentioned, sialic acids must be derivatized for MALDI analysis with possible sample loss and the production of artifacts, particularly with permethylation. Also, many potential biomarkers differ in the extent of galactosylation and/or fucosylation and produce glycans with these markers distributed among glycans in different sialylated states, thus splitting the signal, both within the spectrum and between different charge states if spectra are recorded by ESI MS. Consequently, removal of the sialic acids to give neutral glycans was considered preferable to examination of free sialylated glycans or their derivatized counterparts as a rapid method for biomarker detection where the biomarker did not involve differences in sialylation. MALDI spectra. Tests were conducted to determine the minimum number of laser shots necessary to achieve good precision and the best method of peak measurement (smoothing isotope peaks, measuring only the monoisotopic peak, or adding the peak height of all isotopes). MALDI spectra of the desialylated glycans were recorded for 100 scans (200 s with a laser rate of 5 Hz) from a set of six human serum samples derived from a pool of serum from normal individuals with the MALDI laser beam continually scanning over the target surface to minimize target inhomogeneities. Means, standard deviations, and coefficients of variation were measured for the eight most abundant ions. The best results were obtained by smoothing the isotope peaks (Savitzky Golay 30 2) and little change was observed with greater than 16 spectra (32 s, 160 laser shots) averaged. Changing the laser power from that at which ionization just occurred to that when the major peak began to saturate made no difference to the measurements, consistent with no laser-induced decomposition of the neutral glycans. It is sometimes thought that fucose is unstable under these conditions but these results indicate otherwise. Allowing for target change, etc., the speed of this analysis would equate to about 2500 samples/day. ESI spectra. Groups of 10 spectra were averaged and peak heights for the same eight ions as above were measured from (a) the monoisotopic peak, (b) all of the isotope peaks, and (c) the smoothed peak (Savitzky Golay 100 2). The averages, standard deviations, and coefficients of variation were then evaluated for the 10 measurements from each peak (rows 1 3 in Table 2) and compared with the same measurement made by averaging all of the spectra per run rather than averaging each group of 10 spectra (row 4 in Table 2). Results were very similar although the measurements made from only the monoisotopic peak showed a slight bias toward the low-mass glycans as would be expected. When the results of the averaged data were compared with the measurements made by MALDI-TOF, the results were remarkably consistent (Table 2 and Fig. 6) even though the measurements were made with different instruments in different laboratories (Oxford, UK and Dublin, Ireland). Both MALDI-TOF and ESI measurements, therefore, appear to give reliable measurements of glycan quantitation for neutral glycans in samples of this type. However, it could well be that samples containing different types of glycan may give different results as a consequence of differences in ionization efficiency. This point is currently being addressed but the required samples in a sufficient state of purity are not yet available. The earlier work [8,9], however, demonstrated equivalence in ionization efficiency of complex glycans of the type involved here when ionized by MALDI. Protocol The current protocol for N-linked glycan analysis is as follows: Glycans are released from the glycoproteins using one of the many methods available (e.g. [38,57]. Residual contamination is removed with a Nafion 117 membrane [39] as described under Materials and methods. The glycans are then analyzed by MALDI mass spectrometry to provide a glycan profile and, if sialylation is detected (usually by the presence of abundant broad metastable ions [58], the glycans are reacted with methanol in the presence of DMT-MM to stabilize them and a second MALDI spectrum is acquired to determine the sialic acid linkage. Desialylated (enzymatic (A. ureafaciens) or mild acid treatment), neutral glycans are then examined by negative ion MS/MS with nano-spray sample introduction for structural identification. Although it would be possible to use LC/MS in this context, we prefer the simple infusion technique because spectral acquisition can be tailored to the concentration of individual constituents. Spectra of abundant constituents can be recorded in a few seconds whereas the instrument can be left to acquire spectra from minor components until a satisfactory signal:noise ratio is reached. Typically, samples representing 0.25% of the glycans obtained from about 5 lg of glycoprotein and dissolved in 5 8 ll of MeOH:H 2 O can be sprayed for up to 2 h and usable spectra acquired from constituents that are hardly visible in the glycan profile. It is estimated that only a few fem-
10 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Table 2 Comparison of averaged measurements for the three measurement methods in ESI and measurements from MALDI spectra Measurement m/z ESI, One sample, different methods of measurement Monoisotopic Total isotopes Average mass Average of 3 methods ESI, Average from six samples Av SD CV(%) MALDI, Average of six samples Av SD CV(%) Fig. 6. Comparison of the concentrations of the eight major ions from desialylated N-glycans obtained from human serum glycoproteins. The colors represent measurements from each of six samples taken from pooled human serum; the first column of each color is the MALDI-TOF measurement and the second is the measurement made by ESI. An, number of antennae; Gn, number of galactose residues; F, presence of a fucose residue; B, presence of a bisecting GlcNAc. tomoles are required to eventually produce an interpretable spectrum. Applications IgG. Fig. 7a shows the MALDI spectrum of desialylated N-glycans released from human IgG (most glycans are not sialylated in this glycoprotein). Negative ion fragmentation spectra of the three major constituents with compositions of Hex 3-5 HexNAc 4 dhex 1 are shown in Figs. 7b d. In all three spectra, the 2,4 A R ion was formed by loss of 307+HX mass units, showing that the fucose residue was located at the 6-position of the reducing-terminal GlcNAc. The spectra of the three major compounds were consistent with those of biantennary glycans with zero, one, and two galactose residues as is well established for IgG glycans [59,60]. Fig. 7b shows the spectrum of the glycan with no galactose residues as reflected by the single C 1 ion at m/z
