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.     

A comprehensive and comparative analysis for MALDI FTMS lipid and phospholipid profiles from biological samples

Described here is a computationally automated method for translating complex accurate mass spectra into biologically relevant and meaningful data. Rapid profiling of detailed high resolution mass spectra resulting from direct analysis of whole cells and tissues by matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) is discussed. Lipid and phospholipid ions create complex spectra containing multiple m/z values corresponding to the same fundamental chemical species. A computational approach is employed to sort ions, with mass to charge ratios lower than m/z 1000, into groups of similar lipid and phospholipid compositions for comprehensive and rapid analysis. By sorting or binning ions in this manner, variations in the degree of cation exchange can be avoided, thus increasing the comparability of the data. The result is displayed as a histogram that is easily interpretable and comparable with similar analyses and is particularly useful for direct comparison of similar tissues. Spectra of leaves from a healthy Prunus persica (peach) tree are compared with those from leaves infected by the fungus Taphrina deformans. Although the infection can be seen as a difference in leaf structure and by visual inspection of the mass spectra, the method described here details the chemical difference in phospholipid compositions and their relative abundances.     

Comparision of FAB and MALDI Mass Spectrometry of Ginsenosides

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252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.     

Disappearance of interfering alkali-metal adducted peaks from matrix-assisted laser desorption/ionization mass spectra of peptides with serine addition to alpha-cyano-4-hydroxycinnamic acid matrix

It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H](+) and [M-H](-). The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na](+) and [M+K](+), and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra.     

The fragmentation of ethoxylated surfactants by AP-MALDI-QIT

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important technique to characterize the chemical structure of industrial polymer materials. MALDI methods have been developed to address a broad variety of different polymer materials containing different chemistries. One of the key aspects of the typical MALDI experiment is the generation of intact ions. The development of Atmospheric Pressure (AP) MALDI quadrupole ion trap (QIT) instruments has opened another channel to obtaining MS/MS experiments for polymer samples. These experiments provide a new method to obtain chemical structure information from MALDI experiments. Collision-Induced Dissociation (CID) provides an improved MALDI MS/MS experiment that can be done on readily available mass spectrometers. AP MALDI QIT techniques have been successfully applied to a variety of synthetic polymers. This work explores the applicability of AP MALDI QIT methods to relatively low molecular weight ethoxylated surfactants. In these experiments we show the CID fragmentation mass spectra on some ethoxylated surfactants, and demonstrate the existence of analyte matrix clusters.     

Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.     

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.     

Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

Mass spectral studies on aryl-substituted N-carbamoyl/N-thiocarbamoyl narcotine and related compounds

Positive ion mass spectral fragmentation of new N-carbamoyl/N-thiocarbamoyl derivatives of narcotine and compounds closely related to it are reported and discussed. The techniques used include electron impact (EI), fast-atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Prominent peaks in the mass spectra of these compounds appear to involve C-C bond cleavage beta to the amine nitrogen with loss of the 4,5-dimethoxy(1H)isobenzofuranone moiety from their molecular ions, along with another prominent peak at m/z 382. No molecular ion peaks of these compounds were recorded in EI, whereas intense [M + H]+ ion peaks were observed in FAB and ESI spectra. MALDI also yielded [M + H]+ ion peaks in good agreement with FAB and ESI studies.     

Characterization of bacterial lipooligosaccharides by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) with a time-of-flight analyzer has been used to analyze bacterial lipooligosaccharides (LOS). Crude LOS preparations from pathogenic strains of Haemophilus influenzae and Haemophilus ducreyi and a commercial preparation of lipopolysaccharide from Salmonella typhimurium were treated with hydrazine to remove O-linked fatty acids on the lipid A moiety. The resulting O-deacylated LOS forms were water soluble and more amenable to cocrystallization with standard MALDI matrices such as 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline. Under continuous extraction conditions, O-deacylated LOS yielded broad peaks with abundant salt adducts as well as forming prompt fragments through β-elimination of phosphoric acid, that is, [M-H₃PO₄-H]. However, when a time delay was used between ionization and extraction (“delayed extraction”) a significant improvement was seen in both mass resolution and the stability of the molecular ions against β-elimination of phosphoric acid, especially in the negative-ion mode. Both an external two-point calibration and an internal single-point calibration were used to assign masses, the latter of which provided the highest degree of accuracy (better than 0.01% in most cases). At higher laser powers, the LOS molecules cleave readily between the oligosaccharide and lipid A moieties yielding a number of prompt fragments. Postsource decay (PSD) analysis of selected molecular ions provided a set of fragments similar to those seen in the linear spectra, although they were more limited in number because they were derived from a single LOS-glycoform. Both the prompt and PSD fragments provided important structural information, especially in assigning the phosphate and phosphoethanolamine substitution pattern of the lipid A and oligosaccharide portions of LOS. Last, with the addition of ethylenediaminetetraacetic acid followed by pulsed sonication, the relatively insoluble (and impure) LOS preparations yielded MALDI spectra similar to the O-deacylated LOS, although these intact LOS preparations required higher laser powers to ionize and were generally more affected by competing impurities.     

