Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry

The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned.     

Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).     

Structural analysis of oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino)ethyl ester by matrix-assisted laser desorption/ionization mass spectrometry

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans.     

Direct mass spectrometric analysis of ozonides: application to unsaturated glycerophosphocholine lipids

The reaction of ozone with double bonds present in glycerophosphocholine lipids results in formation of ozonides that can be directly analyzed by mass spectrometry as either positive or negative molecular ion species generated by electrospray ionization. Polyunsaturated fatty acyl groups esterified to the phospholipid yielded a mixture of ozonide species with the maximum number of ozone molecules added equal to the total number of double bonds. Ozonide decomposition resulted in omega-aldehyde and omega-carboxylic acid products as revealed by ESI-MS. Collisional activation of the ozone adducts for mono- and polyunsaturated phospholipids gave rise to fragment ions indicative of the position of the double bonds in these molecules. The major decomposition pathway for either positive or negative ozonide ion species involved charge remote fragmentation of the ozonide initiated by homolytic cleavage of the peroxide bridge followed by rearrangement to form the omega-aldehyde and omega-carboxylate acyl species. The reaction of ozone with phospholipids containing polyunsaturated fatty acyl groups is a useful method to probe the position of double bonds by electrospray ionization mass spectrometry.     

Applications of in-source fragmentation of protein ions for direct sequence analysis by delayed extraction MALDI-TOF mass spectrometry

In matrix-assisted laser desorption/ionization of proteins, there exists a certain amount of fast metastable decay immediately after laser irradiation. The fragment ions thus formed can be resolved and their m/z values measured accurately by employing delayed extraction linear time-of-flight mass spectrometry. At higher than threshold laser fluences, proteins exhibit a series of fragment ions providing useful sequence information. We also observe that when moderate amounts of salts are present in the sample with sinapinic acid being the matrix, the intensities of cn ions (N-terminal fragments) are enhanced compared to other types of fragment ions. This enhancement in cn ion signals allows direct sequencing of proteins. The cn ions are completely absent when Xxx-Pro bonds are encountered and are of lower intensity when Xxx-Gly bonds are involved. Further, the cn ion series is interrupted at Xxx-Cys, when the cysteine is involved in a disulfide bond. Upon reduction of the disulfide bonds, the series continues and information is available for longer stretches. Using 10-20 pmol of recombinant proteins, sometimes contiguous sequence information up to 70 residues is obtained in a matter of minutes. Applications of the technique to some recombinant proteins with intra- or interchain disulfide linkages are presented.     

Comparison of fragmentation modes for the structural determination of complex oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry

Fragment ions from underivatized N-linked oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry were obtained by spontaneous fragmentation on a magnetic sector mass spectrometer, by post-source decay (PSD) on a reflectron time-of-flight (TOF) instrument and by collision-induced dissociation on a magnetic sector instrument fitted with an orthagonal-TOF analyser. Spontaneous fragmentation on the magnetic sector instrument produced ions mainly by glycosidic cleavage together with two abundant ions formed by cross-ring cleavage of the reducing-terminal residue. The PSD spectra were similar, the majority of ions being formed by glycosidic cleavage. Internal fragment ions were abundant. High-energy collision-induced dissociation spectra recorded with the orthagonal-TOF analyser, differed considerably from the other types of spectra, particularly in the appearance of major fragment ions produced by cross-ring cleavages of most of the constituent monosaccharide residues. These ions allowed much sequence and branching information to be obtained from the oligosaccharide.     

Mass spectrometric analysis of permethylated glycosphingolipids II. Comparative studies on different blood-group active and related erythrocyte membrane glycosphingolipids

