Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.     

New fragmentation mechanisms in matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates

The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Differentiating structural isomers of sialylated glycans by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry

Using model acidic glycans, we demonstrate the benefits of permethylation for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) tandem mass spectrometry. With both the linear and branched structures, extensive cross-ring fragmentation product ions were generated, yielding valuable information on sugar linkages. Elimination of the negative charges commonly associated with sialylated structures through permethylation allowed their structural analysis in the positive ion mode. Extensive A- and X-type ions were observed for the linear structures, and slightly weaker signals for the branched sialylated structures. The diagnostic cross-ring fragments, permitting a distinction between alpha2-3 and alpha2-6 linkages of the sialic acid residues, were seen in abundance. Importantly, the cross-ring fragmentation with the branched structures provides adequate information to assign sialic acid residues, with a specific linkage, to a particular antenna.     

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.     

Liquid secondary ionization,tandem and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric characterization of glycosphingolipid derivatives

Permethylated, peracetylated and perbenzoylated derivatives of glycosphingolipids (GSLs) were prepared to compare their liquid secondary ionization mass spectrometric (LSIMS) and collision-induced dissociation tandem mass spectrometric (CID/MS/MS) fragmentation patterns and also to determine sensitivity improvement in LSIMS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) relative to the native species. Permethylation was carried out in the liquid phase, whereas peracetylation and perbenzoylation could be effected using either liquid (bulk)-phase or gas-phase procedures. Lower amounts of starting material were required for the gas-phase derivatization (? 100 pmol) compared with the bulk phase (?1 nmol), because the former method permits more efficient sample handling. All three types of derivatives yielded sensitivity improvements of at least two orders of magnitude over the native species in both LSIMS and MALDI-TOFMS. The behavior of the permethylated compounds was used as the benchmark for GSL structural information content in normal and tandem mass spectra. Fragments present in spectra of the three types of derivatives generated complementary information. Permethylated GSLs favored the formation of ions related to the ceramide moieties, whereas peracetylation enhanced the production of carbohydrate-related ions. The LSI mass spectra of perbenzoylated GSLs contained information on both ceramide and sugar portions of the molecules. Each of the LSIMS, MS/MS and MALDI-TOFMS techniques proved to be complementary to the others in this study; the use of all three is recommended for the generation of complete structural information.     

Specific recognition of DNA bulge structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study reports a novel approach utilizing an octahedral CoII(HAPP)(TFA)2 reagent in the presence of H2O2 with analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to serve as an efficient probe for bulged DNA structures. Elucidation of DNA bulge-specific recognition pathways and cleavage mechanisms is demonstrated by characterization of bulge-specific cleavage products and other backbone lesion fragments. The cleavage specificity of CoII(HAPP)(TFA)2/H2O2 arises from sugar oxidative strand scission, for which the position of the abstracted hydrogen is unambiguously determined as the 4'-H of the deoxyribose moiety. Furthermore, differentiation between bulge-specific recognition and diffusion-controlled non-selective cleavage can be clarified through time-dependent MALDI-TOFMS studies. The present results demonstrate that MALDI-TOFMS can be a sensitive and efficient technique for complex mechanistic studies of this kind, providing information for future rational drug design targeting bulged DNA structures.     

Covariance mapping in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A novel method for acquisition and numerical analysis of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectral data is described. The digitized ion current transient from each consecutive laser shot is first acquired and stored independently. Subsequently, statistical correlation parameters between all stored transients are computed. We illustrate the uses of this event-by-event analysis method for studies of sample surface heterogeneity as well as for elucidating the mechanisms of ion formation in MALDI. Other potential applications of the method are also outlined.     