11 54 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Fig. 7. Analysis of N-glycans from human IgG. (a) Positive ion reflectron-maldi-tof mass spectrum of N-glycans from human IgG. (b) Negative ion MS/MS spectrum of the ungalactosylated glycan producing the ion at m/z 1485 in a. (c) Negative ion MS/MS spectrum of the mixture of monogalactosylated glycans (m/z 1647 in a). (d) Negative ion MS/MS spectrum of the digalactosylated glycan producing the ion at m/z 1809 in a. (e) Negative ion MS/MS spectrum of the bisected glycan producing the ion at m/z 2012 in Fig. 8a. 220 and the F-type ion at m/z 262 (GlcNAc+59). The D and [D-18] ions at m/z 526 and 508 show only GlcNAc in the 6-antenna. Fig. 7c shows the negative ion MS/MS spectrum of the mixture of monogalactosylated glycans (m/z 1647 in Fig. 7a). The additional galactose residue on one of the antennae produces two C 1 ions at m/z 179 (Gal) and m/z 220 (GlcNAc). The presence of two sets of D and [D-18] ions at m/z 526 and 508 and at m/z 688 and 670 and two F ions at m/z 262 and 424 are consistent with the galactose being attached to either antenna, in line with previous observations [60]. Fig. 7d shows the negative ion MS/MS spectrum of the digalactosylated glycan producing the ion at m/z 1809 in Fig. 7a. With two antennae of the same composition, only single C 1 (m/z 179), F (m/z 424), D (m/z 688), and [D-18] (m/z 670) ions are produced. Fig. 7e shows the negative ion MS/MS spectrum of the bisected biantennary glycan with the prominent [D-221] - ion at m/z 670, indicating the presence of the bisecting Glc- NAc residue. Single C 1 and F ions are present as in Spectrum 7d. N-Glycans released from human serum glycoproteins. Reaction of the released glycans with methanol in the presence of DMT-MM stabilized the sialic acids as methyl esters (a2?6-linked) or lactones (a2?3-linked), allowing them to be profiled by MALDI-TOF mass spectrometry (Fig. 8a). Sialic acids were then removed from the glycoproteins with A. ureafaciens sialidase and the sample was re-examined by MALDI-TOF MS (Fig. 8b) and negative ion nanospray MS/MS. Structures were assigned to all peaks from the neutral glycans (Table 3) and the results from the MALDI-TOF spectrum of the derivatized sialylated glycans allowed the amount of a2?3- and a2?6- linked sialic acid to be assigned to each. As an example of the spectral interpretation, Fig. 8c shows the negative ion MS/MS spectrum of a mixture of two fucosylated triantennary glycans as their phosphate adducts. One of these glycans is substituted with fucose on the reducing-terminal GlcNAc, whereas the other is substituted with fucose on a GlcNAc residue of the 3-antenna (as shown by the prominent E ion at m/z 977 and the F ion at m/z 570). Although the negative ion spectra did not allow the branch of the 3- antenna that contains the fucose to be determined, it is depicted on the 1-4-linked GlcNAc to be consistent with earlier reports on the known structure of the acute-phase serum glycoproteins, a1-acid glycoprotein [61] and a1-antitrypsin [62]. In this spectrum, the 2,4 A R fragment at m/z 1843 (loss of mass units) shows fucose at the 6- position of the reducing-terminal GlcNAc and the corresponding fragment at m/z 1989 (loss of units) shows the presence of the other isomer which is substituted with fucose on an antenna. The presence of a prominent E ion at m/z 977 (m/z 831 plus 146 from fucose) locates the fucose to the 3-antenna. The position of the fucose on an antenna was further reflected by the F-type ion at m/z 570 (m/z fucose) and its position on the GlcNAc residue was confirmed by the absence of a C 1 ion at m/z 325
12 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Fig. 8. Analysis of N-glycans from human serum. (a) Positive ion MALDI-TOF spectrum of total glycans following reaction with methanol in the presence of DMT-MM. Major compounds only are annotated; for a more detailed analysis, see the paper by Royle et al. submitted to this journal [36]. (b) Positive ion MALDI-TOF spectrum of total glycans following desialylation with Arthrobacter ureafaciens. (c) Negative ion MS/MS spectrum of the ion at m/z 2174 in spectrum b indicates the presence of two different fucosylated triantennary glycans. Spectral interpretation is described in the main text. (galactose + fucose). D and [D-18] ions were observed only at m/z 688 and 670, respectively, consistent with the absence of fucosylation on the 6-antenna and the structure of the triantennary glycan as having a branched 3-antenna. The ratio of the 2,4 A R ions (m/z 1843 and 1989) provides a rapid measurement of the relative amounts of each isomer in the mixture and, by extrapolation, to the amounts in the total glycan profile. The MALDI-TOF spectrum of the sialylated glycans showed that approximately half of the sialic acid in these fucosylated triantennary compounds was a2?3-linked (ion at m/z 2914), whereas in the corresponding biantennary glycan most sialic acid was a2?6-linked. The a2?3-linked sialic acid is consistent with the presence of the sialyl-lewis x epitope that is known to occur in a1-acid glycoprotein [63]. The methods described above have been in use for some time and have contributed to studies of N-glycans released from serum glycoproteins in several disease states such as cancer [64 66], arthritis [67], and pancreatitis. In several studies, the mass spectrometric techniques, particularly those based on negative ion fragmentation, enabled rapid structural identification of isomeric compounds in cases where classical methods proved laborious. Discussion Negative ion MS/MS provides a powerful method for structural analysis of neutral N-linked glycans because each structural feature gives rise to a specific fragment ion and, moreover, fragments are generally not produced by competing mechanisms as in the dissociation of positive ions. Cross-ring cleavages supplying specific structural information on branching and linkage are much more abundant than in the spectra of [M + Na] + and particularly [M + H] + ions. Also, rearrangement ions that can give rise to ambiguous information in positive ion spectra have not been detected. The approach that we use to obtain structural identification is first to obtain profiles of the glycan mixture by MALDI-TOF MS on desialylated and/or derivatized samples and to assign compositions from the measured masses with software developed in-house. Equivalent software is also available on the internet, e.g., at (requires password) and Structural features can then be assigned by negative ion MS/MS of the neutral (desialylated) glycans and the occurrence of sialylated forms of the glycans can be deduced using data from the MALDI experiments and the results of derivatization with DMT-MM.