Three isomeric ceramide tetrasaccharides--P blood-group active globoside, lacto-N-neotetraosyl ceramide as ABH blood-group precursor, both isolated from human erythrocytes and "asialo ganglioside" from human brain as reference standard--and two ceramide pentasaccharides--H blood-group active glycosphingolipid, obtained from blood-group B active ceramide hexasaccharide of human B erythrocytes after alpha-galactosidase treatment and ceramide pentasaccharide from rabbit erythrocytes with B-like blood-group activity--were investigated by mass spectrometry after permethylation. The carbohydrate moiety exhibits differences not only concerning the sugar sequence but also with regard to the position of some glycosidic linkages: Oligosaccharides containing N-acetylhexosamine substituted at position 4 produce spectra that are distinctly different from those containing C-3 substituted N-acetylhexosamines, thus allowing the differentation between type 1 and type 2 carbohydrate chains. Moreover, oligosaccharide ions with a hexose at the cleavage site exhibit a fragmentation pattern different from those with a N-acetylhexosamine at the "reducing terminal". The intensity ratio between parent ion and parent ion -32 mass units is Q greater than or equal to 3 in the first case, whereas in the latter case Q is less than 1. The Q-values are given for 14 oligosaccharide ions. Differences in the composition of the ceramide residues can also be deduced from the mass spectra.     

A MALDI probe for mass spectrometers

A new MALDI probe has been designed that uses transmission geometry. This geometry allows the probe to be fashioned after typical EI/CI solid probes which enables it to be introduced into spatially constrained ion source regions such as encountered in quadrupole ion trap mass spectrometers. In the probe design demonstrated here, light from a fiber optic irradiates the backside of a sample through a small piece of quartz on which the sample has been directly deposited. The performance characteristics exhibited by utilizing this probe for MALDI on a quadrupole ion trap mass spectrometer are similar to those which can be obtained through the traditional methods of implementing MALDI. Spectra have been obtained from 50 fmol of total loading of bombesin, MS/MS has been performed on 5 pmol of des-Arg9-bradykinin, and the analyte ion signal is shown to last for over 2500 laser shots for 2 pmol of bombesin. Optical micrographs showing the crystal distribution of a sample containing 2 pmol of bombesin have been obtained as a function of the number of laser shots for a single sample loading. Although this probe was designed for use with the quadrupole ion trap, it can be adapted for use with all types of mass spectrometers. Thus, with only one laser, one fiber optic, and this probe, MALDI can be performed on multiple instruments in a lab.     

Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone

Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.     

Oligonucleotide sequence and composition determined by matrix-assisted laser desorption/ionization

Molecular weight measurements of several oligonucleotides ranging in size from 12 to 60 bases were performed by matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer (MALDI-TOF). In each case, the mass accuracy was better than 0.1%. Sequences for two 12-base oligonucleotides and a 24-base oligonucleotide were determined using calf spleen phosphodiesterase to sequentially cleave from the 5' end. A MALDI-TOF spectrum of the digest mixture shortly after the addition of the enzyme produced a characteristic oligonucleotide ladder. Molecular ions in the mass spectrum corresponded to the products of enzymatic cleavage, and the mass differences between these peaks identified the individual nucleotides. The resolution and mass accuracy of MALDI-TOF were sufficient to unambiguously identify the individual nucleotides in the 12- and 24-base strands.     

Applications of 1.06-micron IR laser desorption on a Fourier transform mass spectrometer

Various sugars, peptides, and lipids were analyzed on a Fourier transform mass spectrometer using laser desorption and ionization with and without the assistance of matrixes. A compact Nd:YAG laser with an output at 1.06 microns corresponding to fundamental frequency was employed. Gram-negative and Gram-positive bacteria were also subjected to laser desorption mass spectrometry. Characteristics ions of conjugated lipid, formed by attachment of alkali metal cations, endogenous to the cells, were observed. Particle/liquid matrixes (e.g., cobalt in glycerol) proved to be useful with the 1.06-micron laser. The particles absorb efficiently laser radiation in a broad wavelength range. The liquid provides the same advantages as in fast atom bombardment: increased signal-to-noise ratios and enhanced sample lifetimes. The effect of laser power on total ion current was shown to differ for samples with and without the particle/liquid matrix. The Fourier transform analyzer provides MS/MS capability for both positive and negative ions from complex mixtures. Ions desorbed externally are introduced into the cell via a quadrupole ion guide with a lower mass cutoff. Such a setup allows matrix ions to be excluded and thus provides excellent signal-to-noise ratios for lower mass range fragment ions formed inside the cell.     