Evaluation of combined matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry experiments for peptide mass fingerprinting analysis

Peptide Mass Fingerprinting (PMF) is still of significant interest in proteomics because it allows a large number of complex samples to be rapidly screened and characterized. The main part of post-translational modifications is generally preserved. In some specific cases, PMF suffers from ambiguous or unsuccessful identification. In order to improve its reliability, a combined approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) was evaluated. The study was carried out on bovine serum albumin (BSA) digest. The influence of several important parameters (the matrix, the sample preparation method, the amount of the analyte) on the MOWSE score and the protein sequence coverage were evaluated to allow the identification of specific effects. A careful investigation of the sequence coverage obtained by each kind of experiment ensured the detection of specific peptides for each experimental condition. Results highlighted that DHB-FTICRMS and DHB- or CHCA-TOFMS are the most suited combinations of experimental conditions to achieve PMF analysis. The association (convolution) of the data obtained by each of these techniques ensured a significant increase in the MOWSE score and the protein sequence coverage.     

Rapid identification of environmental bacterial strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In recent years, various mass spectrometry procedures have been developed for bacterial identification. The accuracy and speed with which data can be obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) could make this a powerful tool for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. Therefore, the first objectives of this study were the adjustment and the optimization of experimental parameters allowing a rapid identification of whole bacterial cells without laborious sample preparation. The tested experimental parameters were matrix, extraction solvent, salt content, deposition method, culture medium and incubation time. This standardized protocol was applied to identify reference and environmental bacterial strains of Escherichia coli, Salmonella and Acinetobacter. The environmental bacterial strains were isolated from sewage sludge using an original microextraction procedure based on repeated sonications and enzymatic treatments. The bacterial identification was realized by the observation of the respective genus-, species- and strain-specific biomarkers. This bacterial taxonomy could be completed within one hour, with minimal sample preparation, provided that sufficient bacteria had been collected prior to MALDI-TOF analysis.     

A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance

A matrix-assisted laser desorption/ionization (MALDI) source has been coupled to a tandem quadrupole/time-of-flight (QqTOF) mass spectrometer by means of a collisional damping interface. Mass resolving power of about 10,000 (FWHM) and accuracy in the range of 10 ppm are observed in both single-MS mode and MS/MS mode. Sub-femtomole sensitivity is obtained in single-MS mode, and a few femtomoles in MS/MS mode. Both peptide mass mapping and collision-induced dissociation (CID) analysis of tryptic peptides can be performed from the same MALDI target. Rapid spectral acquisition (a few seconds per spectrum) can be achieved in both modes, so high throughput protein identification is possible. Some information about fragmentation patterns was obtained from a study of the CID spectra of singly charged peptides from a tryptic digest of E. coli citrate synthase. Reasonably successful automatic sequence prediction (>90%) is possible from the CID spectra of singly charged peptides using the SCIEX Predict Sequence routine. Ion production at pressures near 1 Torr (rather than in vacuum) is found to give reduced metastable fragmentation, particularly for higher mass molecular ions. Copyright 2000 John Wiley & Sons, Ltd.     

Identification of isoenzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The identification of isoforms is one of the great challenges in proteomics due to the large number of identical amino acids preventing their separations by two-dimensional electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become a rapid and sensitive tool in proteomics, notably with the new instrumental improvements. In this study, we used several acquisition modes of MALDI-TOFMS to identify isoforms of porcine glutathiones S-transferase. The use of multiple proteases coupled to the different acquisition modes of MALDI-TOFMS (linear, reflectron, post-source decay (PSD) and in-source decay, positive and negative modes) allowed the identification of two sequences. Moreover, a third sequence is pointed out from a PSD study of a tryptic ion revealing the modification of the amino acid tyrosine 146 to phenylalanine.     

