Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry

The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique described to date has proven to be a convenient and rapid method for identification and characterizations of proteins. However, the general MALDI-TOF MS analysis of complex carbohydrates and glycopeptides still entails special consideration of ionization and the fragmentation characteristics of labile carbohydrate moieties. In this study, an efficient and practical method we termed the matrix-dependent selective fragmentation (MDSF) technique of MALDI-TOF/TOF MS, which allows highly sensitive and reliable fragmentation of oligosaccharides and N-glycopeptides. Results from application of the MDSF technique to TOF/TOF MS analysis demonstrated that in comparison to the conventional postsource decay up to 170 times more sensitive product ion peaks could be obtained. It was also suggested that MDSF generates desired structural information based on the controlled cleavage of the singly charged precursor ion with different electronic excited states made by this method. Ideal product ion peaks observed by MDSF in TOF/TOF MS facilitated structural characterization of complex oligosaccharide derivatives including unstable Neu5Ac and Fuc residues and N-glycopeptides.     

Characterizing oligosaccharides using injected-ion mobility/mass spectrometry

Injected-ion mobility/mass spectrometry techniques have been used to measure the reduced ion mobilities for negatively charged raffinose, melezitose and α-, β-, and γ-cyclodextrins formed by electrospray ionization. At low injection energies, the mass spectra are dominated by negatively charged (deprotonated) parent ions. At high injection energies, the mass spectra recorded for the cyclodextrins and raffinose display peaks that result from cross-ring cleavage of individual sugar units. Melezitose dissociates by cleavage of the glycosidic bonds. The ion mobility distributions can be used to distinguish between different isomeric forms of parent and fragment ions having the same mass-to-charge ratios.     

Characterization of Enterobacteria using MALDI-TOF mass spectrometry

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.     

Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography-MALDI-TOF/TOF tandem mass spectrometry

The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.     

Structural characterization of chemically derivatized oligosaccharides by nanoflow electrospray ionization mass spectrometry.

Oligosaccharides released from several glycoproteins were derivatized with either 4-aminobenzoic acid 2-(diethylamino)ethyl ester (ABDEAE) (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) or 2-aminopyridine. The resulting derivatives were analyzed on a nanoflow electrospray ionization (ESI) quadrupole-inlet time-of-flight mass spectrometer using the low-energy collision-induced dissociation technique. In the MS/MS spectra, the oxonium (b or internal series) and y series ions, which are derived from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were observed in the low-mass region, but that were not found in the PSD analyses (Mo, W.; et al. Anal. Chem. 1998, 70, 4520-4526), rendered a more detailed structural insight. The oxonium ions at m/z 512.2, which are derived from the fucosylated oligosaccharides of immunoglobulin Y and thyroglobulin, were observed, suggesting that fucosylation had occurred proximal to the outer nonreducing terminus. In addition, the data herein show that structural elucidation can be routinely achieved at a low sample concentration. For the case of ABDEAE derivatives, this can be achieved at the 50 fmol/microL level and with the actual sample consumption at the attomole level using nanoflow ESI MS/MS.     

Decomposition of ESR spectra using MALDI-TOF mass spectrometry

ESR (or EPR) spectroscopy on spin-labeled site-directed cysteine mutants is ideally suited for structural studies of membrane proteins due to its high sensitivity and its low demands with respect to sample purity and preparation. Many features can be inferred from the spectral line shape of an ESR spectrum, but the analysis of ESR spectra is complicated when multiple sites with different line shapes are present. Here, we present a method to decompose the spectrum of a doubly labeled peptide that is composed of a singly labeled, noninteracting component and a doubly labeled, dipolar-broadened component using a combination of optical and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The effect on the interspin distance calculation based on the dipolar broadening is quantified and discussed.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

Fragmentation characteristics of collision-induced dissociation in MALDI TOF/TOF mass spectrometry

The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.     

Automated tandem mass spectrometry by orthogonal acceleration TOF data acquisition and simultaneous magnet scanning for the characterization of petroleum mixtures

The automated acquisition of the product ion spectra of all precursor ions in a selected mass range by using a magnetic sector/orthogonal acceleration time-of-flight (oa-TOF) tandem mass spectrometer for the characterization of complex petroleum mixtures is reported. Product ion spectra are obtained by rapid oa-TOF data acquisition and simultaneous scanning of the magnet. An analog signal generator is used for the scanning of the magnet. Slow magnet scanning rates permit the accurate profiling of precursor ion peaks and the acquisition of product ion spectra for all isobaric ion species. The ability of the instrument to perform both high- and low-energy collisional activation experiments provides access to a large number of dissociation pathways useful for the characterization of precursor ions. Examples are given that illustrate the capability of the method for the characterization of representative petroleum mixtures. The structural information obtained by the automated MS/MS experiment is used in combination with high-resolution accurate mass measurement results to characterize unknown components in a polar extract of a refinery product. The exhaustive mapping of all precursor ions in representative naphtha and middle-distillate fractions is presented. Sets of isobaric ion species are separated and their structures are identified by interpretation from first principles or by comparison with standard 70-eV EI libraries of spectra. The utility of the method increases with the complexity of the samples.     

