MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis of N-Linked Glycans

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.     

Travelling‐wave ion mobility and negative ion fragmentation of high‐mannose N‐glycans

The isomeric structure of high‐mannose N‐glycans can significantly impact biological recognition events. Here, the utility of travelling‐wave ion mobility mass spectrometry for isomer separation of high‐mannose N‐glycans is investigated. Negative ion fragmentation using collision‐induced dissociation gave more informative spectra than positive ion spectra with mass‐different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling‐wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N‐glycans released from the well‐characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross‐sectional data, details of the negative ion collision‐induced dissociation spectra of all resolved isomers are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Travelling‐wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N‐glycans

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1‐acid glycoprotein and thyroglobulin were measured with a travelling‐wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man₃GlcNAc₃ from chicken ovalbumin and Man₃GlcNAc₃Fuc₁ from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision‐induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high‐mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high‐mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility‐m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of Minor N-linked Glycans on Antibodies Using Endo H Release and MALDI–Mass Spectrometry

Abstract

A MALDI mass spectrometry method using Bruker Daltonic's LIFT technology for MS/MS analysis has been developed for profiling and characterizing low abundant N-glycans from recombinant immunoglobulin G (IgG) antibodies. In this method, Endoglycosidase H (Endo H) released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2-AB) and separated by normal phase chromatography. Endo H hydrolyses the bond between the two GlcNAc residues of the trimannosyl core of high mannose and hybrid N-linked glycans, leaving the core GlcNAc attached to the protein. High mannose and hybrid type N-glycans are released from the glycoprotein whereas the more abundant, complex biantennary type oligosaccharide structures are unaffected. Analysis of Endo H treated glycan moieties by MALDI mass spectrometry identified several minor species of high mannose and hybrid type glycans. Subsequent MALDI TOF MS/MS analysis of the resulting products yielded information about structural features of the high mannose and hybrid type glycans. This study involving Endo H treatment followed by MALDI mass spectrometry coupled with LIFT technology for MS/MS analysis offers a specific and sensitive technique for visualizing, and characterizing minor glycan species.     

Fragmentation of negative ions from N‐linked carbohydrates,Part 4. Fragmentation of complex glycans lacking substitution on the 6‐antenna

Negative ion CID spectra of N‐linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N‐glycans from a patient lacking the enzyme N‐acetylglucosaminyltransferase‐2 provided an opportunity to investigate fragmentation of glycans lacking a 6‐antenna. These glycans contained one or two galactose‐N‐acetylglucosamine‐chains attached to the 3‐linked mannose residue of the trimannosyl‐chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna‐specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme‐deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

Characteristic ion clusters as determinants for the identification of pyrrolizidine alkaloid N‐oxides in pyrrolizidine alkaloid–containing natural products using HPLC–MS analysis

Pyrrolizidine alkaloid (PA)–containing plants are widely distributed in the world. PAs are hepatotoxic, affecting livestock and humans. PA N‐oxides are often present together with PAs in plants and also exhibit hepatotoxicity but with less potency. HPLC–MS is generally used to analyze PA‐containing herbs, although PA references are unavailable in most cases. However, to date, without reference standards, HPLC–MS methodology cannot distinguish PA N‐oxides from PAs because they both produce the same characteristic ions in mass spectra. In the present study, the mass spectra of 10 PA N‐oxides and the corresponding PAs were systemically investigated using HPLC–MS to define the characteristic mass fragment ions specific to PAs and PA N‐oxides. Mass spectra of toxic retronecine‐type PA N‐oxides exhibited two characteristic ion clusters at m/z 118–120 and 136–138. These ion clusters were produced by three unique fragmentation pathways of PA N‐oxides and were not found in their corresponding PAs. Similarly, the nontoxic platynecine‐type PA N‐oxides also fragmented via three similar pathways to form two characteristic ion clusters at m/z 120–122 and 138–140. Further application of using these characteristic ion clusters allowed successful and rapid identification of PAs and PA N‐oxides in two PA‐containing herbal plants. Our results demonstrated, for the first time, that these characteristic ion clusters are unique determinants to discriminate PA N‐oxides from PAs even without the availability of reference samples. Our findings provide a novel and specific method to differentiate PA N‐oxides from PAs in PA‐containing natural products, which is crucial for the assessment of their intoxication. Copyright © 2012 John Wiley & Sons, Ltd.     

Structural Studies of Fucosylated <Emphasis Type="Italic">N</Emphasis>-Glycans by Ion Mobility Mass Spectrometry and Collision-Induced Fragmentation of Negative Ions

There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewis^x and Lewis^y epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone.

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Ionisation and fragmentation of complex glycans with a quadrupole time-of-flight mass spectrometer fitted with a matrix-assisted laser desorption/ionisation ion source

This paper reports the use of an experimental matrix-assisted laser desorption/ionisation (MALDI) ion source fitted to a quadrupole time-of-flight (Q-Tof) mass spectrometer for the analysis of carbohydrates, particularly the N-linked glycans from glycoproteins. Earlier work on the Q-Tof instrument, using electrospray ionisation, gave excellent MS/MS spectra, particularly from the [M + Na]+ ions, but suffered from the major disadvantages that the signal was often split between singly and multiply charged ions and that sensitivity fell dramatically as the molecular weight of the carbohydrate rose. The MALDI ion source did not suffer from these problems and the instrument produced excellent MS and MS/MS spectra from small amounts of complex, underivatised glycans as well as those derivatised at the reducing terminus. Positive ion MS spectra of sialylated glycans recorded on the new instrument were much less complex than those recorded with a conventional MALDI-TOF instrument because of the absence of ions resulting from metastable (post-source decay, (PSD)) fragmentations occurring in the flight tube. However, considerable fragmentation by loss of sialic acid still occurred. MS/MS spectra of the [M + Na]+ ions from all compounds were almost identical to those recorded earlier with the electrospray-Q-Tof combination and far superior to MALDI-PSD spectra recorded with reflectron-TOF instruments. Spectra are shown for neutral and sialylated N-linked glycans from chicken ovalbumin, riboflavin binding protein, alpha1-acid glycoprotein, bovine fetuin and ribonuclease B, both as free glycans and as those derivatised at their reducing termini. The technique was applied to the structural determination of N-linked glycans from human secretory IgA and Apo-B 100 from human low-density lipoprotein.     