Off-line hyphenation of boronate affinity monolith-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for efficient analysis of glycoproteins/glycopeptides

Boronate affinity materials have attracted increasing attentions as sample enrichment platforms for glycoproteomic analysis in recent years. However, most of the boronate affinity materials that have already employed for proteomic analysis are suffering from apparent disadvantages, such as alkaline pH for binding, weak affinity, and relatively poor selectivity. Benzoboroxoles are a unique class of boronic acids which have showed excellent binding properties for the recognition of cis-diol-containing compounds. Recently, a 3-carboxy-benzoboroxole-functionalized monolithic column had been reported and it had exhibited the best selectivity and affinity as well as the lowest binding pH among all reported boronate affinity monolithic columns. In this study, an off-line hyphenation of this boronate affinity monolithic column-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and the powerfulness of this hyphenated approach in the analysis of glycoproteins and glycopeptides in complex samples was investigated. The approach was first applied to the analysis of glycopeptides in the tryptic digest of horseradish peroxidase (HRP). Totally 22 glycopeptides were identified. To the best of our knowledge, this is the best performance among all the boronic acid-functionalized materials. We further employed this approach to the analysis of intact proteins in human saliva. Totally 6 intact glycoproteins were successfully identified. As comparison, when the samples were analyzed without extraction, only a few glycopeptides were identified from the tryptic digest of HRP while no glycoproteins were found from the saliva samples.     

On-target endoglycosidase digestion matrix-assisted laser desorption/ionization mass spectrometry of glycopeptides.

The digestion of glycopeptides with endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 degrees C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The endoglycosidases used for digestion were endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger.     

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small molecules

A variety of surfactants have been tested as matrix-ion suppressors for the analysis of small molecules by matrix-assisted laser desorption/ionization time-of flight mass spectrometry. Their addition to the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) greatly reduces the presence of matrix-related ions when added at the appropriate mole ratio of CHCA/surfactant, while still allowing the analyte signal to be observed. A range of cationic quaternary ammonium surfactants, as well as a neutral and anionic surfactant, was tested for the analysis of phenolics, phenolic acids, peptides and caffeine. It was found that the cationic surfactants, particularly cetyltrimethylammonium bromide (CTAB), were suitable for the analysis of acidic analytes. The anionic surfactant, sodium dodecyl sulfate, showed promise for peptide analysis. For trialanine, the detection limit was observed to be in the 100 femtomole range. The final matrix/surfactant mole ratio was a critical parameter for matrix ion suppression and resulting intensity of analyte signal. It was also found that the mass resolution of analytes was improved by 25-75%. Depth profiling of sample spots, by varying the number of laser shots, revealed that the surfactants tend to migrate toward the top of the droplet during crystallization, and that it is likely that the analyte is also enriched in this surface region. Here, higher analyte/surfactant concentration would reduce matrix-matrix interactions (known to be a source of matrix-derived ions).     

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides.     

Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation

We explored matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry for the analysis of N-glycosylated peptides, using horseradish peroxidase (HRP) as a test case. Two different types of cleavage were observed in the TOF/TOF fragmentation spectra: Firstly, cleavages of peptide bonds yielded fragments with the attached N-glycans staying intact, thus revealing information on peptide sequence and glycan attachment site. Secondly, fragmentation of the glycan moiety was characterized by cleavage of glycosidic bonds as well as a (0,2)X-ring fragmentation of the innermost N-acetylglucosamine of the chitobiose core. Loss of the complete N-glycan moiety occurred by cleavage of both the N-glycosidic bond and the side-chain amide group of the N-glycosylated asparagine, yielding a characteristic peak doublet with a mass difference of 17 Da, which revealed the individual masses of the N-glycan and the peptide moiety. Analysis of a HRP tryptic digest at the sub-picomole level allowed the characterization of various N-glycosylated peptides including those with internal disulfide linkages, a glycopeptide linked via a disulfide bond to another peptide, and a 5 kDa glycopeptide carrying two N-glycans. The potential of our approach was illustrated by the detection of the following novel features of HRP glycosylation: (i) The conjugation of a xylosylated trimannosyl N-glycan without core-fucosylation to site Asn316, showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine1deoxyhexose1 attached to N-glycosylation sites Asn285 and Asn298, which might represent a Fuc(alpha1-3)GlcNAc- moiety arising from the processing of N-glycans by a horse-radish endoglycosidase during biosynthesis of HRP.     

Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.