Matrix-assisted laser desorption ionization (MALDI) has become an enabling technology for the fields of protein mass spectrometry (MS) and proteomics. Despite its widespread use, for example, in protein identification via peptide mass fingerprinting, a comprehensive model for the generation of free gas-phase ions has not yet been developed. All matrices in use today, such as ␣-cyano-4-hydroxycinnamic acid (CHCA), have been found empirically and stem from the early days of MALDI. By systematic and targeted variation of the functional groups of the ␣-cyanocinnamic acid core unit, 4-chloro-␣-cyanocinnamic acid (Cl-CCA) was selected and synthesized, and it exhibited outstanding matrix properties. Key features are a substantial increase in sensitivity and a considerably enhanced peptide recovery in proteomic analyses because of a much more uniform response to peptides of different basicity. Using Cl-CCA as a matrix for a 1 fmol bovine serum albumin (BSA) in-solution digest, the sequence coverage is raised to 48%, compared with 4% for CHCA. For a gel band containing 25 fmol of BSA, unambiguous protein identification becomes possible with Cl-CCA. These findings also imply ion formation via a chemical ionization mechanism with proton transfer from a reactive protonated matrix species to the peptide analytes. The considerable increase in performance promises to have a strong impact on future analytical applications of MALDI, because current sensitivity limits are overcome and more comprehensive analyses come into reach.ionization mechanism ͉ proton transfer M atrix-assisted laser desorption ionization (MALDI) (1-3) relies on a cocrystallization of the analytes of interest with an excess of small organic compounds, the matrix, which is accomplished by either simply drying Ϸ 1-l droplet of a solution containing matrix and analyte (dried-droplet preparation) (2, 3) or by depositing a droplet of the analyte solution onto a prespotted matrix layer (surface preparation) (4). The matrix exhibits a strong absorption at the laser wavelength used (typically 337 nm or 355 nm in the UV), which, after irradiation with a short pulse of laser light, leads to the ablation of a shallow surface region into the vacuum of the mass spectrometer, the release of intact gaseous matrix and analyte molecules, and their partial ionization. Since its introduction in the 1980s, MALDI has found widespread use in the mass spectrometric analysis of biological macromolecules but recently also has been used increasingly for small-molecule analysis (5, 6).However, the main field of application of MALDI mass spectrometry (MS) today is protein identification in proteomic schemes by using the peptide mass fingerprint (PMF) approach (7), often supplemented by MALDI-time-of-f light/time-offlight (TOF/TOF) analyses. ␣-Cyano-4-hydroxycinnamic acid (CHCA) (8) as a matrix (in positive-ion mode) allows the researcher to identify proteins via MS or MS/MS analysis of their proteolytic peptide products (typically generated by trypsin) and by using protein or DNA seque...

This work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model. Experimental differentiation between the predicted mechanisms becomes possible by the use of deuterated matrix esters as MALDI matrices, which are stable under typical sample preparation conditions and generate deuteronated reagent ions, including the deuterated and deuteronated free matrix acid, only upon laser irradiation in the MALDI process. While the generation of deuteronated analyte ions proves the gas phase protonation model, the detection of protonated analytes by application of deuterated matrix compounds without acidic hydrogens proves the survival of analytes precharged from solution in accordance with the predictions from the Lucky Survivor model. The observed ratio of the two analyte ionization processes depends on the applied experimental parameters as well as the nature of analyte and matrix. Increasing laser fluences and lower matrix proton affinities favor gas phase protonation, whereas more quantitative analyte protonation in solution and intramolecular ion stabilization leads to more Lucky Survivors. The presented results allow for a deeper understanding of the fundamental processes causing analyte ionization in MALDI and may alleviate future efforts for increasing the analyte ion yield.

Thirteen novel PAX (peptide-antimicrobial-Xenorhabdus) peptides were identified in Xenorhabdus nematophila HGB081. Their structures including the absolute configuration were elucidated using a combination of labeling experiments, detailed MS/MS experiments, the advanced Marfey's method, and a detailed analysis of the biosynthesis gene cluster, which was identified as well.

Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029T . Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029 T secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.

The laser wavelength constitutes a key parameter in ultraviolet-matrix-assisted laser desorption ionization-mass spectrometry (UV-MALDI-MS). Optimal analytical results are only achieved at laser wavelengths that correspond to a high optical absorption of the matrix. In the presented work, the wavelength dependence and the contribution of matrix proton affinity to the MALDI process were investigated. A tunable dye laser was used to examine the wavelength range between 280 and 355 nm. The peptide and matrix ion signals recorded as a function of these irradiation parameters are displayed in the form of heat maps, a data representation that furnishes multidimensional data interpretation. Matrixes with a range of proton affinities from 809 to 866 kJ/mol were investigated. Among those selected are the standard matrixes 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) as well as five halogen-substituted cinnamic acid derivatives, including the recently introduced 4-chloro-α-cyanocinnamic acid (ClCCA) and α-cyano-2,4-difluorocinnamic acid (DiFCCA) matrixes. With the exception of DHB, the highest analyte ion signals were obtained toward the red side of the peak optical absorption in the solid state. A stronger decline of the molecular analyte ion signals generated from the matrixes was consistently observed at the low wavelength side of the peak absorption. This effect is mainly the result of increased fragmentation of both analyte and matrix ions. Optimal use of multiply halogenated matrixes requires adjustment of the excitation wavelength to values below that of the standard MALDI lasers emitting at 355 (Nd:YAG) or 337 nm (N(2) laser). The combined data provide new insights into the UV-MALDI desorption/ionization processes and indicate ways to improve the analytical sensitivity.

The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches. In these cases higher effective temperatures could be measured accompanied by increased analyte intensities, which can be explained by stronger volatilization processes caused by a hindered heat dissipation resulting in a raised analyte transfer into the gas phase. For all sublimed compounds, a strong increase of the initial ion velocity compared with DD preparations could be measured. Higher initial ion velocities correlate with a decrease in internal ion energy which might be attributed to the very uniform crystal morphology exhibited by sublimed compounds. For sublimed matrices without reduced grain size, at least slightly higher analyte intensities could be detected at raised laser fluences. Analyte accumulation in the uppermost matrix layers or the detected higher ion stability can be explanations for these results. Ever increasing numbers of samples call for automation in high-throughput workflows. One of the key areas for progress in automated MALDI analyses consists of improved sample preparation techniques. Numerous efforts have been conducted for improving the preparation step, e.g., the generation of thin uniform matrix layers with high shot-to-shot reproducibility using the aerospray technique by combination of matrix and nitrocellulose [4] or for analysis of synthetic polymers [5]. Also, electrospray deposition and vacuum deposition were reported to yield homogeneous sample preparations with increased analyte intensities [6 -9]. Matrix deposition by sublimation for MALDI imaging has been reported to yield more intense signals with less alkali adducts compared with the electrospray technique [10]. Additionally, sublimed matrix spots deposited on extremely hydrophobic surfaces described in [11][12][13][14] offer a variety of beneficial properties compared with standard dried droplet (DD) preparations: The main advantage of sublimed matrix spots compared with standard DD preparations are higher analyte sensitivities [14 -17]. This is likely related to the formation of considerably smaller crystals and narrow size distribution of thin-film sublimed matrices, such as ␣-cyano-4-hydroxycinnamic acid (CHCA), compared with DD preparations with comparatively large particle sizes and broader size distribution [15]. As a consequence of this, the specific matrix surface area as well as the amount of potential binding places increases compared with standard DD precipitates, which will result in increased analyte binding properties. Additionally, th...