Molecular weight determination of megadalton DNA electrospray ions using charge detection time-of-flight mass spectrometry

We present results obtained with a novel mass spectrometer capable of determining the mass of multiply charged electrospray ions generated from samples of macromolecules in the megadalton (MDa) size range. The instrument utilizes a sensitive amplifier which can detect the charge on a single ion as it passes through a tube detector. A velocity measurement of an ion with known electrostatic energy provides the ion's mass-to-charge ratio. Simultaneous detection of the ion charge permits a mass assignment to be made for each ion. Electrospray ions of DNA and polymer molecules with masses greater than 1 x 10(6) Da and charge numbers (z) in excess of 425 e(-) are readily detected in this mass spectrometer. The weights of small particles were also measured. The on-axis single-ion detection configuration provides a duty cycle of nearly 100% and extends the practical application of electrospray mass spectrometry to the analysis of very large molecules with relatively inexpensive instrumentation.     

De novo peptide sequencing in an ion trap mass spectrometer with 18O labeling

De novo peptide sequencing in an ion trap mass spectrometer coupled on-line with a capillary HPLC using 18O labeling provides a viable alternative to the method using the combination of nanospray, 18O labeling and a quadrupole/time-of-flight mass spectrometer. Seven to sixteen amino acid residues can be sequenced from the liquid chromatography/randem mass spectrometry (LC/MS/MS) spectra. This approach combines the benefit of capillary LC and the high sensitivity of the ion trap operated in the MS/MS mode. The wide availability of the LCQ mass spectrometer makes this approach readily adaptable to the biological mass spectrometry community.     

DNA damage by tert-butoxyl radicals generated in the photolysis of a water-soluble, DNA-binding peroxyester acting as a radical source

The photolysis of the water-soluble perester 1 leads to tert-butoxyl radicals as confirmed by EPR studies with the spin trap 5, 5-dimethylpyrroline N-oxide (DMPO). In the presence of DNA, oxidative cleavage of the latter was demonstrated by the formation of strand breaks in supercoiled pBR 322 DNA and by a substantial decrease of the melting temperature of salmon testes DNA. Guanidine, released from, for example, oxazolone and oxoimidazolidine on base treatment, was observed with calf thymus DNA and 2'-deoxyguanosine. These DNA modifications were effectively inhibited by the radical scavenger di-tert-butylcresol or the hydrogen atom donor glutathione. Photosensitization by the arene chromophore was excluded since the corresponding ester 2 caused no DNA damage, nor were the photoproducts of the perester 1 active. The efficacy of the perester 1 in oxidizing DNA derives from the fact that the tert-butoxyl radicals are photolytically generated in the immediate vicinity of the DNA, due to electrostatic binding of the cationic perester to the DNA, as confirmed by fluorescence measurements. These results demonstrate that the photolysis of perester 1 provides a suitable source of tert-butoxyl radicals in aqueous media, a necessary prerequisite for biochemical investigations.     

An external source 7 T Fourier transform ion cyclotron resonance mass spectrometer with electrostatic ion guide

An external source 7 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer offers three main novel features. First, a 9-way ion-source cross allows for mounting of up to three ionization sources simultaneously, thereby minimizing 'downtime' for changing ion sources. Second, an electrostatic (wire-in-cylinder) ion guide transports the ions approximately 1.5 m from the ion source to the ion trap for mass analysis, through a large magnetic field gradient. Third, the system operates from a modular data system described elsewhere in this issue. Luteinizing hormone-releasing hormone (LH-RH) matrix-assisted laser desorption/ionization (MALDI) FTICR positive-ion mass spectra exhibit signal-to-noise ratio greater than 1000:1 and mass resolving power, m/delta m 50% > 100,000. Laser-induced fragmentation of bradykinin demonstrates the ability of the ion guide to transmit both molecular and fragment ions simultaneously. Ultra-high resolution (average resolving power approximately 400,000) was achieved for poly(ethylene glycol) of specified number-average molecular weight, Mn approximately 3400. Future installation of an electrospray source to the ion-source cross should allow for better characterization of the performance of the ion guide.     

