Direct comparison of Au<Stack><Subscript>3</Subscript><Superscript>+</Superscript></Stack> and C<Stack><Subscript>60</Subscript><Superscript>+</Superscript></Stack> cluster projectiles in SIMS molecular depth profiling

The sputtering properties of two representative cluster ion beams in secondary ion mass spectrometry (SIMS), C(60)(+) and Au(3)(+), have been directly compared. Organic thin films consisting of trehalose and dipalmitoylphosphatidylcholine (DPPC) are employed as prototypical targets. The strategy is to make direct comparison of the response of a molecular solid to each type of the bombarding cluster by overlapping the two ion beams onto the same area of the sample surface. The ion beams alternately erode the sample while keeping the same projectile for spectral acquisition. The results from these experiments are important to further optimize the use of cluster projectiles for SIMS molecular depth profiling experiments. For example, Au(3)(+) bombardment is found to induce more chemical damage as well as Au implantation when compared with C(60)(+). Moreover, C(60)(+) is found to be able to remove the damage and the implanted Au effectively. Discussions are also presented on strategies of enhancing sensitivity for imaging applications with cluster SIMS.     

MALDI‐MS profiling of serum O‐glycosylation and N‐glycosylation in COG5‐CDG

Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)‐CDG are genetic diseases due to defects of the COG complex subunits 1–8 causing N‐glycan and O‐glycan processing abnormalities. In COG‐CDG, isoelectric focusing separation of undersialylated glycoforms of serum transferrin and apolipoprotein C‐III (apoC‐III) allows to detect N‐glycosylation and O‐glycosylation defects, respectively. COG5‐CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix‐assisted laser desorption/ionization‐MS for a high‐throughput screening of differential serum O‐glycoform and N‐ glycoform in five patients with COG5‐CDG. When compared with age‐matched controls, COG5‐CDG showed a significant increase of apoC‐III_0a (aglycosylated glycoform), whereas apoC‐III₁ (mono‐sialylated glycoform) decreased significantly. Serum N‐glycome of COG5‐CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high‐mannose structures and hybrid glycans. Using advanced and well‐established MS‐based approaches, the present findings reveal novel aspects on O‐glycan and N‐glycan profiling in COG5‐CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis. Copyright © 2017 John Wiley & Sons, Ltd.     

Mass spectrometry in the detection and diagnosis of congenital disorders of glycosylation

Mass spectrometry (MS) of large molecules such as proteins and oligosaccharides has not been employed in clinical practices, while that of small metabolites is widely used for the screening and diagnosis of various congenital diseases. Congenital disorders of glycosylation (CDG) is a newly recognized group of diseases derived from defects in the biosynthetic pathway of protein glycosylation and the patients are never decisively diagnosed unless the glycoprotein molecules are analyzed. We have constructed a diagnostic system where MS of glycoproteins and glycopeptides identifies abnormalities in their glycan moieties. This program is anticipated to reveal the prevalence of CDG and to demonstrate the essential role of MS in the emerging field of medicine, disease glycomics and glycoproteomics.     

Ion Mobility Mass Spectrometry for Extracting Spectra <Emphasis Type="Italic">of N</Emphasis>-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact,Folded HIV gp120

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.     

Structural analysis of oligosaccharides by atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry.

An ion source incorporating a fibre optic interface has been constructed for atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry. The configuration has been applied to the study of linear and complex oligosaccharides. Multi-stage tandem mass spectrometry (MSn, n = 2-4) experiments carried out in the ion trap enable extended fragmentation pathways to be investigated that yield structural information. Collisional activation of sodiated oligosaccharides, as demonstrated on the model compound maltoheptaose, produces primarily B and Y fragments resulting from cleavage of glycosidic bonds; fragments from cross-ring cleavages are also observed following further stages of tandem mass spectrometry, providing additional linkage information. The analyses of mixtures of complex oligosaccharides are demonstrated for N-linked glycans from chicken egg glycoproteins and a ribonuclease glycan mixture. Mass spectrometric and tandem mass spectrometric data for sugars with molecular weights up to 4000 Da is shown for mixtures of linear dextrans and N-linked glycans. The use of MSn (n = 3, 4) on these complex molecules enabled structural information to be elucidated that confirms data observed in the MS/MS spectra.     

Sensitive LC MS quantitative analysis of carbohydrates by Cs<Superscript>+</Superscript> attachment

The development of a sensitive assay for the quantitative analysis of carbohydrates from human plasma using LC/MS/MS is described in this paper. After sample preparation, carbohydrates were cationized by Cs(+) after their separation by normal phase liquid chromatography on an amino based column. Cesium is capable of forming a quasi-molecular ion [M + Cs](+) with neutral carbohydrate molecules in the positive ion mode of electrospray ionization mass spectrometry. The mass spectrometer was operated in multiple reaction monitoring mode, and transitions [M + 133] --> 133 were monitored (M, carbohydrate molecular weight). The new method is robust, highly sensitive, rapid, and does not require postcolumn addition or derivatization. It is useful in clinical research for measurement of carbohydrate molecules by isotope dilution assay.     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct analysis in real time (DART) mass spectrometry of nucleotides and nucleosides: elucidation of a novel fragment [C<Subscript>5</Subscript>H<Subscript>5</Subscript>O]<Superscript>+</Superscript> and its in-source adducts