Site-specific protein glycosylation analysis with glycan isomer differentiation

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.     

Identification of N‐glycans from Ebola virus glycoproteins by matrix‐assisted laser desorption/ionisation time‐of‐flight and negative ion electrospray tandem mass spectrometry

The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N‐glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Identification of the glycans was made with normal‐phase high‐performance liquid chromatography (HPLC), matrix‐assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi‐, tri‐ and tetra‐antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6‐antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high‐mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP. Copyright © 2010 John Wiley & Sons, Ltd.     

Resolving the problem of chromatographic overlap by 3D cross correlation (3DCC) processing of LC, MS and NMR data for characterization of complex glycan mixtures

Chromatographic overlap is a common problem in the analysis of complex mixtures. As a result, it is not possible to identify the components because each resulting NMR or MS spectrum contains multiple components. We introduce three-dimensional cross correlation (3DCC) that dissects NMR spectra of a mixture into spectra of the individual components without actually separating them. Correlation of peaks from MS and NMR profiles along a common LC time domain yields 3DCC NMR spectra of pure components correlated with a mass and a retention time. The method requires an LC run followed by fractionation and recording of MS and NMR spectra. The method is applicable to mixtures of any classes of molecules. Here, we demonstrate its application to a mixture of complex glycans obtained from a glycoprotein. Fourteen glycans eluting within only 3 min showed heavy overlap in the chromatographic run. 3DCC allowed their direct characterization without separation. Some of these structures from the glycoprotein bovine fibrinogen had not previously been described. The 3DCC procedure has been implemented in standard software. Actually, 3DCC can be used for any combination of separation techniques, like LC or GC, combined with two characterization methods like UV, IR, Raman, NMR or MS.     

Qualitative and quantitative comparison of N‐glycans between pig endothelial and islet cells by high‐performance liquid chromatography and mass spectrometry‐based strategy

N‐glycan structures released from miniature pig endothelial and islet cells were determined by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF), negative ion electrospray ionization (ESI) MS/MS and normal‐phase high performance liquid chromatography (NP‐HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N‐glycans including 129 sialylated and 18 α‐galactosylated glycans from pig endothelial cells and 80 N‐glycans including 41 sialylated and one α‐galactosylated glycans from pig islet cells. The quantity of the α‐galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc‐terminated N‐glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using α 1,3‐galactosyltransferase gene‐knockout (GalT‐KO) pig. Copyright © 2009 John Wiley & Sons, Ltd.     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

Derivatization of sialic acids for stabilization in matrix‐assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2 → 3)‐ and α(2 → 6)‐isomers

Sialylated carbohydrates usually decompose by loss of sialic acid when ionized by matrix‐assisted laser desorption/ionization (MALDI) as the result of the labile carboxylic proton. Stabilization has previously been achieved by forming methyl esters with methyl iodide, a procedure that eliminates the labile proton. In this paper, we describe an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the MALDI spectrum. The sugars were desalted, dissolved in methanol, and treated with 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMT‐MM). After removal of the solvent, the products were transferred directly to the MALDI target and examined from 2,5‐dihydroxybenzoic acid. Small amounts of N‐glycans derived from biological sources benefited from an additional clean‐up stage involving Nafion 117. α(2 → 6)‐Linked sialic acid produced only methyl esters whereas α(2 → 3)‐linked sialic acids were converted into their lactones providing a 32 Da difference in mass. Negative ion collision‐induced decomposition (CID) mass spectra of these neutralized glycans provided information, in many cases, on the antenna of N‐linked glycans to which the variously linked sialic acids were attached. The method was applied to N‐linked glycans released from bovine fetuin and porcine thyroglobulin. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-MS

Ion mobility-mass spectrometry (IMS-MS) and molecular modeling techniques have been used to characterize ovalbumin N-linked glycans. Some glycans from this glycoprotein exist as multiple isomeric forms. The gas-phase separation makes it possible to resolve some isomers before MS analysis. Comparisons of experimental cross sections for selected glycan isomers with values that are calculated for iterative structures generated by molecular modeling techniques allow the assignment of sharp features to specific isomers. We focus here on an example glycan set, each having a m/z value of 1046.52 with formula [H₅N₄+2Na]²⁺, where H corresponds to a hexose, and N to a N-acetylglucosamine. This glycan appears to exist as three different isomeric forms that are assignable based on comparisons of measured and calculated cross sections. We estimate the relative ratios of the abundances of the three isomers to be in the range of ∼1.0:1.35:0.85 to ∼1.0:1.5:0.80. In total, IMS-MS analysis of ovalbumin N-linked glycans provides evidence for 19 different glycan structures corresponding to high-mannose and hybrid type carbohydrates with a total of 42 distinct features related to isomers and/or conformers.     

Isomer Information from Ion Mobility Separation of High-Mannose Glycan Fragments

Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man₈GlcNAc₂ isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated.

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Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry

The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with ¹⁵N and uniformly with ¹³C. The pattern of isotope labeling of second-generation fragment ions derived via a _n and b _n ions (where n=4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b _n ions where n=8 or 9.     

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