Post-source decay fragmentation analyses of linkage isomers of Lewis-type oligosaccharides in curved-field reflectron matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: combined in-source decay/post-source decay experiments and relative ion abundance analysis

Linkage isomers of Lewis(X) trisaccharide (Le(X)) and Lewis(a) trisaccharide (Le(a)) were distinguished by the post-source decay (PSD) fragment spectra obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without permethylation. Both Y- and Z-type fragmentations were observed at the C-3 position of N-acetylhexosamine. beta-Elimination at C-3 of the reducing-end N-acetylglucosamine in Le(X) formed a double bond, which conjugated to an N-acetyl group, making the chemical species stable. In contrast, the double bond formed in the reducing end glucose of 3-fucosyllactose was unstable owing to the lack of a conjugated system. Therefore, beta-elimination of N-acetylglucosamine occurred predominantly rather than that of hexose in MALDI-PSD fragmentation. The measurements of the PSD fragment mass spectra using pseudo precursor ions originating from in-source decay were useful for the analyses of the fragmentation mechanisms and for the assignments of the chemical species of the fragment ions. The combined in-source decay/post-source decay experiments revealed the formation of a double bond between C-2 and C-3 in N-acetylglucosamine of Le(X). Abundance analysis of the PSD ions indicated that the 1-3 glycosyl linkage cleaves more easily than does the 1-4 linkage in MALDI-PSD fragmentation. Ion abundance analyses were useful in estimating the degree of Y- and Z-type fragmentation at the C-3 position of hexose and N-acetylhexosamine. The analysis of the relative ion abundances was a powerful tool for the assignments of the chemical species of the PSD ions.     

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

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

A parallel approach to post source decay MALDI-TOF analysis

We present a novel enhancement to matrix-assisted laser desorption ionization (MALDI) post-source decay (PSD) analysis whereby fragment ions from multiple precursor ions are acquired into the same spectrum without employing a timed ion gate to preselect each parent ion. Fragment ions are matched to their corresponding precursor ions by comparing spectra acquired at slightly different reflectron electric fields. By measuring the difference in time-of-flight (TOF) between the two spectra for each fragment, it is possible to calculate the mass of the fragment ion and its parent. This new "parallel PSD" technique reduces analysis time and consumes less sample than conventional PSD, which requires an ion gate for serial preselection of precursor ions.     

An experimental comparison of electrospray ion-trap and matrix-assisted laser desorption/ionization post-source decay mass spectra for the characterization of small drug molecules

An experimental comparison of product ion spectra produced by matrix-assisted laser desorption/ionization (MALDI) and electrospray ion-trap MS( n) for a group of small drug molecules is presented in this paper. The goal of the study was to demonstrate the usefulness of MALDI-MS with post-source decay (PSD) and collision-induced dissociation (CID) for the structural analysis of small drug molecules in the drug discovery process, where traditionally electrospray LC/MS methods are used. PSD and PSD/CID gave diverse product ions that were highly indicative of the structure of the drugs investigated (a group of 4-quinolone antibiotics and oleandomycin). In addition, the number of different product ions generated with MALDI-MS was always higher than with electrospray ion-trap MS( n) (with n < or =4) for the drug molecules studied. This investigation also showed that the choice of a suitable MALDI matrix for the analysis of low molecular weight compounds is quite important. It was found that of the three matrices examined, alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) produced the most intense fragmentation levels while TiO2, with its advantage of virtually no low mass background signals, did not generate quite the same amount of information.     

Enhancement of matrix-assisted laser desorption/ionization photodissociation tandem time-of-flight mass spectra; spectral reduction and cleanup of isotopomeric contamination

Weak signal intensity and poor precursor ion selection are the major difficulties in tandem time-of-flight (TOF) mass spectrometry of ions generated by matrix-assisted laser desorption/ionization (MALDI). Even though the latter can be overcome in photodissociation (PD) tandem TOF mass spectrometry via ion pulse-PD laser pulse synchronization, clean monoisotopic selection of precursor ions of high m/z can often be difficult for various reasons. A considerable enhancement of post-source decay (PSD) and PD tandem mass spectra has been achieved in this work via single-ion detection and post-acquisition reduction of the spectra. Also, an algorithm has been developed to clean up isotopomeric contamination when the resolution for precursor ion selection is less than adequate. A high-quality tandem TOF mass spectrum which results from PD of virtually monoisotopic precursor ions has been obtained.     