Phosphatidylethanolamines (PEs) are abundant lipid constituents of the cellular membrane. The amino group of PEs exhibits high reactivity with hypochlorous acid that is generated under inflammatory conditions in vivo. The analysis of the resulting PE mono-and dichloramines is of significant interest since these species represent important mediators of lipid peroxidation. We have shown in a previous communication that mass spectrometric detection of PE chloramines is only possible with ESI MS, whereas MALDI-TOF MS fails to detect these products if standard matrices are used.In this work we demonstrate that the detection of PE chloramines is also possible by MALDI-TOF MS if 4-chloro-␣-cyanocinnamic acid is used as matrix. The underlying processes ␣ leading to ionization of these species will be discussed in detail. Both, experimental and theoretical studies taking into account possible intramolecular rearrangements were performed to clarify these aspects. ; sulfhydryl and thioether groups exhibit highest reactivity, followed by amino groups. Although olefinic residues of, e.g., lipids also react with HOCl, the corresponding second-order rate constants are relatively small [4].Since MPO is secreted from the neutrophils [5], the largest amount of HOCl is generated extracellularly. As a result of the permeability of the cellular membrane for HOCl [1], the phospholipids of the inner leaflet are also affected by this compound. The low reactive double bonds of the acyl side chains [4] are the only interaction possibility of phosphatidylcholines (PCs) with HOCl, whereas highly reactive amino groups of phosphatidylethanolamines (PEs) allow for much faster reaction [3]. Therefore, PEs are most likely the first molecules that react with HOCl. Mono-and dichloramines are generated as the primary products. Due to the instability of these compounds, further secondary products such as aldehydes and nitriles are also observed [4]. It has been recently shown that PE-derived chloramines mediate further events of lipid peroxidation [6] and are, therefore, of high interest.Electrospray ionization mass spectrometry (ESI MS) is an established and widely used tool for the analysis of chlorinated amines and amino acids [7]. PE-derived chloramines were successfully detected as sodiated cations and as deprotonated species with ESI MS [6,7]. In contrast, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS [8] is less suitable for the analysis of chloramines: We have shown in a recent communication that MALDI-TOF MS fails to detect PE-derived chloramines if standard matrices as 2,5-dihydroxybenzoic acid (DHB) or ␣-cyano-4-hydroxycinnamic acid (CHCA) are ␣ ␣ used [9]. Reactions between the matrix and the chloramines as well as the cleavage of the nitrogen-chlorine bond by UV laser irradiation were both assumed to contribute to this detection problem [9]. Recent investigations have shown that the newly developed matrix 4-chloro-␣-cyanocinnamic acid ␣ ␣ (ClCCA) easily enables the detection of chlorinated PE species an...

The success of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as a widely employed analytical tool in the biomolecular sciences builds strongly on an effective laser-material interaction that is resulting in a soft co-desorption and ionization of matrix and imbedded biomolecules. To obtain a maximized ion yield for the analyte(s) of interest, in general both wavelength and fluence need to be tuned to match the specific optical absorption profile of the used matrix. However, commonly only lasers with fixed emission wavelengths of either 337 or 355 nm are used for MALDI-MS. Here, we employed a wavelength-tunable dye laser and recorded both the neutral material ejection and the MS ion data in a wide wavelength and fluence range between 280 and 377.5 nm. α-Cyano-4-hydroxycinnamic acid (HCCA), 4-chloro-α-cyanocinnamic acid (ClCCA), α-cyano-2,4-difluorocinnamic acid (DiFCCA), and 2,5-dihydroxybenzoic acid (DHB) were investigated as matrices, and several peptides as analytes. Recording of the material ejection was achieved by adopting a photoacoustic approach. Relative ion yields were derived by division of photoacoustic and ion signals. In this way, distinct wavelength/fluence regions can be identified for which maximum ion yields were obtained. For the tested matrices, optimal results were achieved for wavelengths corresponding to areas of high optical absorption of the respective matrix and at fluences about a factor of 2-3 above the matrix- and wavelength-dependent ion detection threshold fluences. The material ejection as probed by the photoacoustic method is excellently fitted by the quasithermal model, while a sigmoidal function allows for an empirical description of the ion signal-fluence relationship.