Negative ion MALDI‐TOF MS,ISD and PSD of neutral underivatized oligosaccharides without anionic dopant strategies,using 2,5‐DHAP as a matrix

Oligosaccharides represent complex class of analytes for mass spectrometric analysis due to the high variety of structural isomers concerning glycosidic linkages and possible branching. A systematic study of the negative ion mode matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry of various neutral oligosaccharides under selection of an appropriate matrix, like 2,5‐dihydroxyacetophenone (2,5‐DHAP) is reported here, without commonly used anion dopant strategies. Nevertheless, we were able to generate relevant in‐source decay (ISD) cross‐ring fragment ions, typically obtained in the negative ion mode. Data observed indicate that the intrinsic property of the terminal non‐reduced aldose is crucial for this behavior. A systematic study of the post source decay (PSD) of molecular, pseudomolecular and ISD cross‐ring cleavage precursor ions is reported here. A direct comparison of the positive and negative ion mode MALDI MS1 and PSD behavior of neutral oligosaccharides could also be performed under the use of the same matrix preparation, because 2,5‐DHAP is fully compatible with positive ion mode acquisition. We found that PSD spectra of deprotonated neutral oligosaccharides obtained in the negative ion mode are richer, because they contained both glycosidic and cross‐ring fragment ions. However, we also found that cross‐ring fragment ions are readily produced in the positive ion mode when potassiated precursor ions were selected. In addition, we show evidence that non‐anionic dopants and specific instrumental parameters can also significantly influence the ISD fragmentation. Taken together, our results should increase our understanding of oligosaccharide behavior in the negative ion mode as well as increase our knowledge regarding many aspects of in‐source MALDI chemistry. Copyright © 2016 John Wiley & Sons, Ltd.     

Enhanced in-source fragmentation in MALDI-TOF-MS of oligonucleotides using 1,5-diaminonapthalene

The capability to rapidly and confidently determine or confirm the sequences of short oligonucleotides, including native and chemically-modified DNA and RNA, is important for a number of fields. While matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been used previously to sequence short oligonucleotides, the typically low fragmentation efficiency of in-source or post-source decay processes necessitates the accumulation of a large number of spectra, thus limiting the throughput of these methods. Here we introduce a novel matrix, 1,5-diaminonapthalene (DAN), for facile in-source decay (ISD) of DNA and RNA molecular anions, which allows for rapid sequence confirmation. d-, w-, and y-series ions are prominent in the spectra, complementary to the (a-B)- and w- ions that are typically produced by MALDI post-source decay (PSD). Results are shown for several model DNA and RNA oligonucleotides, including combinations of DAN-induced fragmentation with true tandem TOF MS (MS/MS) for pseudo-MS³ and “activated-ion PSD.”     

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.     

A new strategy to determine the protein mutation site using matrix-assisted laser desorption ionization in-source decay: Derivatization by ionic liquid

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) can be considered as state of the art in the field of proteins and peptides analysis. In this work, we have designed an ionic liquid derivative strategy to obtain abundant fragment ions in MALDI in-source decay (ISD) and used the analysis of angiogenin with mutation in the fortieth (K40I) as an instance. Firstly, we have synthesized two types of ionic liquids, 3-allyl-4-methyl-1H-imidazol-3-ium and 4-methyl-3-(pent-4-yn-1-yl)-1H-imidazol-3-ium. Then in the light-catalyzed reaction, the alkenyl ionic liquid can open the disulfide bond of K40I protein and add to the thiol. And the derived protein can process in-source decay under the effect of ionic liquid group to produce c–z type ions. Additionally this fragmentation is potentiated to support widely range of fragment ions which can cover the location of mutation. Our results have supplied a new top-down method about how to analyze the mutation or even post-translational modification of proteins in MALDI mass spectrometry.     