Flavonoid–matrix cluster ions in MALDI mass spectrometry

The behaviour of 2,5‐dihydroxybenzoic acid (2,5‐DHB) matrix under matrix‐assisted laser desorption/ionisation (MALDI) conditions was investigated, and the formation of 2,5‐DHB cluster ions, mainly dehydrated 2,5‐DHB ions, is reported. Interestingly, in the mass spectra of this compound, besides dimers and trimers, protonated tetramers, pentamers, hexamers and heptamers were also found with significant abundance. The MALDI behaviour of four flavonoids, quercetin, myricetin, luteolin and kaempferol, using 2,5‐DHB as matrix, was also investigated. The mass spectra of the flavonoids studied revealed a number of flavonoid–2,5‐DHB cluster ions (mainly with the dehydrated 2,5‐DHB). The number of clusters formed is dependent on the structure of the analyte. For luteolin and kaempferol, in particular, evidence was found for the formation of cluster ions involving retro Diels Alder fragments and intact flavonoids molecules, as well as the corresponding protonated retro Diels Alder fragments with dehydrated DHB molecules. All ion compositions were attributed taking into account high accuracy mass measurements and tandem mass spectrometry experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of Hanford-Related Organics Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI/TOF-MS) was used for the analysis of low-molecular phosphate compounds found in Hanford tank wastes. The mass spectra of these compounds indicate protonated peaks as well as sodium adducts. Analytical methods presently utilized for the analysis of the phosphate-related organics are both time consuming and labor intensive. A promising alternative is MALDI/TOFMS. The MALDI process produces both positive and negative ions directly and very little sample is required. In addition, there is limited sample preparation and minimal hazardous waste production.     

Dissociation of biomolecules using a ultraviolet matrix-assisted laser desorption/ionisation time-of-flight/curved field reflectron tandem mass spectrometer equipped with a differential-pumped collision cell

A commercial matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) instrument equipped with a curved field reflectron (CFR) was modified in order to perform collision-induced dissociation (CID) on a variety of biomolecules. The incorporation of a high-resolution ion gate together with a collision cell within the field-free region allowed tandem mass analysis (MS/MS), without the necessity to decelerate the precursor ions prior to activation. The simultaneous detection of all product ions remained possible by using the CFR. To test the MS/MS performances, ACTH (fragment 1-17), a complex high mannose carbohydrate (Man)(8)(GlcNac)(2) and a lysophosphatidylcholine lipid (18:1) were analysed on the modified instrument. Direct comparison with the low-energy product ion spectra, acquired on a MALDI quadrupole ion trap (QIT) two-stage reflectron time-of flight (ReToF) mass spectrometer, showed significant differences in the types of product ions observed. The additional ions detected were a clear indication of the high-energy fragmentation processes occurring in the collision cell.     