Sequence determination of sulfated carrageenan-derived oligosaccharides by high-sensitivity negative-ion electrospray tandem mass spectrometry

Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation is assessed for sequence determination of multiply sulfated oligosaccharide fragments of carrageenan obtained from partial depolymerization of the polysaccharides by either enzymatic digestion or mild acid hydrolysis. Carrageenan oligosaccharides with homogeneous disaccharide compositions were used to establish their fragmentation pattern, which was then applied to sequence determination of unusual oligosaccharides with either "hybrid" biose compositions or odd-numbered residues. As sulfate groups are labile, sulfate loss during collision-induced association was prevented by sodium adduction. The product ion spectra of [M - Na]- (where M represents the sodium salt of oligosaccharides) feature an extensive series of B- and C-type glycosidic cleavages, whereas the Y-type cleavage occurs mainly at the sulfated residues. The assignment of reducing or nonreducing terminal fragments was assisted by oligosaccharide reduction and the product ion spectra of the derived alditols. Due to the anionic nature of the sulfate present, high-sensitivity detection (1-5 pmol, using a hexasaccharide as an example) was obtained.     

Organic ion imaging using tandem mass spectrometry.

A triple-quadrupole mass spectrometer has been interfaced with a wide-angle secondary ion microprobe. The combination permits acquisition of data necessary to determine the distribution of targeted organic analytes even in the presence of overwhelming isobaric interference. Micrographs generated from secondary ion intensity alone are compared to those generated using secondary ionization with tandem mass spectrometry (MS/MS), both for image reference and to show the improvement in image quality that can be attained when MS/MS is employed. Inhomogeneous mixtures of glycerol, KCl, and asparagine on 1-cm-diameter aluminum targets were used to demonstrate the instrument's selectivity. Secondary ions generated from samples of this system include isobaric 133Cs+ implanted from the primary ion beam, the 41K(+)-glycerol adduct, and protonated asparagine.     

Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, Y162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162YnLYEIAR168 and the corresponding decrease in the 162YLYEIAR168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK75, also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.     

Aflatoxin screening by MALDI-TOF mass spectrometry

Efficient detection of aflatoxins B1, B2, G1, and G2 has been performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a UV-absorbing ionic liquid matrix to obtain "matrix-free" mass spectra and addition of NaCl to enhance sensitivity via Na+ cationization. Using ionic alpha-cyano-4-hydroxycinnamic acid (Et3N-alpha-CHCA) as the matrix, matrix-free mass spectra in the m/z range of interest are acquired, and the B1, B2, G1, and G2 aflatoxins are readily detected with an LOD as low as 50 fmol. The technique is fast, requires little sample preparation and no derivatization or chromatographic separation, and seems therefore to be suitable for high-throughput aflatoxin screening. It should be easily extended to other micotoxins and provide an attractive technique to control the quality of major crops subjected to huge world commercial trades such as peanuts, corn, and rice as well as to monitor bioterrorism threats by micotoxin poisoning.     

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Measuring complete isotopomer distribution of aspartate using gas chromatography/tandem mass spectrometry

We have developed a simple and accurate method for determining the complete positional isotopomer distribution of aspartate carbon atoms by gas chromatography/tandem mass spectrometry for (13)C-metabolic flux analysis. First, we screened tandem mass spectrometry (MS) spectra of the tert-butyldimethylsilyl (TBDMS) derivative of aspartate for daughter fragments with the necessary carbon atom fragmentations to fully resolve all 16 isotopomers of aspartate. Tandem MS scanning parameters were optimized for each daughter fragment, and the accuracy of tandem MS measurements were evaluated. We selected five accurate fragments that provided a redundant set of 47 labeling measurements to quantify the complete isotopomer distribution of aspartate by least-squares regression. The validity of the approach was demonstrated using six (13)C-labeled aspartate standards and natural aspartate.     

Sequencing of sulfated oligosaccharides from mucins by liquid chromatography and electrospray ionization tandem mass spectrometry

As part of a strategy for profiling diverse mixtures of sulfated mucin-derived oligosaccharides, liquid chromatography coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative ion mode has been explored. Two mixtures of sulfated oligosaccharide alditols from porcine stomach and large intestine were analyzed by straight phase chromatography using an amino-bonded column connected to a Q-TOF instrument. Nine sulfated mucin-derived oligosaccharide alditols from porcine stomach underwent extensive fragmentation allowing determination of their sequence. The fragmentation generated primary, secondary, and tertiary fragment ions informative for the elucidation of the saccharide sequence and localization of the sulfate group. From a single chromatographic analysis, the sequences of 28 different sulfated mucin oligosaccharide alditols purified from porcine large intestine were elucidated, revealing information concerning prominent core sequences and terminal blood group-type epitopes. Analysis of these two sulfated oligosaccharide mixtures demonstrated the usefulness of HPLC-ESI-MS/MS: the on-line separation of multiple isomeric suffated oligosaccharides as present in biological samples, informative fragmentation allowing the identification of the sequence of nonderivatized oligosaccharides, and a sensitivity sufficient for the analysis of quantities as obtained from natural sources.     

Quantitative thin-layer chromatography/mass spectrometry analysis of caffeine using a surface sampling probe electrospray ionization tandem mass spectrometry system

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 mum/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 muL) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by approximately 8% or more) than the literature values.