13 56 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Table 3 Masses, compositions, and structures for neutral glycans obtained from human serum after sialic acid removal MALDI (m/z [M + Na] + ) ESI (m/z [M + H 2 PO 4 ] - ) Composition Structure Found Calc. Found Calc. Hex HexNAc dhex
14 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Table 3 (continued) MALDI (m/z [M + Na] + ) ESI (m/z [M + H 2 PO 4 ] - ) Composition Structure Found Calc. Found Calc. Hex HexNAc dhex (continued on next page)
15 58 N-glycan analysis by MALDI and ESI mass spectrometry / D.J. Harvey et al. / Anal. Biochem. 376 (2008) Table 3 (continued) MALDI (m/z [M + Na] + ) ESI (m/z [M + H 2 PO 4 ] - ) Composition Structure Found Calc. Found Calc. Hex HexNAc dhex As with all techniques, some structural features are not revealed. The most serious of these is the nature of the individual sugar residues. However, if the enzymology of the organism from which the glycans were obtained is known, the conserved nature of these N-linked glycans means that it is fairly safe to assume that these residues follow a consistent pattern. However, if there is any doubt, more traditional methods such as exoglycosidase digestion or combined gas-chromatography/mass spectrometry linkage analysis [68] can be used to supply the missing information. The products of exoglycosidase digestions can be monitored by MALDI-TOF MS (without derivatization) and their negative ion MS/MS spectra used to add increasing confidence to the structural assignments. The negative ion spectra of isomeric glycans contain easily detected massdifferent ions that allow structures to be assigned, thus avoiding the necessity for chromatographic separation. Nevertheless, for very complex mixtures and detection of minor isomers, chromatography is still advisable. Although the negative ion fragmentation method is compatible with the analysis of glycans derivatized at the reducing terminus with neutral derivatives such as 2-aminobenzamide used for chromatographic detection, the use of such derivatives is not necessary and, indeed, has a detrimental effect on the analysis. In particular, the 2,4 A R ions that are necessary for confirming the nature of the negative ion adduct and the presence or absence of fucose substitution on the reducing-terminal GlcNAc are missing as the result of the opened reducing-terminal ring that prevents the electron shifts of the type shown in Fig. 3. Also, the Nafion membrane clean-up procedure is inappropriate for these labeled glycans because of the tendency of the label to stick to the membrane. The use of negatively charged derivatives such as 2-aminobenzoic acid drastically reduces the information content of the spectra because of charge localization on the acid function of the derivative. Consequently, the method works best with underivatized glycans and we believe it to be among the most powerful yet for determining the structure of N-linked glycans. Acknowledgments This project was funded by the Oxford Glycobiology Institute Endowment. We thank the Wellcome Trust and the Biotechnology and Biological Sciences Research Council for equipment grants to purchase the Q-Tof mass and Tof- Spec MALDI-TOF mass spectrometers, respectively. We also thank the Department of Chemistry, Oxford for use of the Waters-Micromass MALDI micro mass spectrometer. References [1] S. Zamze, D.R. Wing, M.R. Wormald, A.P. Hunter, R.A. Dwek, D.J. Harvey, A family of novel, acidic N-glycans in Bowes melanoma tissue plasminogen activator have L2/HNK-1-bearing antennae, many with sulfation of the fucosylated chitobiose core, Eur. J. Biochem. 268 (2001) [2] C.W. Sutton, J.A. O Neill, J.S. Cottrell, Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry, Anal. Biochem. 218 (1994) [3] D.J. Harvey, P.M. Rudd, R.H. Bateman, R.S. Bordoli, K. Howes, J.B. Hoyes, R.G. Vickers, Examination of complex oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments, Org. Mass Spectrom. 29 (1994) [4] D.J. Harvey, Matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides and glycoconjugates, J. Chromatogr., A 720 (1996) [5] D.J. Harvey, Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates, Mass Spectrom. Rev. 18 (1999) [6] D.J. Harvey, Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates and glycoconjugates, Int. J. Mass Spectrom. 226 (2003) [7] D.J. Harvey, Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update covering the period , Mass Spectrom. Rev. 25 (2006) [8] D.J. Harvey, Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides, Rapid Commun. Mass Spectrom. 7 (1993) [9] T.J.P. Naven, D.J. Harvey, Effect of structure on the signal strength of oligosaccharides in matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments, Rapid Commun. Mass Spectrom. 10 (1996)
Certificate of Analysis
Certificate of Analysis Human IgG Glycoprotein Standard Cat. #: GCP-IGG-50U Batch: B13T-06 Nominal size: 50μg Expiry: Dec 2020 Description: A glycoprotein standard for use during glycan release and labeling.
More informationStructural Elucidation of N-glycans Originating From Ovarian Cancer Cells Using High-Vacuum MALDI Mass Spectrometry
PO-CON1347E Structural Elucidation of N-glycans Originating From Ovarian Cancer Cells Using High-Vacuum MALDI Mass Spectrometry ASMS 2013 TP-708 Matthew S. F. Choo 1,3 ; Roberto Castangia 2 ; Matthew E.
