Analysis of the allergenic proteins in black tiger prawn (Penaeus monodon) and characterization of the major allergen tropomyosin using mass spectrometry

Crustaceans are the third most prevalent cause of food‐induced anaphylaxis after peanuts and tree nuts. The severity of the allergenic proteins depends mainly on the amino acid sequence that induces production of IgE antibodies. In black tiger prawn (Penaeus monodon), the crude protein extract was profiled and its allergenic potency was examined against patient's sera. Proteins having strong immunoreactivity with patient's IgE were characterized using peptide mass fingerprinting (PMF). Tropomyosin (TM) (33 kDa), myosin light chain (20 kDa), and arginine kinase (40 kDa) were identified as allergenic proteins. Tropomyosin, the most abundant and potent allergen, was purified using ion‐exchange chromatography for de novo sequencing experiments. Using bottom up tandem mass spectrometry, the full amino acid sequence was achieved by a combination of matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass spectrometry (QqToF). Myosin light chain and arginine kinase were also characterized, and their related peptides were de novo sequenced using the same approach. The immunological reactivity of the crude prawn extracts and purified TM samples were analyzed using a large number of patients' sera. A signature peptide was assigned for the TM protein for future quantification work of black tiger prawn TM levels in different matrices (i.e. water, air, food) in the seafood industry. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer

This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in‐gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). A characteristic band around a molecular mass of 185 kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI‐MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI‐MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one‐step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high‐performance liquid chromatography (HPLC) coupled in‐line to an electrospray quadrupole‐hexapole‐quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0 → 1020.4 and 750.0 → 1205.4) and (754.8 → 1028.6 and 754.8 → 1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish. Copyright © 2009 John Wiley & Sons, Ltd.     

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

Toward milk speciation through the monitoring of casein proteotypic peptides

The possibility of detecting extraneous milk in singles species cheese‐milk has been explored. A mass spectrometry (MS)‐based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the α_s1‐casein (CN) f8‐22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4‐22 peptide was selected as a marker for the two caprine α_s1‐CN A and B variants, which differ by a Pro¹⁶ (B)‐>Leu¹⁶ (A) substitution. MALDI analysis of the digest allowed the detection of α_s1‐CN f8‐22 and caprine α_s1‐CN f4‐22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)‐MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the α_s1‐CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI‐MS method. The isotopic‐label‐free quantification of isoform‐ or variant‐specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Dansyl‐peptides matrix‐assisted laser desorption/ionization mass spectrometric (MALDI‐MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI‐MS/MS analysis of the proteome

Peptide tagging is a useful tool to improve matrix‐assisted laser desorption/ionization tandem mass spectrometric (MALDI‐MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS‐Cl). DNS‐Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI‐MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N‐terminal DNS‐Cl sulfonation improves the peptide fragmentation and promotes the generation of b‐fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI‐MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage. Copyright © 2010 John Wiley & Sons, Ltd.     

The effect of sodium dodecyl sulfate and anion‐exchange silica gel on matrix‐assisted laser desorption/ionization mass spectrometric analysis of proteins

Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), this surfactant can often cause signal suppression. We have previously reported an on‐probe sample preparation method using a suspension of anion‐exchange silica gel and sinapinic acid (i.e., gel‐SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on‐probe sample preparation method using both SDS and the gel‐SA suspension improved the detection limit of protein samples in the MALDI‐MS analysis by about ten‐fold as compared to that of protein samples without SDS and the gel‐SA suspension. This method can be applied not only to the MALDI‐MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. Copyright © 2009 John Wiley & Sons, Ltd.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

Acid hydrolysis followed by matrix‐assisted laser desorption/ionization mass spectrometry for the rapid diagnosis of serum protein biomarkers in patients with major depression

We have developed a technique combining acid hydrolysis with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) for the rapid study of the changes in the levels of positive and negative acute phase protein biomarkers in the sera of patients suffering from major depression. The serum proteins were first precipitated using an organic solvent; after separation, they were subjected to hydrochloric acid hydrolysis (6 M HCl) for 10 min. The resulting peptides were characterized using MALDI‐TOF MS. Short‐term treatment of the serum proteins with HCl efficiently removed interference from the abundant protein – albumin – and produced abundant peptide ion signals in the range of m/z 4000–10 000. This approach allowed us to rapidly detect the peptide ions originating from transferrin (a common negative acute phase protein) and fibrinogen (a common positive acute phase protein). The average ratios and (standard deviations) of the ion signals derived from transferrin/fibrinogen were 3.58 (±1.93) for the healthy control subjects and 1.02 (±0.52) for the patients suffering from major depression. The differences in transferrin/fibrinogen ratios between healthy controls and patients suggest that major depression will induce internal inflammation and cause either an increase in the level of fibrinogen or a decrease in the level of transferrin. Copyright © 2009 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.     

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.     

Controlling matrix suppression for matrix‐assisted laser desorption/ionization analysis of small molecules

The need for high‐throughput methodologies providing both qualitative and quantitative information has grown substantially in the pharmaceutical laboratory in recent years. Currently, tandem mass spectrometry (MS/MS) using quadrupole technology offers analysis in the minutes time scale. The use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) offers the advantage of speed and automation and enables analysis in the seconds time scale with accurate mass capabilities that are not typically found in quadrupole MS/MS. However, one of the limitations of MALDI for the analysis of small molecules is the abundance of interfering matrix peaks in the low molecular weight region of the mass spectrum. Described herein is an evaluation of a pre‐prepared MALDI target plate that has been coated with a thin layer of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and nitrocellulose. This modified plate has been shown to suppress or eliminate CHCA matrix signals without any significant loss of analyte sensitivity when compared with analysis of the same sample using an unmodified target plate. Copyright © 2004 John Wiley & Sons, Ltd.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Aniline/α‐cyano‐4‐hydroxycinnamic acid is a highly versatile ionic liquid for matrix‐assisted laser desorption/ionization mass spectrometry

The performance of a matrix‐assisted laser desorption/ionization (MALDI) ionic liquid matrix (ILM) consisting of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and aniline (ANI) was evaluated to assess whether it could offer possible advantages over conventional matrices. Ultraviolet (UV), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and laser desorption/ionization mass spectrometry (LDI‐MS) experiments were carried out with the aim of confirming the structure of the ANI‐CHCA ILM. Different model analytes such as amino acids, peptides, proteins, lipids, phospholipids, synthetic polymers, and sugars were tested. Mass spectra with similar or improved signal‐to‐noise (S/N) ratio (compared to CHCA) were invariably obtained demonstrating the potential of this ILM as a general purpose matrix. Furthermore, protein identification by peptide mass fingerprinting (PMF) and database search was facilitated compared to CHCA since higher scores and increased sequence coverage were observed. Finally, a complex lipid mixture (i.e. a raw extract of a milk sample) analysed by MALDI‐MS showed improved S/N ratio, a reduced chemical noise and a limited formation of matrix‐clusters. Copyright © 2009 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.     

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.     

Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI)

In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization‐quadrupole‐time of flight (ESI‐Q‐TOF) and matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2‐oxazoline via a living cationic ring‐opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI‐Q‐TOF and MALDI‐TOF MS in combination with collision‐induced dissociation (CID) experiments. In ESI‐MS as well as MALDI‐MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2‐hydride shift with a charge‐remote rearrangement via a four‐membered cyclic transition state, as well as charge‐induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4‐hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks. Copyright © 2012 John Wiley & Sons, Ltd.