Matrix-assisted laser desorption/ionization in-source decay combined with tandem time-of-flight mass spectrometry of permethylated oligosaccharides: targeted characterization of specific parts of the glycan structure

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) has been introduced in recent years as a valuable tool for the structural characterization of permethylated oligosaccharides. In this report, we describe the combination of MALDI in-source decay (ISD) with the subsequent TOF/TOF-MS analyses of specific fragments, allowing the detailed characterization of the selected part of the oligosaccharide molecule. Part of the second-generation fragment ions were different from those observed in conventional MALDI-TOF/TOF-MS experiments. Other fragments, which had already been observed in conventional MALDI-TOF/TOF-MS and again showed up in second-generation fragment analysis, could be assigned to specific parts of the molecule. Our approach disclosed different structural features of the oligosaccharides: due to permethylation, the glycosidic linkage fragments allowed the distinction between terminal, monosubstituted and disubstituted monosaccharides and indicated the oligosaccharide sequence. Moreover, substitution positions were deduced based on characteristic cross-ring fragmentation by high-energy collision-induced fragmentation. In conclusion, combination of MALDI-ISD with TOF/TOF-MS allows the detailed characterization of specific moieties of permethylated oligosaccharides and is, therefore, a powerful technique for structural glycomics.     

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.     

Enhanced post-source decay and cross-ring fragmentation of oligosaccharides facilitated by conversion to amino derivatives

Post-source decay (PSD) fragmentation of chemically or enzymatically produced aminoglycans has been evaluated through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Conversion of native glycans to their respective aminoglycan derivatives improved detection sensitivity of the usual fragments and promoted cross-ring fragmentation of linear oligosaccharides, facilitating linkage recognition. The cross-ring fragmentations for both dextrin and dextran oligosaccharides were not limited to the reducing-end glucose moiety, as they were extended throughout the entire molecule. When the amino group was generated for N-glycans derived from three different glycoproteins, an enhancement of PSD was observed, without a significant extent of cross-ring fragmentation.     

Gas-phase fragmentation of oligosaccharides in MALDI laser-enhanced in-source decay induced by thermal hydrogen radicals

Laser-enhanced in-source decay (LEISD) MALDI MS recently proposed for structural analysis of oligosaccharides was used to systematically investigate ISD fragmentation of oligosaccharides, which was found to be mediated by thermal hydrogen radicals from a matrix and underwent a charge-induced process, depending on the nature of the matrix and the structure of an oligosaccharide.     

Post-source decay mass spectrometry: optimized calibration procedure and structural characterization of permethylated oligosaccharides

Permethylated oligosaccharides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) using a reflectron time-of-flight instrument in the post-source decay (PSD) mode. Under these ionization conditions, such derivatives yield intense signals corresponding to sodium or potassium cationized molecular species. Fragments observed in the PSD spectra result exclusively from cleavage of glycosidic bonds, preferentially at N-acetylhexosamine residues. A systematic study was carried out on a series of permethylated oligosaccharides to allow rationalization of the fragmentation processes. Fragments originating from both the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, glycosyl residues linked in position 3 of HexNAc units give rise to a highly specific elimination process, which allows unambiguous assignment of (1-3) interglycosidic linkages. Special attention was paid to the structural analysis of oligosaccharides carrying the commonly encountered fucosyl and sialyl end-caps. In the case of sialylated residues, a targeted methodology involving desialylation and specific CD3-labeling of the nascent free hydroxyl groups was developed to mark the initial location of sialic acid residues along the oligosaccharide backbone. As accurate mass determination of fragment ions is essential for their assignment, a simplified protocol for the calibration in the PSD mode is described. This procedure allows the determination of the correction function parameters required to process the data for an instrument that employs post-acceleration detection. MALDI/PSD-MS of permethylated oligosaccharides, by providing structural information at the low picomole level, appears to be a valuable complement, or an alternative, to the techniques currently in use for carbohydrate structural analysis.     