More informationFetuin Glycoprotein Standard
Certificate of Analysis Fetuin Glycoprotein tandard Cat. #: GCP-FET-U-X (GCP-FET-U B7K- *) Batch: BC- Nominal size: * μg Expiry Date: Apr Description: A glycoprotein standard for use during glycan release
More informationThe extensive literature on the mass spectrometry
Fragmentation of Negative Ions from Carbohydrates: Part 2. Fragmentation of High-Mannose N-Linked Glycans David J. Harvey Department of Biochemistry, Glycobiology Institute, University of Oxford, Oxford,
More informationDavid J. Harvey Department of Biochemistry, Glycobiology Institute, University of Oxford, Oxford, United Kingdom
Fragmentation of Negative Ions from Carbohydrates: Part 1. Use of Nitrate and Other Anionic Adducts for the Production of Negative Ion Electrospray Spectra from N-linked Carbohydrates David J. Harvey Department
More informationLudger Guide to Sialylation: II. Highly Sialylated Glycoproteins
Ludger Guide to Sialylation: II Highly Sialylated Glycoproteins Ludger has over 15 years experience providing products and services for the biopharmaceutical industry and in that time we have noticed that
More informationSupporting information
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2014 Supporting information Glycan Reductive Isotope-coded Amino Acid Labeling (GRIAL) for Mass Spectrometry-based
More informationCERTIFICATE OF ANALYSIS
CERTIFICATE OF ANALYSIS PRODUCT NAME: PRODUCT CODE: LOT NUMBER: PACK SIZE: PURITY: FORM: STORAGE: EXPIRATION: GLYKO ASIALO, GALACTOSYLATED, BIANTENNARY COMPLEX N-GLYCAN, CORE-SUBSTITUTED WITH FUCOSE (NA2F)
More informationTECHNICAL BULLETIN. R 2 GlcNAcβ1 4GlcNAcβ1 Asn
GlycoProfile II Enzymatic In-Solution N-Deglycosylation Kit Product Code PP0201 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description Glycosylation is one of the most common posttranslational
More informationIsomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High- Temperatures
Supplementary Information Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High- Temperatures Shiyue Zhou 1, Yifan Huang 1, Xue Dong 1, Wenjing Peng 1, Lucas Veillon
More informationRAPID SAMPLE PREPARATION METHODS FOR THE ANALYSIS OF N-LINKED GLYCANS
RAPID SAMPLE PREPARATION METHODS FOR THE ANALYSIS OF N-LINKED GLYCANS Zoltan Szabo, András Guttman, Tomas Rejtar and Barry L. Karger Barnett Institute, Boston, MA, USA PCT Workshop,Boston, 21 May, 2010.
More informationAnalysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS
Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS Ying Qing Yu Waters Corporation, Milford, MA, U.S. A P P L I C AT ION B E N E F I T
More informationLudgerPure TM APTS Labelled IgG Glycan Library
Certificate of Analysis LudgerPure TM APTS Labelled IgG Glycan Library Cat. #: CAPTS-IgG-0 Batch #. B-0 Size: approx. 0 pmol Description and: Source A mixture of APTS labelled fucosylated bi-antennary
More informationHigh fidelity glycan sequencing using a combination of capillary electrophoresis and exoglycosidase digestion
High fidelity glycan sequencing using a combination of capillary electrophoresis and exoglycosidase digestion Andras Guttman Senior Manager Applications, Separations SCIEX, Brea, CA 92822 The rapidly increasing
More informationGlycanPac AXR-1 Columns
CHRMATGRAPHY GlycanPac AXR- Columns For High Resolution Glycan Analysis Product Specifications The Thermo Scientific GlycanPac AXR- columns are highperformance, silica-based HPLC columns for simultaneous
More informationDr Mark Hilliard, NIBRT. Waters THE SCIENCE OF WHAT S POSSIBLE TM
RFMS Glycan Characterization Techniques for Biotherapeutics Dr Mark Hilliard, NIBRT Waters THE SCIENCE OF WHAT S POSSIBLE TM The Complexity of Glycosylation Glycosylation is the most common posttranslational
More informationAutomating Mass Spectrometry-Based Quantitative Glycomics using Tandem Mass Tag (TMT) Reagents with SimGlycan
PREMIER Biosoft Automating Mass Spectrometry-Based Quantitative Glycomics using Tandem Mass Tag (TMT) Reagents with SimGlycan Ne uaca2-3galb1-4glc NAcb1 6 Gal NAca -Thr 3 Ne uaca2-3galb1 Ningombam Sanjib
More informationApplying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis
Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis Ying Qing Yu Waters Corporation, Milford, MA, USA APPLICATION BENEFITS
More informationIsomer Separation of Positively Labeled N-glycans by CE-ESI-MS
Isomer Separation of Positively Labeled N-glycans by CE-ESI-MS G.S.M. Kammeijer Center for Proteomics and Metabolomics CE IN THE BIOTECHNOLOGY & PHARMACEUTICAL INDUSTRIES 19 TH SYMPOSIUM ON THE PRACTICAL
More informationApplication Note. Abstract. Author. Biotherapeutics & Biosimilars. Sonja Schneider Agilent Technologies, Inc. Waldbronn, Germany
Sensitive and Reproducible Glycan Analysis of Human Immunoglobulin G The Agilent 1260 Infi nity Bio-inert Quaternary LC System with an Agilent AdvanceBio 2.7 µm Glycan Mapping Column and Fluorescence Detection
More informationSupporting Information for MassyTools-assisted data analysis of total serum N-glycome changes associated with pregnancy
Supporting Information for MassyTools-assisted data analysis of total serum N-glycome changes associated with pregnancy Bas C. Jansen 1, Albert Bondt 1,2, Karli R. Reiding 1, Coen J. de Jong 1, David Falck
More informationBIOCHEMICAL DIAGNOSIS OF DISEASES CAUSING OLIGOSACCHARIDURIA BY MASS SPECTROMETRY
BIOCHEMICAL DIAGNOSIS OF DISEASES CAUSING OLIGOSACCHARIDURIA BY MASS SPECTROMETRY Pedro Ruiz-Sala, Isaac Ferrer-López Centro de Diagnóstico de Enfermedades Moleculares CEDEM, Universidad Autónoma de Madrid,
More informationElectronic Supplementary Information
