Direct identification of trypanosomatids by matrix‐assisted laser desorption ionization–time of flight mass spectrometry (DIT MALDI‐TOF MS)

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix‐assisted laser desorption ionization/time of flight mass spectrometry (MALDI‐TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI‐TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI‐TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI‐TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software‐assisted identification at the strain level. Overall, this study shows the importance of MALDI‐TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry‐based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of MALDI‐TOF mass spectra with microsatellite length polymorphisms in Candida albicans

Candida albicans is the most frequent yeast involved in human infections. Its population structure can be divided into several genetic clades, some of which have been associated with antifungal susceptibility. Therefore, detecting and monitoring fungal clones in a routine laboratory setting would be a major epidemiological advance. Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectra results are now widely used as bar codes to identify microorganisms in clinical microbiology laboratories. This study aimed at testing MALDI‐TOF mass spectra bar codes to identify clades among a set of C. albicans isolates. Accordingly, 102 clinical strains were genotyped using 10 microsatellite markers and analyzed via MALDI‐TOF mass spectrometry. The mass spectra were compared with a reference spectral library including 33 well‐characterized collection strains, using a Microflex^TM system and Biotyper^TM software, to test the capacity of the spectrum of a given isolate to match with the reference mass spectrum of an isolate from the same genetic clade. Despite high confidence species identification, the spectra failed to significantly match with the corresponding clade (p = 0.74). This was confirmed with the MALDI‐TOF spectra similarity dendrogram, in which the strains were dispersed irrespective of their genetic clade. Various attempts to improve intra‐clade spectra recognition were unsuccessful. In conclusion, MALDI‐TOF mass spectra bar code analysis failed to reliably recognize genetically related C. albicans isolates. Further studies are warranted to develop alternative MALDI‐TOF mass spectra analytical approaches to identify and monitor C. albicans clades in the routine clinical laboratory. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection of pathogenic Verticillium spp. using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Verticillium spp. have been listed by the European and Mediterranean Plant Protection Organization (EPPO) and China as plant quarantine pests. Although attempts have been made to develop a simple routine laboratory assay to detect these organisms, none are routinely used. We describe for the first time a robust assay for reliable identification of Verticillium spp. using protein fingerprinting data obtained by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry(MALDI‐TOF‐MS). Several sample preparation methods and matrices were investigated to improve mass spectra for the routine identification of six species of Verticillium spp.(Verticillium dahiliae, V. alboatrum, V. fungicola, V. nigrescens, and V. lecanii) by MALDI‐TOF‐MS. Using the optimized experimental method, we constructed a protein fingerprint database for six species of Verticillium and established a analysis criteria of log(Score). This MALDI‐TOF‐MS protocol should prove useful as a rapid and reliable assay for distinguishing different Verticillium spp. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and classification of seafood‐borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI‐TOF MS fingerprinting

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI‐TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood‐borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI‐TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database ( http://www.spectrabank.org ), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI‐TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI‐TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains.     

Evaluation of matrix‐assisted laser desorption/ionization (MALDI) preparation techniques for surface characterization of intact Fusarium spores by MALDI linear time‐of‐flight mass spectrometry

Unambiguous identification of mycotoxin‐producing fungal species as Fusarium is of great relevance to agriculture and the food‐producing industry as well as in medicine. Protein profiles of intact fungal spores, such as Penicillium, Aspergillus and Trichoderma, derived from matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) were shown to provide a rapid and straightforward method for species identification and characterization. In this study, we applied this approach to five different Fusarium spp. strains which are known to affect the growth of different grain plants. To obtain a suitable MALDI matrix system and sample preparation method, thin‐layer, dried‐droplet and sandwich methods and several MALDI matrices, namely CHCA, DHB, FA, SA and THAP dissolved in various solvent mixtures (organic solvents such as ACN, MeOH, EtOH and iPrOH and for the aqueous phase water and 0.1% TFA), were evaluated in terms of mass spectrometric pattern and signal intensities. The most significant peptide/protein profiles were obtained with 10 mg ferulic acid (FA) in 1 mL ACN/0.1% TFA (7:3, v/v) used as matrix system. Mixing the spores with the matrix solution directly on the MALDI target (dried‐droplet technique) resulted in an evenly distributed spores/matrix crystal layer, yielding highly reproducible peptide/protein profiles from the spore surfaces. Numerous abundant ions throughout the investigated m/z range (m/z 1500–15 000) could be detected. Differences in the obtained mass spectral patterns allowed the differentiation of spores of various Fusarium species. Copyright © 2009 John Wiley & Sons, Ltd.     

