Differentiation of enantiomers using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has successfully been used to differentiate pseudo-enantiomeric (isotopically labelled) amino acids by using cyclodextrin as complexing host. By using different pseudo-enantiomeric mixtures (i.e. R(Dn) + S; and R + S(Dn)), it has been demonstrated that the preference of cyclodextrin for S-enantiomers is not due to the size differences caused by the hydrogen/deuterium substitution. It is postulated that this method can be extended to differentiate enantiomers (and determine enantiomeric excess) by using a pair of enantiomeric hosts, as demonstrated previously using other ionization techniques, but with much higher sensitivity.     

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.     

On-line atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A new technique is presented for the coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry with liquid delivery systems. Mass measurements of polymers and peptides are demonstrated using a co-dissolved matrix, e.g. alpha-cyano-4-hydroxycinnamic acid (HCCA). Improvements in terms of sensitivity are achieved by optimizing the shape und control of the exit capillary and by using a laser (355 nm) at a 1 kHz repetition rate. Two calibration experiments promise a good applicability of the presented coupling method for quantitative measurements. The limit of detection achieved so far is 500 nM for peptides in methanol solution containing 25 mM HCCA.     

Characterization of tetrathiofulvalene compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tetrathiofulvalene compounds are important components of charge-transfer complexes, which may be applied in various fields of scientific research and practical applications. Some of these compounds cannot be characterized by mass spectrometry. Here, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for the characterization of tetrathiofulvalenes. The samples could be easily desorbed and ionized to form singly charged ions, and mass spectra with isotopic resolution readily obtained. The mass spectrometric results for 26 compounds have shown that MALDI-TOF is more effective and convenient than other mass spectrometry methods, and resolves the problem of mass spectrometric characterization of tetrathiofulvalene compounds.     

Investigation of the profiling depth in matrix-assisted laser desorption/ionization imaging mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is generally considered to be a surface analysis technique. In this report, the profiling depth of imaging mass spectrometry was examined. MALDI matrix solution was found to be able to gain access to the tissue interior and extract analyte molecules to the tissue surface. As a consequence, prazosin, a small molecule pharmaceutical compound, located as deep as 40 microm away from the surface was readily detected after matrix application. Likewise, cytochrome c, a 12 kDa protein, was also detectable from the tissue interior. Moreover, for prazosin, not only the extent of matrix effect, but also the extraction efficiency of the matrix solvent appeared to be dependent on the type of tissue. These results indicated that experimental conditions that decrease the matrix solvent evaporation during matrix application may increase analyte extraction efficiency and hence sensitivity of the analysis. Furthermore, thin sections should be used to avoid differential extraction efficiency of matrix solvent in different tissues for whole-body analysis.     

Investigation of diterpenoid isosteviol ammonium derivatives by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra of diterpenoid isosteviol ammonium derivatives have been analyzed. The compounds were synthesized as antibacterial agents. It has been found that peaks of salt cations were detected in positive ion MALDI mass spectra of mono-quaternary derivatives. The fragmentation of bis-quaternary salts depended on the distance between the ammonium centers. Clusters containing dication and acidic matrix anion have been observed for compounds with two quaternized nitrogen atoms connected by a nonanomethylene or a longer spacer. Similar clusters did not form for compounds containing pentamethylene or a shorter spacer between two quaternized nitrogen atoms.     

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.     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Chemical degradation methods combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and post-source decay (PSD)-MALDI reflex TOF mass spectrometry (MS) were used to determine the sequence of a peptide branched on to a known peptide backbone. This study was applied to a branched peptide model (derivative of substance P). The branched peptide mimics a digest of a membrane receptor on to which a derivative of substance P was photochemically linked. Chemical degradation based on N-terminal ladder sequencing in combination with MALDI-TOF-MS gave only partial sequence information. Although single PSD mass spectra still remain difficult to interpret unambiguously, PSD-MALDI-TOF-MS was combined with on-target acetylation and H -- D exchange to give a better and successful approach to the unambiguous determination of the complete amino acid side-chain sequence. This study shows the capability of MALDI-TOF-MS to help in characterizing ligand-receptor interactions.     