Direct analysis in real time (DART) mass spectrometry is a recently developed innovative technology, which has shown broad applications for fast and convenient analysis of complex samples. Due to the ease of sample preparation, we have recently initiated an investigation of the feasibility of detecting nucleotides and nucleosides using the DART-AccuTOF instrument, which we will refer to as the DART mass spectrometer. Our experimental results reveal that the ions representing the intact molecules of nucleotides are not detectable in either positive-ion or negative-ion mode. Instead, all four natural nucleotides fragment in the DART ion source, and a common fragment ion, [C₅H₅O]⁺ (1), is observed, which is probably formed via multiple-elimination reactions. Interestingly, 1 can form adducts with nucleobases in different molar ratios in the DART ion source. In contrast to nucleotides, the ions representing the intact molecules of nucleosides are detected in both positive-ion and negative-ion mode using DART mass spectrometry. Surprisingly, the fragmentation pattern of nucleosides is different from that of nucleotides in the DART ion source. In the cases of nucleosides (under positive-ion conditions), the production of 1 is not observed, indicating that the phosphate group plays an important role for the multiple eliminations observed in the spectra of nucleotides. The in-source reactions described in the present work show the complexity of the conditions in the DART ion source, and we hope that our results illustrate a better understanding about DART mass spectrometry.     

Matrix-assisted laser desorption/ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of <Emphasis Type="Italic">N</Emphasis>-linked oligosaccharides from ovalbumin

N-linked oligosaccharides were released from hen ovalbumin by PNGase F and derivatized with phenylhydrazine. They were then examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Phenylhydrazones of N-glycans under MALDI-tandem mass spectrometry (MS/MS) and post-source decay (PSD) conditions produced relatively similar fragmentation patterns; however, more cross-ring cleavages and fragment ions corresponding to low abundance isomeric structures were detected by MS/MS and not in PSD. Most fragment ions corresponded to glycosidic cleavages with preferential loss of residues from the chitobiose core and the 3-antenna. Sialylated phenylhydrazone-N-glycans, characterized here for the first time in ovalbumin by tandem mass spectrometry, underwent losses of sialic acid residues followed the same fragmentation pathways observed with neutral derivatized glycans. The relative abundances of some fragment ions indicated the linkage position of sialic acid and provided information on the number of residues attached to the 6-antenna. Also, new structures of ovalbumin glycans were observed as part of this study and are reported here.     

Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MS<Superscript>n</Superscript>.

Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were separated and detected by negative ion LC-MS and LC-MS(2). The resolution of the chromatography and the information obtained from collision induced dissociation of detected [M - H](-) ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nonasaccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Z(i) and Y(i)), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAcbeta1-3GalNAcol or Gal/GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and A(i) type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution ((0,2)A(i) - H(2)O cleavage) and disubstitution of C-3 and C-4 (Z(i)/Z(i) cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Galbeta1-3/4GlcNAcbeta1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, negative ion LC-MS(2) was able to generate extensive data for understanding the overall glycosylation pattern of a sample, especially when only a limited amount of material is available.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by <Superscript>13</Superscript>C-/<Superscript>12</Superscript>C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by ¹³C-dansyl and ¹²C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.     

Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans

Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n = 2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N-acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1 Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1-type cross-ring cleavage (83.1 Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6-type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6-type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary.     

The role of mobile protons in negative ion CID of oligosaccharides

Carbohydrates of all classes consist of glycoform mixtures built on common core units. Determination of compositions and structures of such mixtures relies heavily on tandem mass spectrometric data. Analysis of native glycans is often necessary for samples available in very low quantities and for sulfated glycan classes. Negative tandem mass spectrometry (MS) provides useful product ion profiles for neutral oligosaccharides and is preferred for acidic classes. In previous work from this laboratory, site-specific influences of sialylation on product ion profiles in the negative mode were elucidated. The present results show how the interplay of two other acidic groups, uronic acids and sulfates, determines product ion patterns for chondroitin sulfate oligosaccharides. Unsulfated chondroitin oligosaccharides dissociate to form C-type ions almost exclusively. Chondroitin sulfate oligosaccharides produce abundant B- and Y-type ions from glycosidic bond cleavage with C- and Z-types in low abundances. These observations are explained in terms of competing proton transfer reactions that occur during the collisional heating process. Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy.     