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.     

Impact of updating the MALDI‐TOF MS database on the identification of nontuberculous mycobacteria

Conventional identification of mycobacteria species is slow, laborious and has low discriminatory power. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has proved highly effective for identifying conventional bacteria, and it may also be useful for identifying mycobacteria. The aim of this study was to evaluate and compare MALDI‐TOF MS with currently recommended molecular methods for the identification of nontuberculous mycobacteria (NTM), applying Mycobacteria Libraries v3.0 (ML3.0) and v2.0 (ML2.0). A total of 240 clinical isolates of 41 NTM species grown on solid media were analysed: 132 isolates of slow‐growing mycobacteria and 108 of rapid‐growing mycobacteria. MALDI‐TOF MS, using ML3.0, identified 192 (80%) NTM isolates with a score ≥1.7, encompassing 35 (85.4%) different species, that is, 17 (7.1%; p  = 0.0863) isolates and 15 (36.6%; p  = 0.0339) species more than currently recommended molecular techniques (polymerase chain reaction reverse hybridization). All these isolates were correctly identified according to molecular identification methods. The application of ML3.0 also identified 15 (6.2%) NTM isolates more than ML2.0 (p  < 0.01). The scores obtained with MALDI‐TOF MS using ML3.0 (mean score: 1.960) were higher in 147 (61.2%) isolates than when using ML2.0 (mean score: 1.797; p  < 0.01). Three of the species analysed were not included in either database, so they were not recognized by this system. In conclusion, MALDI‐TOF MS identified more isolates and species than the recommended polymerase chain reaction reverse hybridization assays. Although the new ML3.0 is not the definitive database, it yielded better results than ML2.0. This shows that the updating of the MALDI‐TOF MS database plays an essential role in mycobacterial identification. Copyright © 2017 John Wiley & Sons, Ltd.     

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI‐TOF MS‐based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer‐based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10⁹ spores per ml. The best peptide/protein profiles (in terms of signal‐to‐noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.     

MALDI‐TOF mass spectrometry,an efficient technique for in situ detection and characterization of actinomycins

An extensive study of actinomycins was performed using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF MS). Actinomycins represent a well‐known family of peptidolactone chromopeptides with potent cytostatic and antibiotic properties. Using five well‐characterized streptomycete strains, we introduced MALDI‐TOF MS as an efficient technique for rapid in situ detection of actinomycins in surface extracts of cells picked from agar plates. By this procedure, actinomycin complexes can be investigated with high sensitivity and accuracy in a minimum of time. These studies were complemented by mass spectrometric investigation of actinomycins obtained from culture filtrate extracts and purified by high‐performance liquid chromatography to detect yet unknown actinomycin species. By feeding experiments, C‐demethyl‐actinomycins from Streptomyces chrysomallus and Streptomyces parvulus as well as hemi‐actinomycins from Streptomyces antibioticus lacking one of the two pentapeptide lactone rings were isolated and characterized as novel variants for structure–activity relationship studies. Structural characterization of the investigated actinomycins was performed by post source decay MALDI‐TOF MS. The specific features of the fragmentation patterns of the protonated and cationized forms of selected actinomycins were investigated in detail. Copyright © 2014 John Wiley & Sons, Ltd.     

MALDI‐TOF MS characterization of dihydroxy telechelic polyisobutylene

MALDI‐TOF MS characterizations of dihydroxy telechelic polyisobutylene is reported. Dichloro telechelic polyisobutylene (Cl—PIB—Cl) was synthesized by means of living cationic polymerization using p‐dicumyl chloride/BCl₃/DMSO initiating systems. The resulting polymer was functionalized by polymer analogous reactions to yield dihydroxy telechelic polyisobutylene (HO—PIB—OH). It was then investigated by MALDI‐TOF MS in the cation mode using 1,8‐dihidroxy‐9(10H)‐anthracenone (dithranol)/CF₃COOAg matrix. The MALDI TOF MS spectra showed an increase in mass by 56 amu units attributed to the isobutylene monomer increment. The endgroups of HO—PIB—OH were determined. A good agreement was also found between the calculated isotope distributions and the isotope distributions determined by means of MALDI.     