Electronic Supplementary Information Microwave-assisted Kochetkov amination followed by permanent charge derivatization: A facile strategy for glycomics Xin Liu a,b, Guisen Zhang* a, Kenneth Chan b and
More information2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry
Dr. Sanjeeva Srivastava 1. Fundamental of Mass Spectrometry Role of MS and basic concepts 2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry 2 1 MS basic concepts Mass spectrometry - technique
More informationThank you for joining us! Our session will begin shortly Waters Corporation 1
UPLC and HPLC Separation Strategies for Successful Characterization of Glycans Derived from Therapeutic Proteins Thank you for joining us! Our session will begin shortly 2013 Waters Corporation 1 Friendly
More informationOligosaccharide Analysis by High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection
Oligosaccharide Analysis by High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection Jeff Rohrer, Ph.D. Director, Applications Development, Dionex Products 1 The world leader
More informationBarry Boyes 1,2, Shujuan Tao 2, and Ron Orlando 2
Barry Boyes 1,2, Shujuan Tao 2, and Ron Orlando 2 1 Advanced Materials Technology, Inc. Wilmington, DE USA 2 Complex Carbohydrate Research Center University of Georgia, Athens, GA USA bboyes@advanced-materials-tech.com
More informationMALDI-TOF. Introduction. Schematic and Theory of MALDI
MALDI-TOF Proteins and peptides have been characterized by high pressure liquid chromatography (HPLC) or SDS PAGE by generating peptide maps. These peptide maps have been used as fingerprints of protein
More informationComparison of mass spectrometers performances
Comparison of mass spectrometers performances Instrument Mass Mass Sensitivity resolution accuracy Quadrupole 1 x 10 3 0.1 Da* 0.5-1.0 pmol DE-MALDI 2 x 10 4 20 ppm 1-10 fmol peptide 1-5 pmol protein Ion
More informationSupporting information
Supporting information Figure legends Supplementary Table 1. Specific product ions obtained from fragmentation of lithium adducts in the positive ion mode comparing the different positional isomers of
More informationDevelopment of a Glycan Database for Waters ACQUITY UPLC Systems
Mark Hilliard, 1 Weston Struwe, 1 Barbara Adamczyk, 1 Radka Saldova, 1 Ying Qing Yu, 2 John O Rourke, 1 Giorgio Carta, 1 and Pauline Rudd 1 1 National Institute for Bioprocessing Research & Training (NIBRT),
More informationOligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA)
Oligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA) Elisabeth A. Kast and Elizabeth A. Higgins GlycoSolutions Corporation, Worcester, MA Data originally presented
More informationBiosynthesis of N and O Glycans
TechNote #TNGL101 Biosynthesis of N and O Glycans These suggestions and data are based on information we believe to be reliable. They are offered in good faith, but without guarantee, as conditions and
More informationSimGlycan. A high-throughput glycan and glycopeptide data analysis tool for LC-, MALDI-, ESI- Mass Spectrometry workflows.
PREMIER Biosoft SimGlycan A high-throughput glycan and glycopeptide data analysis tool for LC-, MALDI-, ESI- Mass Spectrometry workflows SimGlycan software processes and interprets the MS/MS and higher
More informationSmall Scale Preparative Isolation of Corticosteroid Degradation Products Using Mass-Based Fraction Collection Application
Small Scale Preparative Isolation of Corticosteroid Degradation Products Using Mass-Based Fraction Collection Application Pharmaceutical Author Cliff Woodward Agilent Technologies, Inc. 285 Centerville
More informationCHM 424L Organic Laboratory, Dr. Laurie S. Starkey Introduction to Mass Spectrometry
CM 424L rganic Laboratory, Dr. Laurie S. Starkey Introduction to Mass Spectrometry Mass spectrometry is used to determine a sample's molecular mass and molecular formula. Some structural information can
More informationN-Glycan Sequencing Kit
PROTEIN TOOLS N-Glycan Sequencing Kit Instruction Manual NEB #E577S 2 reactions Version 1. 1/18 be INSPIRED drive DISCOVERY stay GENUINE This product is intended for research purposes only. This product
More informationBiological Mass Spectrometry. April 30, 2014
Biological Mass Spectrometry April 30, 2014 Mass Spectrometry Has become the method of choice for precise protein and nucleic acid mass determination in a very wide mass range peptide and nucleotide sequencing
More informationComparison of Relative Quantification of Monoclonal Antibody N-glycans Using Fluorescence and MS Detection
Comparison of Relative Quantification of Monoclonal ntibody N-glycans Using Fluorescence and MS Detection pplication Note iotherapeutics & iologics uthors scar Potter and Gregory Staples gilent Technologies,
More informationBiomolecular Mass Spectrometry
Lipids ot different than other organic small molecules Carbohydrates Polymers of monosaccharides linked via glycosidic bonds (acetals/ ketals) many different combinationsvery interesting no time ucleic
More informationA Fully Integrated Workflow for LC-MS/MS Analysis of Labeled and Native N-Linked Glycans Released From Proteins
A Fully Integrated Workflow for LC-MS/MS Analysis of Labeled and Native N-Linked Glycans Released From Proteins Udayanath Aich, 1 Julian Saba, 2 Xiaodong Liu, 1 Srinivasa Rao, 1 Yury Agroskin, 1 and Chris
More informationDetailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit
be INSPIRED drive DISCOVERY stay GENUINE APPLICATION NOTE Detailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit Beth McLeod, New England Biolabs, Inc. Materials Remicade
More informationLecture 3. Tandem MS & Protein Sequencing
Lecture 3 Tandem MS & Protein Sequencing Nancy Allbritton, M.D., Ph.D. Department of Physiology & Biophysics 824-9137 (office) nlallbri@uci.edu Office- Rm D349 Medical Science D Bldg. Tandem MS Steps:
More informationGlycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren
Glycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren 2012 Waters Corporation 1 Requests and Questions Optimised sample prep protocol to reduce sample preparation time How can I detect
More informationMass Spectrometry. Actual Instrumentation
Mass Spectrometry Actual Instrumentation August 2017 See also http://www.uni-bielefeld.de/chemie/analytik/ms f additional infmation 1. MALDI TOF MASS SPECTROMETRY ON THE ULTRAFLEX 2 2. ESI MASS SPECTROMETRY
More informationSupporting Information
Supporting Information Wiley-VC 2007 69451 Weinheim, Germany Electrospray Ionization Mass Spectrometric Study on the irect rganocatalytic α-alogenation of Aldehydes Cesar A. Marquez, Francesco Fabbretti
More informationGlycoFragment and GlycoSearchMS: web tools to support the interpretation of mass spectra of complex carbohydrates
GlycoFragment and GlycoSearchMS: web tools to support the interpretation of mass spectra of complex carbohydrates Klaus Karl Lohmann* and Claus-W. von der Lieth Nucleic Acids Research, 2004, Vol. 32, Web
More informationMeasuring Lipid Composition LC-MS/MS
Project: Measuring Lipid Composition LC-MS/MS Verification of expected lipid composition in nanomedical controlled release systems by liquid chromatography tandem mass spectrometry AUTHORED BY: DATE: Sven
More informationOligosaccharides, as glycoconjugates, are widely
ARTICLES Targeted Use of Exoglycosidase Digestion for the Structural Elucidation of Neutral O-Linked Oligosaccharides Yongming Xie, Ken Tseng, Carlito B. Lebrilla Department of Chemistry, University of
More informationMethods in Mass Spectrometry. Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University
Methods in Mass Spectrometry Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University Sample Engineering Chemistry Biology Life Science Medicine Industry IDF / Police Sample
More informationMass Spectrometry. - Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications
- Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications Adapted from Mass Spectrometry in Biotechnology Gary Siuzdak,, Academic Press 1996 1 Introduction
More information[ APPLICATION NOTE ] Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY Arc System and ACQUITY QDa Detector
Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY Arc System and ACQUITY QDa Detector Mark Benvenuti, Gareth Cleland, and Jennifer Burgess Waters Corporation, Milford, MA, USA
More informationPhosphorylation of proteins Steve Barnes Feb 19th, 2002 in some cases, proteins are found in a stable, hyperphosphorylated state, e.g.
Phosphorylation of proteins Steve Barnes Feb 19th, 2002 in some cases, proteins are found in a stable, hyperphosphorylated state, e.g., casein more interestingly, in most other cases, it is a transient
More informationRapid, sensitive structure analysis of oligosaccharides
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1629 1633, March 1997 Chemistry Rapid, sensitive structure analysis of oligosaccharides (carbohydrate mass spectrometry structure analysis sequencing) YINGMING ZHAO*,
More informationQuadrupole and Ion Trap Mass Analysers and an introduction to Resolution
Quadrupole and Ion Trap Mass Analysers and an introduction to Resolution A simple definition of a Mass Spectrometer A Mass Spectrometer is an analytical instrument that can separate charged molecules according
More informationProduct Guide for LudgerSep TM C3 anion exchange HPLC Column for Glycan Analysis
Product Guide for LudgerSep TM C3 anion exchange HPLC Column for Glycan Analysis (Ludger Product Code: LS-C3-7.5x75) Ludger Document # LS-C3-Guide-v3.0 Ludger Ltd Culham Science Centre Oxford OX14 3EB
More informationProduct Guide for LudgerSep TM R1 HPLC Column for Glycan Analysis
Product Guide for LudgerSep TM R1 HPLC Column for Glycan Analysis (Ludger Product Code: LS-R1-4.6x150) Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom Tel: +44 1865 408 554 Fax: +44 870
More informationCurrent Glycoprotein Analysis. Glycan Characterization: Oligosaccharides. Glycan Analysis: Sample Preparation. Glycan Analysis: Chromatography
Bio Day DENMARK MARCH 2013 Analysis of N-linked Glycans of GlycoProteins marleen_van_wingerden@waters.com Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides
More informationAdvantages of Ion Mobility Q-TOF for Characterization of Diverse Biological Molecules
Advantages of Ion Mobility Q-TOF for Characterization of Diverse Biological Molecules Add a New Dimension to your Research Capability with Agilent s New Drift Ion Mobility Q-TOF System Overview: 6560 IM
More information189,311, , ,561, ,639, ,679, Ch13; , Carbohydrates
Lecture 31 (12/8/17) Reading: Ch7; 258-267 Ch10; 371-373 Problems: Ch7 (text); 26,27,28 Ch7 (study-guide: applying); 2,5 Ch7 (study-guide: facts); 6 NEXT (LAST!) Reading: Chs4,6,8,10,14,16,17,18; 128-129,
More informationSupporting Information Parsimonious Charge Deconvolution for Native Mass Spectrometry
Supporting Information Parsimonious Charge Deconvolution for Native Mass Spectrometry Marshall Bern* 1, Tomislav Caval 2, Yong J. Kil 1, Wilfred Tang 1, Christopher Becker 1, Eric Carlson 1, Doron Kletter
More informationIonization Methods. Neutral species Charged species. Removal/addition of electron(s) Removal/addition of proton(s)
Ionization Methods Neutral species Charged species Removal/addition of electron(s) M + e - (M +. )* + 2e - electron ionization Removal/addition of proton(s) M + (Matrix)-H MH + + (Matrix) - chemical ionization
More informationProtein Analysis using Electrospray Ionization Mass Spectroscopy *