Identification of non-peptide species in selenized yeast by MALDI mass spectrometry using post-source decay and orthogonal Q-TOF detection

The potential of tandem mass spectrometry following matrix-assisted laser desorption ionization (MALDI) was studied for speciation of selenium. Non-peptide selenium-containing compounds were isolated from a selenized yeast aqueous extract by size-exclusion chromatography. Post-source decay (PSD) was compared with orthogonal quadrupole collision cell dissociation for the purpose of obtaining fragmentation and structural information. In the PSD mode, the use of ion gate covering the whole isotopic cluster of the parent compound allowed the immediate recognition of fragments containing Se and those in which this element was absent. The tandem mass spectra obtained by orthogonal MALDI Q-TOF were equally informative in terms of the number of fragments but suffered from a poorer sensitivity. The mass accuracy was ca. 20 times better in the oMALDI configuration than in the PSD mode. An unknown selenium compound with an m/z 388 was detected with a mass accuracy of 3 ppm according to the proposed empiric formula.     

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.     

Identification of reducing and nonreducing neutral carbohydrates by laser‐enhanced in‐source decay (LEISD) MALDI MS

In this work, laser‐enhanced in‐source decay (LEISD) technique of matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI‐FT‐ICR‐MS) was used to distinguish reducing and nonreducing carbohydrates. Interestingly, easier cleavage of (1 → 2)‐linked glycosidic bonds for nonreducing carbohydrates containing D‐fructofuranosyl units was observed in MALDI‐FT‐ICR‐MS, which was in agreement with the result of theoretical calculation by the software package Gaussian 09. Importantly, no cross‐ring cleavage of fructofuranosyl residues was detected in the LEISD spectra of nonreducing carbohydrates. LEISD method therefore offers an attractive alternative for fast and efficient differentiation of reducing and nonreducing carbohydrates, and the positions of nonreducing monosaccharide residues in a carbohydrate chain could be easily speculated. Copyright © 2013 John Wiley & Sons, Ltd.     

New advances in the understanding of the in-source decay fragmentation of peptides in MALDI-TOF-MS

In-source decay (ISD) is a rapid fragmentation occurring in the matrix-assisted laser desorption/ionization (MALDI) source before the ion extraction. Despite the increasing interest for peptides de novo sequencing by ISD, the influence of the matrix and of the peptide itself is not yet fully understood. Here we compare matrices with high ISD efficiencies to gain deeper insight in the ISD fragmentation process(es). The major ISD fragments are the c- and z-ions, but other types of fragments are also observed, and their origin is studied here. Two main pathways lead to fragmentation in the source: a radical-induced pathway that leads to c-, z-, w-, and d- ions, and a thermally activated pathway that leads to y-, b-, and a-ions. A detailed analysis of the ISD spectra of selected peptides revealed that (1) the extents of the two in-source pathways are differently favored depending on the matrix used, that (2) the presence of a positive/negative charge on the radical-induced fragments is necessary for their observation in positive/negative mode, respectively, and that (3), for a same peptide, the patterns of the different types of fragments differ according to the matrix used.     

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.     

Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z′-ions originating from cleavage at the N–Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z′-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a “mobile proton” are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

Post-source decay time-of-flight study of fragmentation mechanisms of protonated synthetic polymers under matrix-assisted laser desorption/ionization conditions

Post-source decay (PSD) of three different nylon oligomers desorbed under matrix-assisted laser desorption/ionization (MALDI) conditions was studied and their fragmentation pathways were investigated. The fragmentation of the protonated oligomers is very similar to that of peptides. The b(n)(+), y(n)(+) and z(n)(+) series of ions were observed in abundance in the PSD spectrum. The end groups and the length of the spacer in the repeating unit influence the fragmentation of the different polyamides and the relative abundances of the product ions. Competitive dehydration and deamination reactions were observed, and depend on the nature of the end groups and the repeating units. The PSD spectra are very similar to collision-induced dissociation (CID) spectra obtained under low-energy conditions, implying that the selected precursor ions possess similar average internal energies. All the peaks observed in the PSD spectrum can be rationalized by reasonable fragmentation mechanisms.     