Modeling bacteriophage amplification as a predictive tool for optimized MALDI‐TOF MS‐based bacterial detection

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is a valuable tool for rapid bacterial detection and identification but is limited by the need for relatively high cell count samples, which have been grown under strictly controlled conditions. These requirements can be eliminated by the natural infection of a viable bacterial species of interest with a host‐specific phage. This produces a rapid increase in phage protein concentrations in comparison to bacterial concentrations, which can in turn be exploited as a method for signal amplification during MALDI‐TOF MS. One drawback to this approach is the requirement for repetitive, time‐consuming sample preparation and analysis applied over the course of a phage infection to monitor phage concentrations as a function of time to determine the MALDI‐TOF MS detection limit. To reduce the requirement for repeated preparation and analysis, a modified phage therapy model was investigated as a means for predicting the time during a given phage infection when a detectable signal would occur. The modified model used a series of three differential equations composed of predetermined experimental parameters including phage burst size and burst time to predict progeny phage concentrations as a function of time. Using Yersinia pestis with plague diagnostic phage ?A1122 and Escherichia coli with phage MS2 as two separate, well‐characterized model phage–host pairs, we conducted in silico modeling of the infection process and compared it with experimental infections monitored in real time by MALDI‐TOF MS. Significant agreement between mathematically calculated phage growth curves and those experimentally obtained by MALDI‐TOF MS was observed, thus verifying this method's utility for significant time and labor reduction. Copyright © 2012 John Wiley & Sons, Ltd.     

Dehydrogenation and dehalogenation of amines in MALDI‐TOF MS investigated by isotopic labeling

Secondary and tertiary amines have been reported to form [M–H]⁺ that correspond to dehydrogenation in matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS). In this investigation, we studied the dehydrogenation of amines in MALDI‐TOF MS by isotopic labeling. Aliphatic amines were labeled with deuterium on the methylene of an N‐benzyl group, which resulted in the formation of [M–D]⁺ and [M–H]⁺ ions by dedeuteration and dehydrogenation, respectively. This method revealed the proton that was removed. The spectra of most tertiary amines with an N‐benzyl group showed high‐intensity [M–D]⁺ and [M–H]⁺ ion peaks, whereas those of secondary amines showed low‐intensity ion peaks. Ratios between the peak intensities of [M–D]⁺ and [M–H]⁺ greater than 1 suggested chemoselective dehydrogenation at the N‐benzyl groups. The presence of an electron donor group on the N‐benzyl groups enhanced the selectivity. The dehalogenation of amines with an N‐(4‐halobenzyl) group was also observed alongside dehydrogenation. The amino ions from dehalogenation can undergo second dehydrogenation. These results provide the first direct evidence about the position at which dehydrogenation of an amine occurs and the first example of dehalogenation of haloaromatic compounds in MALDI‐TOF MS. These results should be helpful in the structural identification and elucidation of synthetic and natural molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

The effect of temperature on the stability of compounds used as UV‐MALDI‐MS matrix: 2,5‐dihydroxybenzoic acid, 2,4,6‐trihydroxyacetophenone, α‐cyano‐4‐hydroxycinnamic acid, 3,5‐dimethoxy‐4‐hydroxycinnamic acid,nor‐harmane and harmane

The thermal stability of several commonly used crystalline matrix‐assisted ultraviolet laser desorption/ionization mass spectrometry (UV‐MALDI‐MS) matrices, 2,5‐dihydroxybenzoic acid (gentisic acid; GA), 2,4,6‐trihydroxyacetophenone (THA), α‐cyano‐4‐hydroxycinnamic acid (CHC), 3,5‐dimethoxy‐4‐hydroxycinnamic acid (sinapinic acid; SA), 9H‐pirido[3,4‐b]indole (nor‐harmane; nor‐Ho), 1‐methyl‐9H‐pirido[3,4‐b]indole (harmane; Ho), perchlorate of nor‐harmanonium ([nor‐Ho + H]⁺) and perchlorate of harmanonium ([Ho + H]⁺) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI‐MS), ultraviolet laserdesorption/ionization‐time‐of‐flight‐mass spectrometry (UV‐LDI‐TOF‐MS) and electrospray ionization‐time‐of‐flight‐mass spectrometry (ESI‐TOF‐MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV‐absorption, fluorescence emission and excitation spectroscopy) and ¹H nuclear magnetic resonance spectroscopy (¹H‐NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO₂, yielding the trans‐/cis‐4‐hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and ¹H‐NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well‐known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV‐MALDI‐MS. Commercial SA (SA 98%; trans‐SA/cis‐SA 5 : 1) showed mainly cis‐ to‐trans thermal isomerization and, with very poor yield, loss of CO₂, yielding (3′,5′‐dimethoxy‐4′‐hydroxyphenyl)‐1‐ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV‐MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted. Copyright © 2008 John Wiley & Sons, Ltd.     