OpenStax-CNX module: m38341 1 Protein Analysis using Electrospray Ionization Mass Spectroscopy * Wilhelm Kienast Andrew R. Barron This work is produced by OpenStax-CNX and licensed under the Creative Commons
More informationProduct Guide for LudgerSep TM R1 HPLC Column for DMB labelled Sialic Acid Analysis
Product Guide for LudgerSep TM R1 HPLC Column for DMB labelled Sialic Acid Analysis Product # LS-R1-4.6x150 Ludger Document # LS-R1-DMB-Guide-v5.1 Ludger Ltd Culham Science Centre Oxford OX14 3EB United
More informationProduct Guide for LudgerSep TM ur2 UHPLC Column for DMB Sialic Acid Analysis
Product Guide for LudgerSep TM ur2 UHPLC Column for DMB Sialic Acid Analysis Product # LS-UR2-2.1x100 Ludger Document # LS-uR2-DMB-Guide-v2.1 Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom
More informationFundamentals of Soft Ionization and MS Instrumentation
Fundamentals of Soft Ionization and MS Instrumentation Ana Varela Coelho varela@itqb.unl.pt Mass Spectrometry Lab Analytical Services Unit Index Mass spectrometers and its components Ionization methods:
More informationEnvelope glycans of immunodeficiency virions are almost entirely oligomannose antigens
Supporting Information for: Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens Katie J. Doores *1,2, Camille Bonomelli *3, David J. Harvey 3, Snezana Vasiljevic 3, Raymond
More informationMass Spectrometry Introduction
Mass Spectrometry Introduction Chem 744 Spring 2013 What MS is and is not MS is NOT a spectroscopic method. Molecules are not absorbing EM radiation MS is the generation, separation and characterization
More informationBiochemistry: A Short Course
Tymoczko Berg Stryer Biochemistry: A Short Course Second Edition CHAPTER 10 Carbohydrates 2013 W. H. Freeman and Company Chapter 10 Outline Monosaccharides are aldehydes or ketones that contain two or
More informationPeptide sequencing using chemically assisted fragmentation (CAF) and Ettan MALDI-ToF Pro mass spectrometry
application note Ett MALDI-ToF Pro Peptide sequencing using chemically assisted fragmentation (CAF) d Ett MALDI-ToF Pro mass spectrometry Key words: MALDI-ToF, PSD, peptide sequencing, chemically assisted
More informationOzonolysis of phospholipid double bonds during electrospray. ionization: a new tool for structure determination
Ozonolysis of phospholipid double bonds during electrospray ionization: a new tool for structure determination Michael C. Thomas, Todd W. Mitchell, Stephen J. Blanksby Departments of Chemistry and Biomedical
More informationFOCUS: MASS SPECTROMETRY IN GLYCOBIOLOGY AND RELATED FIELDS: RESEARCH ARTICLE
B The Author(s), 2018. This article is an open access publication J. Am. Soc. Mass Spectrom. (2018) 29:1179Y1193 DOI: 10.1007/s13361-018-1950-x FOCUS: MASS SPECTROMETRY IN GLYCOBIOLOGY AND RELATED FIELDS:
More informationIon fragmentation of small molecules in mass spectrometry
Ion fragmentation of small molecules in mass spectrometry Jeevan Prasain jprasain@uab.edu 6-2612 Nomenclature: the main names and acronyms used in mass spectrometry Molecular ion: Ion formed by addition
More informationStructural analyses of carbohydrates and oligosaccharides
A Comparative Study of the Fragmentation of Neutral Lactooligosaccharides in Negative- Ion Mode by UV-MALDI-TOF and UV- MALDI Ion-Trap/TOF Mass Spectrometry Tohru Yamagaki, Hiroaki Suzuki, and Kazuo Tachibana
More informationCovalent attachment of carbohydrates to proteins
The Q-Trap Mass Spectrometer, a Novel Tool in the Study of Protein Glycosylation K. Sandra, B. Devreese, and J. Van Beeumen Laboratory of Protein Biochemistry and Protein Engineering, Ghent University,
More informationIt is estimated that up to 50% of all proteins are. A Glycomics Platform for the Analysis of Permethylated Oligosaccharide Alditols
A Glycomics Platform for the Analysis of Permethylated Oligosaccharide Alditols Catherine E. Costello, a Joy May Contado-Miller, a and John F. Cipollo a,b a Mass Spectrometry Resource, Department of Biochemistry,
More informationSeparation of 15 Underivatized Saccharide and Sialic Acid USP Standards
Application Note Raw Material & Media Analysis Separation of Underivatized Saccharide and Sialic Acid USP Standards Using an Agilent AdvanceBio MS Spent Media Column with TOF MS Detection Author Richard
More informationCharacterization of Noncovalent Complexes of Polyelectrolytes by Mass Spectrometry
The University of Akron IdeaExchange@UAkron Honors Research Projects The Dr. Gary B. and Pamela S. Williams Honors College Spring 2015 Characterization of Noncovalent Complexes of Polyelectrolytes by Mass
More information[ Care and Use Manual ]
MALDI Calibration Kit I. Introduction The MALDI Calibration Kit is a conveniently packaged selection of MALDI matrices and calibration standards (includes high-purity Neg Ion Mode Calibrant for accurate
More informationMass Spectrometry. Mass spectrometer MALDI-TOF ESI/MS/MS. Basic components. Ionization source Mass analyzer Detector
Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic components Ionization source Mass analyzer Detector 1 Principles of Mass Spectrometry Proteins are separated by mass to charge ratio (limit
More informationTechnical Note # TN-31 Redefining MALDI-TOF/TOF Performance
Bruker Daltonics Technical Note # TN-31 Redefining MALDI-TOF/TOF Performance The new ultraflextreme exceeds all current expectations of MALDI-TOF/TOF technology: A proprietary khz smartbeam-ii TM MALDI
More informationN-Glycosidase F Deglycosylation Kit
For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. N-Glycosidase F Deglycosylation Kit Kit for the deglycosylation of asparagine-linked glycan chains on glycoproteins.