Structural characterization of carcinogen-modified oligodeoxynucleotide adducts using matrix-assisted laser desorption/ionization mass spectrometry

The aim of this study was to determine the chemical structure of in vitro 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-modified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization mass spectrometry. A single-stranded 11-mer ODN, 5'-d(CCATCGCTACC), was reacted with N-acetoxy-PhIP, resulting in the formation of one major and eight minor PhIP-ODN adducts. A 10 min treatment of the major and one minor PhIP-ODN adduct with a 3'-exonuclease, bovine intestinal mucosa phosphodiesterase (BIMP), and a 5'-exonuclease, bovine spleen phosphodiesterase, results in inhibition of the primary exonuclease activity at deoxyguanosine (dG) producing 5'-d(CCATCG(PhIP)) and 5'-d(G(PhIP)CTACC) product ions, respectively. Post-source decay (PSD) of these enzymatic end products identifies dG as the sole modification site in two 11-mer ODN-PhIP adducts. PSD of the minor PhIP-ODN adduct digestion end product, 5'-d(CCATCG(PhIP)), also reveals that the PhIP adducted guanine moiety is in an oxidized form. Prolonged treatment of the PhIP-ODN adducts at 37 degrees C with BIMP induces a non-specific, or endonuclease, enzymatic activity culminating in the formation of deoxyguanosine 5'-monophosphate-PhIP (5'-dGMP-PhIP). The PSD fragmentation pattern of the 5'-dGMP-PhIP [M + H](+) ion of the major adduct confirms PhIP binds to the C-8 position of dG. For the minor adduct, PSD results suggest that PhIP binds to the C-8 position of an oxidized guanine, supporting the hypothesis that this adduct arises from oxidative degradation, resulting in a spirobisguanidino structure.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

Probing proteins on functionalized silicon surfaces using matrix-assisted laser desorption/ionization mass spectrometry

Flat H-terminated Si(111) substrates modified with alkyl monolayers terminated with hydrophobic and hydrophilic functional groups were prepared using known surface functionalization methods and characterized by FTIR, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The surfaces were then used for the study of non-specific binding of proteins from complex mixtures (using standard mixture of proteins with average molecular weight approximately 6-66 kDa) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protein adsorption on these surfaces (following on-probe fractionation of the mixture) was found to be dependent on the nature of surface functional groups, and nature and pH of rinsing solutions used. The results obtained in this work demonstrate that simple silicon-based surface modifications can be effective for direct analysis of complex mixtures by MALDI-MS. Preliminary results obtained using similarly functionalized porous silicon substrates proved that such substrates are (due to their increased surface areas) better performing than flat silicon.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.     

Surface-activated and matrix-assisted laser desorption/ionization mass spectrometry of organotellurium compounds

Various types of ionization of organotellurium compounds in mass-spectrometric study are considered, with diphenyl telluroxide as example. The mass spectra of diphenyl telluroxide are presented. The possibility of applying surface-activated and matrix-assisted laser desorption/ionization mass spectrometry to organotellurium compounds is examined.     

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).     

Structural features of polyacylated anthocyanins using matrix-assisted laser desorption/ionization and electrospray ionization time-of-flight mass spectrometry

In our continuing studies to isolate water-soluble vacuolar pigments, we expect to elucidate more structural details using mass spectrometry (MS). Because of its sensitivity, only a small amount of pigment extracted from natural plants is required for MS measurement. Nuclear magnetic resonance is also a useful spectroscopic method for structural determination. In this study, two soft ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), on time-of-flight (TOF) mass spectrometers, were used to analyze five polyacylated anthocyanins with more than two aromatic acid molecules in the side chains. ESI is advantageous for the detection of individual molecular ions, while MALDI is essential for the detection of characteristic fragment ions originating from the anthocyanidin. Although 2,5-dihydroxybenzoic acid (DHBA) is an effective matrix in MALDI-TOFMS to obtain informative fragment ions of polyacylated anthocyanins, α-cyano-4-hydroxycinnamic acid (CHCA) is the preferred matrix for the identification of aglycones. In particular, in measurements of polyacylated anthocyanins with two acylated glycoside chains, fragment ions originating from anthocyanidin can only be observed in MALDI-TOFMS using CHCA as the matrix.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.