Enhanced matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis of bacteriophage major capsid proteins with β‐mercaptoethanol pretreatment

Bacteriophage (phage) proteins have been analyzed previously with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). However, analysis of phage major capsid proteins (MCPs) has been limited by the ability to reproducibly generate ions from MCP monomers. While the acidic conditions of MALDI‐TOF MS sample preparation have been shown to aid in disassembly of some phage capsids, many require further treatment to successfully liberate MCP monomers. The findings presented here suggest that β‐mercaptoethanol reduction of the disulfide bonds linking phage MCPs prior to mass spectrometric analysis results in significantly increased MALDI‐TOF MS sensitivity and reproducibility of Yersinia pestis‐specific phage protein profiles. Copyright © 2009 John Wiley & Sons, Ltd.     

Differential characterization of biogenic amine‐producing bacteria involved in food poisoning using MALDI‐TOF mass fingerprinting

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine‐producing enteric and marine bacteria by means of MALDI‐TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182±1 and 8363±6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635±1 and 7267±2 were specific to both M. morganii and Proteus spp. Biogenic amine‐forming Proteus spp. exhibited three genus‐specific peaks at m/z 3980, 7960±1 and 9584±2. The genus Photobacterium also showed three genus‐specific peaks at m/z 2980±1, 4275±1 and 6578±1. The two histamine‐producing Gram‐positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000–7000 m/z range and could be easily distinguished from biogenic amine‐forming Gram‐negative bacteria. Clustering based on MALDI‐TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI‐TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine‐forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.     

Characterization of different food‐isolated Enterococcus strains by MALDI‐TOF mass fingerprinting

Enterococcus is a controversial genus due to its great variability; this genus includes pathogenic strains, spoilage strains, and apparently safe strains including some probiotic strains. Previous studies focused on the characterization of strains of Enterococcus spp. involved in nosocomial infections. However, little research has been conducted on Enterococcus strains in foodstuffs. In the present work, 36 strains of different species of Enterococcus have been characterized by means of MALDI‐TOF MS, resulting in highly specific mass spectral fingerprints. Characteristic peak masses common to certain bacterial species of Enterococcus have been identified. Thus, a peak at m/z 4426 ± 1 was assigned as a genus‐specific biomarker. In addition, phyloproteomic relationships based on the mass spectral data were compared to the results of a phylogenetic analysis based on the 16S rRNA gene sequence. A better grouping at the species level was observed in the phyloproteomic tree, especially for the Enterococcus faecium group. Presumably, the assortment of some strains or ecotypes could be related to their ecological niche specialization. The approach described in this study leads the way toward the rapid and specific identification of different strains and species of Enterococcus in food based on molecular protein markers, aiming at the early detection of pathogenic strains and strains implicated in food poisoning or food spoilage.     

Heterobifunctional isotope‐labeled amine‐reactive photo‐cross‐linker for structural investigation of proteins by matrix‐assisted laser desorption/ionization tandem time‐of‐flight and electrospray ionization LTQ‐Orbitrap mass spectrometry

Chemical cross‐linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross‐linked products presents an alternative approach to assess low‐resolution protein structures. By covalently connecting pairs of functional groups within a protein or a protein complex a set of structurally defined interactions is built up. We synthesized the heterobifunctional amine‐reactive photo‐cross‐linker N‐succinimidyl p‐benzoyldihydrocinnamate as a non‐deuterated (SBC) and doubly deuterated derivative (SBDC). Applying a 1:1 mixture of SBC and SBDC for cross‐linking experiments aided the identification of cross‐linked amino acids in the mass spectra based on the characteristic isotope patterns of fragment ions. The cross‐linker was applied to the calcium‐binding protein calmodulin with a subsequent analysis of cross‐linked products by nano‐high‐performance liquid chromatography matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (nano‐HPLC/MALDI‐TOF/TOF‐MS) and nano‐HPLC/nano‐electrospray ionization (ESI)‐LTQ‐Orbitrap‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a MALDI two-layer volume sample preparation technique for analysis of colored conidia spores of <Emphasis Type="Italic">Fusarium</Emphasis> by MALDI linear TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level.      

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.