Comparative study of organic dyes with time-of-flight static secondary ion mass spectrometry and related techniques

A series of cationic, zwitterionic and anionic fluorinated carbocyanine dyes, spin-coated on Si substrates, were measured with time-of-flight static secondary ion mass spectrometry (TOF-S-SIMS) under Ga(+) primary ion bombardment. Detailed fragmentation patterns were developed for all dyes measured. In the positive mode, the resulting spectra showed very intense signals for the precursor ions of the cationic dyes, whereas the protonated signals of the anionic dyes were hardly detected. Differences of three orders of magnitude were repeatedly observed for the secondary ion signal intensities of cationic and anionic dyes, respectively. All measured dyes yielded mass spectra containing several characteristic fragment ions. Although the secondary ion yields were still higher for the cationic than the anionic dye fragments, the difference was reduced to a factor of < or =10. This result and the fact that M(+), [M + H](+) or [M + 2H](+) are even-electron species make it very likely that the recorded fragments were not formed directly out of the (protonated) parent ions M(+), [M + H](+) or [M + 2H](+). In the negative mode, none of the recorded spectra contained molecular information. Only signals originating from some characteristic elements of the molecules (F, Cl), the anionic counter ion signal and some low-mass organic ions were detected. A comparative study was made between TOF-S-SIMS, using Ga(+) primary ions, and other mass spectrometric techniques, namely fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). The measurements showed that MALDI, ESI and FAB all give rise to spectra containing molecular ion signals. ESI and FAB produced M(+) and [M + H](+) signals, originating from the cationic and zwitterionic dyes, in the positive mode and M(-) and [M - H](-) signals of the anionic and zwitterionic dyes in the negative mode. With MALDI, molecular ion signals were measured in both modes for all the dyes. Structural fragment ions were detected for FAB, ESI and MALDI in both the positive and negative modes. Compared with the other techniques, TOF-S-SIMS induced a higher degree of fragmentation.     

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.     

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.     

Titanium dioxide anatase as matrix for matrix-assisted laser desorption/ionization analysis of small molecules

The use of inorganic species as assisting materials in matrix-assisted laser desorption/ionization (MALDI) analysis is an alternative approach to avoid interfering matrix ions in the low-mass region of the mass spectra. Reports of the application of inorganic species as matrices in MALDI analysis of small molecules are, however, scarce. Nevertheless, titanium dioxide (TiO(2)) powder has been reported to be a promising matrix medium. In this study we further explore the use of TiO(2) as a matrix for the MALDI analysis of low molecular weight compounds. We present results showing that nanosized TiO(2) anatase and TiO(2) rutile perform better as MALDI matrices than a commercial TiO(2) anatase/rutile mixture. Moreover, when using nanosized TiO(2) anatase as a matrix, high-quality mass spectra can be obtained with strong analyte signals and weak or non-existing matrix interference ions. Furthermore, our results show that the phase type plays an important role in the application of TiO(2) as a MALDI matrix.     

Effects of substrate surfaces in matrix‐assisted laser desorption/ionization mass spectrometry

For matrix‐assisted laser desorption/ionization (MALDI) mass spectra, undesirable ion contamination can occur due to the direct laser excitation of substrate materials (i.e., laser desorption/ionization (LDI)) if the samples do not completely cover the substrate surfaces. In this study, comparison is made of LDI processes on substrates of indium and silver, which easily emit their own ions upon laser irradiation, and conventional materials, stainless steel and gold. A simultaneous decrease of ion intensities with the number of laser pulses is observed as a common feature. By the application of an indium substrate to the MALDI mass spectrometry of alkali salts and alkylammonium salts mixed with matrices, 2,5‐dihydroxybenzoic acid (DHB) or N‐(4‐methoxybenzylidene)‐4‐butylaniline (MBBA), the mixing of LDI processes can be detected by the presence of indium ions in the mass spectra. This method has also been found to be useful for investigating the intrinsic properties of the MALDI matrices: DHB samples show an increase in the abundance of fragment ions of matrix molecules and cesium ions with the number of laser pulses irradiating the same sample spot; MBBA samples reveal a decrease in the level of background noise with an increase in the thickness of the sample layer. Copyright © 2009 John Wiley & Sons, Ltd.     

Sample exposure effects in matrix-assisted laser desorption—ionization mass spectrometry of large biomolecules

Effects of sample exposure to UV laser irradiation on the matrix-assisted laser desorption—ionization (MALDI) mass spectra of different proteins are reported. The exposure is varied by irradiating the same sample spot with a differing number of UV laser pulses. The ion yield, mass resolution and internal energy content of ejected molecular ions are monitored as a function of the sample exposure. Other parameters that influence the MALDI spectra (and related to sample exposure) such as laser fluence, sample thickness, matrix-to-protein molar ratio, total deposited amount, and molecular mass of the protein, are also examined.