Nanospray analysis of the venom of the tarantula Theraphosa leblondi: a powerful method for direct venom mass fingerprinting and toxin sequencing

Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS), and nanospray ionisation/hybrid quadrupole time-of-flight mass spectrometry (nanoESI-QqTOFMS), were applied to characterize by mass fingerprinting the venom of the French Guyanese tarantula Theraphosa leblondi. Of these techniques direct nanoESI-QqTOFMS, which allowed the detection of 65 protonated molecules with high mass accuracy, appeared to give the best results. Three major peptides, TlTx1, TlTx2 and TlTx3, were sequenced using a combination of nanoESI-MS/MS and enzyme digestion/MS and MS/MS experiments. Each sequence was confirmed by automated Edman sequencing. In patch-clamp experiments these peptides were found to have a specific inhibitory effect on the voltage-dependent potassium channel, Kv4.2.     

Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry

Techniques in mass spectrometry (MS) combined with chemical cross-linking have proven to be efficient tools for the rapid determination of low-resolution three-dimensional (3-D) structures of proteins. The general procedure involves chemical cross-linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. We present a method for mapping low-resolution 3-D protein structures by combining chemical cross-linking with high-resolution FTICR (Fourier transform ion-cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo-bifunctional, amine-reactive cross-linking reagents that bridge distances from 6 to 16 A. The non-digested cross-linking reaction mixtures were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to determine the extent of cross-linking. Enzymatically digested reaction mixtures were separated by nano-high-performance liquid chromatography (nano-HPLC) on reverse-phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-ESI (electrospray ionization) source. Cross-linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross-linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross-linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme.     

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethylcyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS.     

Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix-assisted laser desorption/ionisation mass spectrometry and high-performance liquid chromatography/electrospray ionisation mass spectrometry

Structural studies of the high molecular weight (HMW) glutenin subunits 1Dy10 and 1Dy12 of bread wheat were conducted using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS). For both proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 500-540 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of both proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture and analysis of the digests was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimising the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in the coverage of the whole protein sequences except for two short fragments (T1 and T8), which are identical in the two homologous subunits, and for an additional dipeptide (T14) in subunit 1Dy12, which were not detected. It also demonstrated that, in contrast to the gene-derived data, the sequence of subunit 1Dy12 does not include the dipeptide Gly-Gln between residues Gln(454) and Pro(455), and that the lower mass components present in both fractions correspond to the same sequences lacking short peptides that are probably lost from the protein N- or C-termini. Finally, the results obtained provide evidence for the lack of a substantial level of glycosylation or other post-translational modifications of the two subunits, and demonstrate that mass spectrometric mapping is the most useful method presently available for the direct verification of the gene-derived sequences of HMW glutenin subunits and similar proteins.     

Investigation and correction of the gene-derived sequence of glutenin subunit 1Dx2 by matrix-assisted laser desorption/ionisation mass spectrometry

Direct matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) analysis of a mixture of tryptic peptides was used to verify the gene-derived amino acid sequence of the high molecular weight (HMW) subunit 1Dx2 of bread wheat. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, and optimising the matrix and the acquisition parameters for each mass range. This resulted in coverage of the whole sequence except for a short fragment T3 (3 amino acids), which was not detected. It also allowed the insertion of a Pro residue in position 59 to be identified. The results obtained provide evidence for the lack of a substantial level of glycosylation or other post-translational modifications of subunit 1Dx2, and demonstrate that MALDI-MS is the most useful method presently available for the direct verification of the gene-derived sequences of HMW glutenin subunits and similar proteins.     

A novel mass spectrometry cluster for high-throughput quantitative proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.     

Investigation of sulfhydryl groups in cabbage phospholipase D by combination of derivatization methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

All eight cysteine residues in 92 kDa cabbage phospholipase D (PLD), deduced from the cDNA sequence, were shown to have free sulfhydryl groups by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of tryptic peptides of PLD derivatized with p-chloromercurybenzoate, iodoacetic acid, and N-ethylmaleimide, as well as of underivatized PLD. Assignment of sulfhydryl groups by any one method was not conclusive. However, complementary information derived from tryptic peptides derivatized with different reagents made full assignment of sulfhydryl groups possible.     

External accumulation of ions for enhanced electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.     

Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.     

Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.     

A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.     

Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences, e.g., between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a hybrid quadrupole-7 tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer that provides data-dependent ion selection, accumulation, and dissociation external to the ICR trap, and a control software that directs intelligent MS/MS target selection based on LC elution time and m/z ratio. We show that the continuous on-the-fly alignment of the LC elution time during the targeted LC-MS/MS experiment, combined with the high mass resolution of FTICR MS, is crucial for accurate selection of targets, whereas high mass measurement accuracy MS/MS data facilitate unambiguous peptide identifications. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic versus aerobic conditions demonstrates the feasibility of such approach.     

Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence for sex-based variation among the bradykinin-potentiating peptides

Variation in the snake venom proteome is well documented and it is a ubiquitous phenomenon at all taxonomical levels. However, variation in the snake venom peptidome is so far not described. In this work we used mass spectrometry [liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOFMS)] to explore sex-based differences among the venom peptides of eighteen sibling specimens of Bothrops jararaca of a single litter born and raised in the laboratory. MALDI-TOFMS analyses showed individual variability among the bradykinin-potentiating peptides (BPPs), and, interestingly, four new peptides were detected only in female venoms and identified by de novo sequencing as cleaved BPPs lacking the C-terminal Q-I-P-P sequence. Similar results were obtained with venom from wild-caught adult non-sibling specimens of B. jararaca and in this case we were able to identify the gender of the specimen by analyzing the MALDI-TOF profile of the peptide fraction and finding the cleaved peptides only in female venoms. Synthetic replicates of the cleaved BPPs were less potent than the full-length BPP-10c in potentiating the bradykinin hypotensive effect, suggesting that the C-terminus is critical for the interaction of the BPPs with their mammalian molecular targets. This work represents a comprehensive mass spectrometric analysis of the peptide fraction of B. jararaca venom and shows for the first time sex-based differences in the snake venom peptidome of sibling and non-sibling snakes and suggests that the BPPs may follow distinct processing pathways in female and male individuals.     

Protein abundance quantification in embryonic stem cells using incomplete metabolic labelling with 15N amino acids,matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry,and analysis of relative isotopologue abundances of peptides

An isotope dilution method for protein quantification is presented in the context of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and mass fingerprinting experiments, revealing an unappreciated high reproducibility and accuracy of relative peak intensity measurements. Labelled proteins were generated by growing cells in a medium containing (15)N-enriched amino acids, and were mixed with proteins of natural isotopic composition from control cells in ratios of approximately 0:1, 1:7, 1:2, 2:1, 7:1, and 1:0 (labelled/unlabelled). Mixtures were separated by two-dimensional gel electrophoresis and analysed by MALDI-TOFMS using typical experimental conditions. A linear relationship is demonstrated between the relative isotopologue abundances (RIA values) for particular peaks in the isotopic distribution of tryptic peptide fragments of the proteins, and the mole fractions of labelled proteins in the mixture. Analysis of RIA values (ARIA quantification) for peptides of six typical silver-stained protein spots for the various mixtures could reproduce the experimentally contrived ratios with approximate errors between 4% (2:1 mixture) and about 18% (1:7 mixture). A consideration of error and its propagation is discussed. ARIA does not require complete separation of the isotope patterns of labelled and unlabelled peptides, and is therefore advantageous in combination with all kinds of labelling experiments in biological systems, because it is compatible with minimal metabolic incorporation of labelling reagent. Simulations indicate that the minimum required (15)N enrichment of the total amino acid pool sufficient for ARIA is less than 4%. In an accompanying paper in this issue, we apply ARIA to proteins differentially labelled with isotope-coded alkylation reagents.     

Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics

A cycloalkyl aliphatic saccharide, 5-cyclohexyl-1-pentyl-beta-D-maltoside (CYMAL-5), was evaluated as a novel additive in a high-throughput in-gel protein digestion system using 96-well plates. Addition of 0.1% CYMAL-5 (final concentration) during trypsin treatment was compatible with both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, and gave a better digestion efficiency than n-octylglucoside, which we previously reported. In-gel reduction and alkylation of Cys residues under denaturing conditions also improved the sequence coverage of peptides. In-gel tryptic digestion with the optimum combination of 0.5 mm thick gels, negative staining, alkylation under denaturing conditions (6 M guanidine hydrochloride), and digestion in the presence of CYMAL-5, gave excellent performance especially for membrane protein analysis, where recovery of hydrophobic peptides was markedly enhanced. The new protocol is simple and convenient, and should be widely applicable to gel-based proteomics.     

On-column digestion of proteins in aqueous-organic solvents

Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.     