Investigations of the metastable decay of DNA under ultraviolet matrix-assisted laser desorption/ionization conditions with post-source-decay analysis and hydrogen/deuterium exchange

The fragmentation of positive ions of DNA under the conditions of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was investigated by post-source decay (PSD) analysis and hydrogen/deuterium (H/D) exchange. Spectra of five different synthetic 4mer oligonucleotides were recorded. As a main result the hypothesis was confirmed that for these ions all fragment ions result from processes, initiated by protonation/deuteration of a suitable base followed by a loss of this base as a neutral or ion and further backbone cleavages. The three bases adenine, guanine, and cytosine all exhibit comparable lability for fragmentation. The spectra show evidence for an interaction of the adenine base with the phosphate backbone. Signals of fragments containing TT- and CT-cycloadducts were observed in the spectra.     

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.     

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a_n′ and w_n′) produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w_n′ ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an “in situ” derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5′-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3′ and 5′ fragments (w_n−m, b_m, a_m, d_m−1) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site.     

Improved PSD and CID on a MALDI TOFMS

The influence of several instrument-operating parameters on the product-ion resolution and mass accuracy in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) post-source decay (PSD) and collision-induced dissociation (CID) experiments is reported. Voltages commonly applied to the reflectron for PSD and CID experiments were found to be non-ideal; optimization of these voltages resulted in better resolution across each segment of the measured PSD spectrum. Mass resolution, calculated as M/DeltaM (FWHM) for the product-ion peaks, was as high as 2500. Additionally, precursor-ion selection and segment mass range setup were each found to have dramatic influences on product-ion mass accuracy. An understanding of the influence of these variables aided in the interpretation of (a-NH3) and (b - NH3) ions observed in the PSD/CID spectra of a number of peptides. In addition, product ions resulting from coincidence peaks in the precursor-ion selection window were found to be a general problem. With the improvements to resolution and optimization of these mass accuracy variables, the mass accuracy of product ions from MALDI TOF PSD and CID experiments was tested with several reference materials, including the peptides Substance P, bradykinin, angiotensin I, and angiotensin II and the synthetic polymers poly(methyl methacrylate) and polystyrene. The absolute error (Da) for each test material was, on average, below 0.1 Da, demonstrating a significant improvement in mass accuracy using the improved operational parameters and an extension of the use of poly(ethylene glycol) (PEG) as a mass calibrant for the PSD/CID spectra.     

Reflectron MALDI TOF and MALDI TOF/TOF mass spectrometry reveal novel structural details of native lipooligosaccharides

Lipooligosaccharides (LOS) are powerful Gram-negative glycolipids that evade the immune system and invade host animal and vegetal cells. The structural elucidation of LOS is pivotal to understanding the mechanisms of infection at the molecular level. The amphiphilic nature of LOS has been the main obstacle for structural analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Our approach has resolved this important issue and has permitted us to obtain reflectron MALDI mass spectra of LOS to reveal the fine chemical structure with minimal structural variations. The high-quality MALDI mass spectra show LOS species characteristic of molecular ions and defined fragments due to decay in the ion source. The in-source decay yields B-type ions, which correspond to core oligosaccharide(s), and Y-type ions, which are related to lipid A unit(s). MALDI tandem time-of-flight (TOF/TOF) MS of lipid A allowed for the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. These findings constitute a significant advancement in the analysis of such an important biomolecule by MALDI MS.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation of deprotonated d-ribose and d-fructose in MALDI—Comparison with dissociative electron attachment