Identification of<Emphasis Type="Italic"> N</Emphasis>-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform–ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.Abbreviations amu atomic mass units - AspN endoproteinase AspN - CAM cell adhesion molecule - ESI electrospray ionization - FTICR Fourier transform–ion cyclotron resonance - IgSF immunoglobulin superfamily - MALDI-TOF matrix-assisted laser desorption ionization–time of flight - MS mass spectrometry - NCAM neural cell adhesion molecule - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase - PSA polysialic acid - TFA trifluoroacetic acid     

The influence of sialylation on glycan negative ion dissociation and energetics

For the analysis of native glycans using tandem mass spectrometry (MS), it is desirable to choose conditions whereby abundances of cross-ring cleavages indicative of branch positions are maximized. Recently, negative ion tandem mass spectrometry has been shown to produce significantly higher abundances of such ions in glycans compared to the positive ion mode. Much of this prior work has concerned fragmentation patterns in asialo glycans. The present work compares the abundances of critical cross-ring cleavage ions using negative mode tandem mass spectrometry for milk oligosaccharides and N-linked glycans. For comparison, product ion formation was studied for deprotonated and nitrated ions formed from asialo glycans and deprotonated ions from sialylated glycans. Breakdown profiles demonstrate clearly that more energy was required to fragment sialylated compounds to the same extent as either their asialo or nitrate adducted counterparts. The extraction of a proton from a ring hydroxyl group during the ionization process may be viewed, qualitatively, as imparting significantly more energy to the ion than would that from a molecule bearing an acidic group, so that acidic glycans are more stable in the gas phase, as the negative charge resides on the carboxyl group. These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated.     

Global and site-specific detection of human integrin alpha 5 beta 1 glycosylation using tandem mass spectrometry and the StrOligo algorithm

Glycans are oligosaccharides associated with proteins, and are known to confer specific functions and conformations on glycoproteins. As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics. The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions. Glycomics studies most often aim at identifying glycosylation sites, and thus are performed on deglycosylated proteins resulting in loss of site-specific details concerning the glycosylation. In order to obtain such details by mass spectrometry (MS), either whole glycoproteins must be digested and analyzed as mixtures of peptides and glycopeptides, or glycans must be isolated from glycopeptide fractions and analyzed as pools. This article describes parallel experiments involving both approaches, designed to take advantage of the StrOligo algorithm functionalities with the aim of characterizing glycosylation microheterogeneity on a specific site. A hybrid quadrupole-quadrupole-time-of-flight (QqTOF) instrument equipped with a matrix-assisted laser desorption/ionization (MALDI) source was used. Glycosylation of alpha 5 beta 1 subunits of human integrin was studied to test the methodology. The sample was divided in two aliquots, and glycans from the first aliquot were released enzymatically, labelled with 2-aminobenzamide, and identified using tandem mass spectrometry (MS/MS) and the StrOligo program. The other aliquot was digested with trypsin and the resulting peptides separated by reversed-phase high-performance liquid chromatography (HPLC). A specific collected fraction was then analyzed by MS before and after glycan release. These spectra allowed, by comparison, detection of a glycopeptide (several glycoforms) and elucidation of peptide sequence. Compositions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo.     

Evidence for linkage position determination in known feruloylated mono- and disaccharides using electrospray ion trap mass spectrometry

Various feruloylated arabinose- and galactose-containing mono- and disaccharides with known linkage configurations (2-O-(trans-feruloyl)-L-arabinopyranose, 5-O-(trans-feruloyl)-L-arabinofuranose, O-[2-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->5)-L-arabinofuranose, and O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose) were analyzed by electrospray ionization mass spectrometry using an ion trap or a quadrupole time-of-flight (Q-TOF) mass analyzer. Collision-induced dissociation (CID) experiments using the two mass analyzers generated similar tandem mass spectrometric (MS/MS) fragmentation patterns. However, the ester-bond cleavage ions were more abundant using the Q-TOF mass analyzer. Compared with the positive ion mode, the negative ion mode produces simpler and more useful CID product-ion patterns. For arabinose-containing feruloylated compounds, results obtained with both analyzers show that it is possible to assign the location of the feruloyl group to the O-2 or O-5 of arabinosyl residues. In the characterization of the 2-O-feruloyl and 5-O-feruloyl linkages, the relative abundance of the cross-ring fragment ions at m/z 265 (-60 u or -62 u after 18O-labelling) and at m/z 217 (-108 u or -110 u after 18O-labelling) play a relevant role. For galactose-containing feruloylated compounds, losses of 60, 90 and 120 Da observed in MS3 experiment correspond to the production of 0,2A1, 0,3A1 and (0,2A1-60 Da) cross-ring cleavage ions, respectively, fixing the location of feruloyl group at the O-6 of the galactose residue.     

Hydrophobic Derivatization of <Emphasis Type="Italic">N</Emphasis>-linked Glycans for Increased Ion Abundance in Electrospray Ionization Mass Spectrometry

A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in order to increase the ion abundance of N-linked glycans in electrospray ionization mass spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion abundance, a property that can be further exploited in the analysis of glycans. The results of this study will direct the future synthesis of hydrophobic reagents for glycan analysis using the correlation between hydrophobicity and theoretical non-polar surface area calculation to facilitate the development of an optimum tag for glycan derivatization. The compatibility and advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly more abundant, and the detection of negatively charged sialylated glycans is enhanced.     

LC-MS<Superscript><Emphasis Type="BoldItalic">n</Emphasis></Superscript> Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS ⁿ . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS ⁿ fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS ⁿ experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS ⁿ methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.