A strategy for distinguishing modified peptides based on post-digestion 18O labeling and mass spectrometry

The simultaneous identification of multiple different protein modifications, with or without known mass changes, is a challenging application of mass spectrometry. In this contribution, a strategy for distinguishing modified peptides within a large background of unmodified peptides was demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of cytochrome c (Cyt-c) modified with 4-hydroxy-2-nonenal (HNE), based on post-digestion 18O labeling. Labeling of control Cyt-c peptides obtained from in-solution or in-gel digestion with 18O, prior to mixing in the ratio of 1:1 with peptides derived from a modified sample, identified more HNE modifications than a method based on a known mass increment search (Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136), demonstrating the potential of this strategy to enhance the detection of modified peptides by mass spectrometry. A virtue of the strategy is that it obviates the need for isotopic labeling of the modifier, making the method applicable to the detection of modifications occurring in vivo. Additionally, this technique identified protease auto-cleavage peptides by their altered mass isotopomer distribution due to incomplete 18O exchange, and modified peptides containing 'protein carbonyls' by partial 18O exchange, allowing these peptides to be differentiated during data analysis.     

Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial cultures.

Bacillus subtilis synthesizes the lanthionine containing 32-amino-acid peptide antibiotic (lanti-biotic) subtilin from a ribosomally generated 56-amino-acid precursor pre-propeptide by extensive posttranslational modifications. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to monitor the production of matured subtilin within crude samples taken from B. subtilis culture media without prior fractionation. The processing reaction of subtilin was blocked with the serine protease inhibitor phenylmethylsulfonyl fluoride and different subtilin precursor peptides in the molecular mass range up to 6220 were observed. Two of these species were isolated by reversed-phase high-performance liquid chromatography (HPLC) and structurally analyzed by post-source decay MALDI-TOFMS. We provide evidence that the precursor species comprise the posttranslational modified C-terminal part of subtilin to which leader peptide moieties with different chain lengths are attached. These antimicrobial-inactive species could be processed to antibiotic-active subtilin by incubation with culture media of different subtilin-nonproducing B. subtilis strains as indicated by a combination of antimicrobial growth assays and MALDI-TOFMS analyses. These achievements are strong evidence for the sensitivity of MALDI-TOFMS methodology that allows straightforward investigations of analytes even in complex mixtures without time-consuming sample preparations.     

Proteomics of gluten: mapping of the 1Bx7 glutenin subunit in Chinese Spring cultivar by matrix-assisted laser desorption/ionization

The verification of the cDNA-deduced sequence of the high molecular weight glutenin subunit 1Bx7 in Chinese Spring cultivar was achieved by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the tryptic fragments. The published sequence of the 1Bx7 subunit contains 5 Lys and 15 Arg residues but, due to the presence of three Arg-Pro bonds, which are generally resistant to cleavage by trypsin, or cleaved to a very limited extent by trypsin, 19 peptides can be predicted. The identification of the tryptic fragments was achieved by direct MALDI-MS analysis by using three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures in order to obtain the maximum sequence coverage. MALDI analysis of the 1Bx7 tryptic digest resulted in the identification of the expected peptides and additional fragments arising from non-specific cleavages; the fragments that were not detected are peptides with low mass (from 147.2 to 317.4), so we obtained a sequence coverage of 98.8%. The results reported here also indicated that the sequence of the 1Bx7 subunit from cv. Chinese Spring is different from the cDNA-deduced sequence reported in the literature; in particular, a possible insertion of the hexapeptide QPGQGQ within the sequence Gln630-Tyr725 was suggested. Finally, it is possible to rule out glycosylation of the 1Bx7 subunit, or any other post-translational modification, to within the detection limits of the method.     

Investigation into accurate mass capability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, with respect to radical ion species

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been shown to be an effective technique for the characterization of organometallic, coordination, and highly conjugated compounds. The preferred matrix is 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), with radical ions observed. However, MALDI-TOFMS is generally not favored for accurate mass measurement. A specific method had to be developed for such compounds to assure the quality of our accurate mass results. Therefore, in this preliminary study, two methods of data acquisition, and both even-electron (EE+) ion and odd-electron (OE+.) radical ion mass calibration standards, have been investigated to establish the basic measurement technique. The benefit of this technique is demonstrated for a copper compound for which ions were observed by MALDI, but not by electrospray (ESI) or liquid secondary ion mass spectrometry (LSIMS); a mean mass accuracy error of -1.2 ppm was obtained.