We present a detailed, collaborative study on the fragmentation of deprotonated native d-ribose and d-fructose and the isotopically labelled 1-¹³C-d-ribose, 5-¹³C-d-ribose and C-1-d-d-ribose. The fragmentation is studied in a matrix assisted laser desorption/ionization time of flight mass spectrometer (MALDI ToF MS), both in in-source decay (ISD) and post-source decay (PSD) mode and compared with fragmentation through dissociative electron attachment (DEA). Fragmentation of deprotonated monosaccharides formed in the MALDI process, as well as their transient molecular anions formed upon electron attachment are characterized by loss of different numbers of H₂O and CH₂O units. Two different fragmentation pathways leading to cross-ring cleavage are identified. Metastable decay of deprotonated d-ribose proceeds either via an X-type cleavage yielding fragment anions at m/z = 119, 100 and 89, or via an A-type cleavage resulting in m/z = 89, 77 and 71. A fast and early metastable cross-ring cleavage of deprotonated d-ribose observed in in-source decay is dominated by X-type cleavage leading mainly to m/z = 100 and 71. For dissociative electron attachment to d-ribose a sequential dissociation was identified that includes metastable decay of the dehydrogenated molecular anion leading to m/z = 89. All other fragmentation reactions in DEA to d-ribose are likely to proceed directly and on a faster timescale (below 400 ns).     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Do charge-remote fragmentations occur under matrix-assisted laser desorption ionization post-source decompositions and matrix-assisted laser desorption ionization collisionally activated decompositions?

The precursor ions of tetraphenylporphyrins that are substituted with fatty acids can be introduced into the gas phase by matrix-assisted laser desorption ionization (MALDI) and undergo post-source and collisionally activated decompositions (CAD) in a time-of-flight mass spectrometer. The goal of the research is to obtain a better understanding of post-source decompositions (PSD); specifically, we asked the question of whether ions undergoing PSD have sufficient energy to give charge-remote fragmentations along an alkyl chain. We chose the porphyrin macrocycle because we expected it to act as an inert "support," allowing the molecule to be desorbed by MALDI and to be amenable to charge-remote fragmentation. MALDI-PSD and MALDI-CAD spectra are similar to high-energy CAD spectra and considerably more informative than low-energy CAD spectra, showing that charge-remote fragmentations of the fatty acid moieties do occur upon MALDI-PSD and MALDI-CAD.     

Matrix-assisted laser desorption/ionisation mass spectrometry of oligosaccharides and glycoconjugates

The technique of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is described and examples are given of its use for the examination of glycoproteins, glycopeptides, glycolipids and oligosaccharides. Abundant [M + H]⁺ ions are produced by the glycoproteins and glycopeptides, whereas glycolipids and oligosaccharides give mainly [M + Na]⁺ ions. Resolution on time-of-flight (TOF) instruments is poor but improved resolution can be obtained by use of ion cyclotron resonance or magnetic sector instruments. Although the technique gives mainly [M + Na]⁺ ions from neutral, underivatised oligosaccharides, with little fragmentation when implemented on TOF systems, the use of a reflectron enables fragment ions produced by post-source decay to be obtained. Acidic sugars give less satisfactory positive ion spectra with TOF analysers. but generally produce abundant negative ions. Extensive fragmentation is observed with these compounds when the spectra are recorded with magnetic sector instruments. Neutral glycolipids produce strong spectra from several matrices but acidic glycolipids show extensive fragmentation as the result of sialic acid loss.     

Post-source decay matrix-assisted laser desorption/ionization mass spectrometric study of tetracyclic 2,3-dihydro-1,5-benzothiazepines

The fragmentation behavior of six tetracyclic 2,3-dihydro-1,5-benzothiazepine derivatives cationized with protons and silver ions under post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) conditions is reported. The protonated adduct ions decompose into several structurally important fragment ions, including substituted cyclopropane and benzohydrothiazole cations. Elimination of Ag and H and/or AgH from the silver-cationized adduct ions of these ([M+Ag](+)) compounds was observed. It was also found that [M+Ag](+) produced silver-depleted fragment ions exclusively. Based on the PSD results a fragmentation pathway is proposed for the [M+H](+) and [M+Ag](+) precursor ions.     

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides.     

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.