A comparative study of in- and post-source decays of peptide and preformed ions in matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Effective temperature and matrix effect

In-source decay (ISD) and post-source decay (PSD) of a peptide ion ([Y₆ + H]⁺) and a preformed ion (benzyltriphenylphosphonium, BTPP) generated by matrix-assisted laser desorption ionization (MALDI) were investigated with time-of-flight mass spectrometry. α-Cyano-4-hydroxycinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were used as matrices. For both ions, ISD yield was unaffected by delay time, indicating rapid termination of ISD. This was taken as evidence for rapid expansion cooling of hot “early” plume formed in MALDI. CHCA was hotter than DHB for [Y₆ + H]⁺ while the matrix effect was insignificant for BTPP. The “early” plume temperature estimated utilizing previous kinetic results was 800–900 K, versus 400–500 K for “late” plume. The results support our previous finding that the temperature of peptide ions interrogated by tandem mass spectrometry was lower than most rough estimates of MALDI temperature.     

MALDI linear-field reflectron TOF post-source decay analysis of underivatized oligosaccharides: Determination of glycosidic linkages and anomeric configurations using anion attachment

Six different anionic species (fluoride, chloride, bromide, iodide, nitrate, and acetate) are tested for their abilities to form anionic adducts with neutral oligosaccharides that are detectable by MALDI-TOF mass spectrometry. Fluoride and acetate cannot form anionic adducts with the oligosaccharides in significant yields. However, bromide, iodide, and nitrate anionic adducts consistently appear in higher abundances relative to [M - H](-), just like the highly stable chloride adducts. Post-source decay (PSD) decompositions of Br(-), I(-), and NO(3)(-) adducts of oligosaccharides provide no structural information, i.e., they yield the respective anions as the main product ions. However, determination of linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by negative ion PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of pairs of diagnostic fragment ions allow differentiation of anomeric configurations of glycosidic bonds. Thus, simultaneous identification of the linkage types and anomeric configurations of glycosidic bonds is achieved. Our data indicate that negative ion PSD fragmentation patterns of chloride adducts of oligosaccharides are mainly determined by the linkage types. Correlation may exist between the linkage positions and fragmentation mechanisms and/or steric requirements for both cross-ring and glycosidic bond fragmentations. PSD of the chloride adducts of saccharides containing a terminal Glcalpha1-2Fru linkage also yields chlorine-containing fragment ions which appear to be specifically diagnostic for a fructose linked at the 2-position on the reducing end. This also allows differentiation from saccharides with a 1-1 linked pyranose on the same position.     

Changes in post-source decay fragmentation behavior of poly(methyl methacrylate) polymers with increasing molecular weight studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In order to investigate the systematic changes in fragmentation behavior of poly(methyl methacrylate) (PMMA) with increasing molecular weight, alkali-metal cationized PMMA 20-mer, 60-mer and 100-mer were selected for post-source decay (PSD) fragmentation study by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. PMMA polymers were cationized with lithium, potassium and cesium cations to explore the influence of the cation size on the fragmentation behavior of the polymers. All PMMA polymers could be fragmented by MALDI-PSD and fragmentation of the MALDI ionized synthetic polymer of molecular weight 10 kDa is reported here for the first time. It was shown that an increasing molecular weight of the PMMA chain required an increase in the size of the cation to improve the intensity and the number of the fragments in the PSD spectrum. Some instrumental parameters had to be optimized prior to a successful PSD analysis of the largest PMMA polymers.     

Unusual Fragmentation of Peptide and Protein in Matrix-assisted Laser Desorption/lonization Mass Spectrometry

Unusual amine - bond fragmentation on the peptide/protein backbone has been reported using matrix - assisted laser desorption/ionization time - of- flight mass spectrometry (MALDI - TOFMS)The amine - bond cleavage occurred without metastable decay, while the peptide - bond cleavage occurred with metastable decay of peptide ions in a drift region of TOF mass analyzer. It was presumed that the amine - bond cleavage occurred as a non - ergodic process independent of the ionization under MALDI conditions.