Highly efficient photochemical generation of a triple bond: synthesis, properties, and photodecarbonylation of cyclopropenones

UV irradiation of alkyl-, aryl-, and heteroatom-substituted cyclopropenones results in the loss of carbon monoxide and the formation of quantitative yields of corresponding alkynes. The quantum yield of the photochemical decarbonylation reaction ranges from 20% to 30% for alkyl-substituted cyclopropenones to above 70% for the diphenyl- and dinaphthylcyclorpopenones. Rapid formation (<5 ns) and then a somewhat slower decay (ca. 40 ns) of an intermediate in this reaction was observed by using laser flash photolysis. The DFT calculations allowed us to identify this intermediate as a zwitterionic species formed by a cleavage of one of the carbon-carbon bonds of the cyclopropenone ring. The latter then rapidly loses carbon monoxide to produce the ultimate acetylenic product. Despite their high photoreactivity, cyclopropenones were found to be thermally stable compounds with the exception of hydroxy- and methoxy-substituted cyclopropenones. The latter undergo rapid solvolysis in hydroxylic solvents even at room temperature. The application of this reaction to the in situ generation of the enediyne structure was illustrated by the photochemical preparation of benzannulated enediyne 12.     

Caged RNA: photo-control of a ribozyme reaction

We report here the first photo-chemical control of a ribozyme reaction by the site-specific modification of the 2'-hydroxyl nucleophile in the hammerhead system with a caging functionality. Rapid laser photolysis of the O-(2-nitrobenzyl) caging group initiates an efficient and accurate hammerhead-catalyzed cleavage of substrate RNA under native conditions. RNAs in which reactive functionalities or recognition elements are caged in this manner will be useful tools to probe RNA reactivity and dynamics.     

Performance characteristics of a chemical imaging time-of-flight mass spectrometer

A chemical imaging time-of-flight secondary ion mass spectrometer is described. It consists of a liquid metal ion gun, medium energy resolution reflectron mass analyzer, liquid nitrogen cooled sample stage, preparation chamber and dual stage entry port. Unique features include compatibility with laser postionization experiments, large field of view, cryogenic sample handling capability and high incident ion beam current. Instrument performance is illustrated by the characterization of scanning electron microscopy grids, silver and functionalized polystyrene beads and the postionization of an organic overlayer on a gold substrate.     

alpha-d-glucopyranosyl-, d-alanyl- and l-lysylcardiolipin from gram-positive bacteria: analysis by fast atom bombardment mass spectrometry

Cardiolipin species substituted on O2 of the middle glycerol moiety with alpha-d-glucopyranosyl, d-alanyl and l-lysyl residues were isolated from different gram-positive bacteria. There respective structures were elucidated by positive and negative mode fast atom bombardment mass spectrometry. The structural heterogeneity due to different fatty acid combinations was documented by up to seven molecular ions. General structural features were derived from diagnostic fragment ions, generated by single cleavage at the phosphodiester moieties in both positive and negative ion mode. A diagnostically important fragment ion for d-alanylcardiolipin was observed in the positive ion mode. It arose from double cleavage of the phosphodiester moieties yielding [NaH(Na) PO4-CH2.CH(OCO.CHNH2.CH3) CH2+]+. The fatty acid combinations in the phosphatidyl and diacylglycerol ions make it possible to recognize whether saturated and unsaturated fatty acids were selectively or randomly distributed on the two positions of the glycerol moieties. Molecular structures of cardiolipins, derived from mass spectrometric experiments, are in full agreement with those, elucidated by classical chemical analyses.     

一种质谱仪离子透镜电源的研制及应用

质谱仪由很多功能模块组成,比如:离子源、离子透镜系统、检测器以及真空部件等,其中离子透镜系统负责将离子源产生的离子传输至检测器,同时去除产生干扰的分子和光子,影响仪器的灵敏度及检测下限等指标。介绍了一种基于ARM数字控制的离子透镜电源,该电源可实现多通道电源动态设定并实时监控电源输出,结合离子透镜的机械部件可实现离子的传输,同时也能实现透镜电压的自动调节以满足不同荷质比离子的高效传输,提高仪器的灵敏度。通过质谱仪样机实验表明,该系统设计可靠、电源输出稳定,可以满足质谱仪对离子透镜电源的需要。