More informationMASS SPECTROMETRY OF OLIGOSACCHARIDES
Joseph Zaia* Department of Biochemistry, Boston University School of Medicine, 715 Albany St., R-806, Boston, Massachusetts 02118 Received 20 March 2003; received (revised) 6 August 2003; accepted 6 August
More informationSUPPORTING INFORMATION. High Throughput Reaction Screening using Desorption Electrospray Ionization Mass Spectrometry
Electronic Supplementary Material (ESI) for Chemical Science. This journal is The Royal Society of Chemistry 2018 SUPPORTING INFORMATION High Throughput Reaction Screening using Desorption Electrospray
More informationMass Spectrometry at the Laboratory of Food Chemistry. Edwin Bakx Laboratory of Food Chemistry Wageningen University
Mass Spectrometry at the Wageningen University Mass Spectrometry at the 3 UPLC/CE - ESI - Ion trap MS systems UPLC Thermo Acella with a Velos or VelosPro CE Beckman PA800 with a Thermo VelosPro 1 UPLC-
More information1. Sample Introduction to MS Systems:
MS Overview: 9.10.08 1. Sample Introduction to MS Systems:...2 1.1. Chromatography Interfaces:...3 1.2. Electron impact: Used mainly in Protein MS hard ionization source...4 1.3. Electrospray Ioniztion:
More informationTandem mass spectrometry is becoming the
Fragmentation of Phosphopeptides in an Ion Trap Mass Spectrometer Jon P. DeGnore and Jun Qin Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health,
More informationUltra Performance Liquid Chromatography Coupled to Orthogonal Quadrupole TOF MS(MS) for Metabolite Identification
22 SEPARATION SCIENCE REDEFINED MAY 2005 Ultra Performance Liquid Chromatography Coupled to Orthogonal Quadrupole TOF MS(MS) for Metabolite Identification In the drug discovery process the detection and
More informationDienes Derivatization MaxSpec Kit
Dienes Derivatization MaxSpec Kit Item No. 601510 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION
More informationEssential Lipidomics Experiments using the LTQ Orbitrap Hybrid Mass Spectrometer
Application Note: 367 Essential Lipidomics Experiments using the LTQ rbitrap Hybrid Mass Spectrometer Thomas Moehring 1, Michaela Scigelova 2, Christer S. Ejsing 3, Dominik Schwudke 3, Andrej Shevchenko
More informationHCV infects over 170 million people worldwide.
Mass Spectrometric Characterization of Glycosylation of Hepatitis C Virus E2 Envelope Glycoprotein Reveals Extended Microheterogeneity of N-Glycans Roxana E. Iacob, a Irina Perdivara, a,b Michael Przybylski,
More informationNature Biotechnology: doi: /nbt Supplementary Figure 1. RNAseq expression profiling of selected glycosyltransferase genes in CHO.
Supplementary Figure 1 RNAseq expression profiling of selected glycosyltransferase genes in CHO. RNAseq analysis was performed on two common CHO lines (CHO-K1, CHO-GS) and two independent CHO-GS triple
More informationREDOX PROTEOMICS. Roman Zubarev.
REDOX PROTEOMICS Roman Zubarev Roman.Zubarev@ki.se Physiological Chemistry I, Department for Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm What is (RedOx) Proteomics? Proteomics -
More informationThe effect of temperature and incubation time on the analysis of highly sialylated glycans from bovine fetuin
APPLICATION NOTE GlycanAssure Glycan Analysis and Quantitation System The effect of temperature and incubation time on the analysis of highly sialylated glycans from bovine fetuin Abstract This application
More informationThe Automation of Glycopeptide Discovery in High Throughput MS/MS Data
The Automation of Glycopeptide Discovery in High Throughput MS/MS Data Sajani Swamy A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Master of
More informationCharacterization of the Oligosaccharides Associated with the Human Ovarian Tumor Marker CA125*
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 31, Issue of August 1, pp. 28619 28634, 2003 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Characterization
More informationO O H. Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION EXPERIMENTAL. LC /MS conditions
Simplifying Qual/Quan Analysis in Discovery DMPK using UPLC and Xevo TQ MS Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION The determination of the drug metabolism
More informationGlycosylation is a predominant post-translational modification
B American Society for Mass Spectrometry, 2012 J. Am. Soc. Mass Spectrom. (2012) 23:1408Y1418 DOI: 10.1007/s13361-012-0420-0 RESEARCH ARTICLE Linkage and Branch Analysis of High-Mannose Oligosaccharides
More informationCAMAG TLC-MS INTERFACE
CAMAG TLC-MS INTERFACE 93.1 249.2 40 30 97.1 20 10 250.2 0 200 400 m/z WORLD LEADER IN PLANAR-CHROMATOGRAPHY Identification and elucidation of unknown substances by hyphenation of TLC / HPTLC and MS The
More informationBiological Mass spectrometry in Protein Chemistry
Biological Mass spectrometry in Protein Chemistry Tuula Nyman Institute of Biotechnology tuula.nyman@helsinki.fi MASS SPECTROMETRY is an analytical technique that identifies the chemical composition of
More information