Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

MALDI‐MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single‐stranded DNA‐binding protein

The Escherichia coli single‐stranded DNA binding protein (SSB) selectively binds single‐stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB?ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross‐linking, combined with high‐mass matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB?ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19 100) can be detected with standard MALDI‐MS. With chemical cross‐linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79 500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB?ssDNA complex at m/z 94 200 even in the absence of chemical cross‐linking. The stability of the SSB?ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB?ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB?ssDNA complex is how ssDNA binds to SSB, rather than the protein‐to‐DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non‐specific aggregation in the MALDI plume. Copyright © 2012 John Wiley & Sons, Ltd.     

MALDI‐FT‐ICR‐MS for archaeological lipid residue analysis

Soft‐ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little‐used soft ionization method of matrix assisted laser desorption/ionization‐Fourier transform‐ion cyclotron resonance‐mass spectrometry (MALDI‐FT‐ICR‐MS) for the identification of archaeological lipid residues. It is a high‐resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography‐mass spectrometry (GC‐MS) and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI‐FT‐ICR‐MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.     

Application of Z‐sinapinic matrix in peptide MALDI‐MS analysis

Since introduction of sinapinic acid (SA) and α‐cyano‐4‐hydroxycinnamic acid as matrices, successful application of matrix‐assisted laser desorption/ionization mass spectrometry started for protein/polypeptides. Both show some limitations in short peptide analysis because matrix clusters are quite abundant. Cinnamics currently used are E‐cinnamics. Here, Z‐SA as matrix for peptides is studied and compared with E‐SA and α‐cyano‐4‐hydroxycinnamic acid. Minor number of clusters is always observed in the low m/z region allowing the detection of short peptides. The results here described show that this novel matrix is a tool of choice for direct, rapid and sensitive detection of hydrophilic and hydrophobic peptides. Copyright © 2017 John Wiley & Sons, Ltd.     

Analysis of dammar resin with MALDI‐FT‐ICR‐MS and APCI‐FT‐ICR‐MS

Comprehensive analysis of high‐resolution mass spectra of aged natural dammar resin obtained with Fourier transform ion cyclotron resonance mass spectrometer (FT‐ICR‐MS) using matrix‐assisted laser desorption/ionization (MALDI) and atmospheric pressure chemical ionization (APCI) is presented. Dammar resin is one of the most important components of painting varnishes. Dammar resin is a terpenoid resin (dominated by triterpenoids) with intrinsically very complex composition. This complexity further increases with aging. Ten different solvents and two‐component solvent mixtures were tested for sample preparation. The most suitable solvent mixtures for the MALDI‐FT‐ICR‐MS analysis were dichloromethane‐acetone and dichloromethane‐ethanol. The obtained MALDI‐FTMS mass spectrum contains nine clusters of peaks in the m/z range of 420–2200, and the obtained APCI‐FTMS mass spectrum contains three clusters of peaks in the m/z range of 380–910. The peaks in the clusters correspond to the oxygenated derivatives of terpenoids differing by the number of C₁₅H₂₄ units. The clusters, in turn, are composed of subclusters differing by the number of oxygen atoms in the molecules. Thorough analysis and identification of the components (or groups of components) by their accurate m/z ratios was carried out, and molecular formulas (elemental compositions) of all major peaks in the MALDI‐FTMS and APCI‐FTMS spectra were identified (and groups of possible isomeric compounds were proposed). In the MALDI‐FTMS and APCI‐FTMS mass spectrum, besides the oxidized C₃₀, triterpenoids also peaks corresponding to C₂₉ and C₃₁ derivatives of triterpenoids (demethylated and methylated, correspondingly) were detected. MALDI and APCI are complementary ionization sources for the analysis of natural dammar resin. In the MALDI source, preferably polar (extensively oxidized) components of the resin are ionized (mostly as Na⁺ adducts), whereas in the APCI source, preferably nonpolar (hydrocarbon and slightly oxidized) compounds are ionized (by protonation). Either of the two ionization methods, when used alone, gives an incomplete picture of the dammar resin composition. Copyright © 2012 John Wiley & Sons, Ltd.     

Applicability of MALDI‐TOF MS for determination of quinolone residues in fish

MALDI‐TOF MS approach for determination of six quinolones residues in fillets of pangasius (Pangasionodon hypophthalmus) was studied, considering that is a very sensitive analytical technique with simple and high‐throughput operation, contributing to knowledge regarding application of this technique to the determination of small‐molecular‐weight organic compound residues in foods. LIFT‐MS/MS showed to be a successful approach to identify the presence of all quinolone residues in the fish fillet, at their respective MRL level. This study opens an important field of research for the development of simple and high‐throughput bioanalytical screening methods for the determination of veterinary drug residues in foods.     

Nanodiamonds Used as a Platform for Studying Noncovalent Interaction by MALDI‐MS

作为一种新的,易于实现的快速分析方法,纳米金刚石被应用基于基质隔离激光解吸附电离质谱的非共价相互作用的研究中。表面覆盖有“饵分析物”的纳米金刚石被加入到含有“目标分析物”的溶液中,通过离心的方法进行分离,并用水清洗后再利用基质隔离激光解吸附电离质谱进行分析。同时,通过比较加入纳米金刚石前后的溶液的质谱图,亦可得到相关信息。这种平台可应用于分子间非共价相互作用的研究,并可进一步应用于选择性增强的基质隔离激光解吸附电离质谱分析中,一个例子就是表面覆盖聚赖氨酸的纳米金刚石在磷酸化多肽的质谱分析的应用。     

Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

High‐throughput method for on‐target performic acid oxidation of MALDI‐deposited samples

An information‐rich on‐target performic acid oxidation method, which is compatible with alkylation for differentiation of free cysteine versus disulfide‐containing peptides, is described. On‐target oxidation is achieved using performic acid vapor to oxidize disulfide‐containing peptides and/or small proteins on the matrix‐assisted laser desorption/ionization (MALDI) sample deposits. The on‐target oxidation method is preferred over solution‐phase oxidation methods because (1) less sample handing is required, (2) oxidation throughput is drastically increased and (3) ion suppression effects are reduced because performic acid is not added directly to the MALDI spot. The utility of this method is demonstrated by simultaneous oxidation of multiple MALDI sample deposits containing model disulfide‐linked peptides, intact bovine insulin and a bovine ribonuclease A proteolytic digest. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off‐line CE‐UV/MALDI‐MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI‐MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI‐MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE‐UV/MALDI‐MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.     

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     

2,5‐Dihydroxybenzoic acid solution in MALDI‐MS: ageing and use for mass calibration

2,5‐Dihydroxybenzoic acid (DHB) is one of the most widely used and studied matrix compounds in matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. However, the influence of ageing of the DHB solution on the MALDI mass spectra has not been yet systematically studied. In this work, the possible changes occurring in the acidified acetonitrile/water solution of the MALDI matrix compound DHB during 1‐year usage period have been monitored with MALDI‐Fourier transform ion cyclotron resonance mass spectrometer (MALDI‐FT‐ICR‐MS) and attenuated total reflectance Fourier transform infrared (ATR‐FT‐IR) spectroscopy. No significant ageing products have been detected. The ability of the aged DHB solution to act as a MALDI matrix was tested with two materials widely used in art and conservation – bone glue (a proteinaceous material) and shellac resin (a resinous material) – and good results were obtained. A number of peaks in the mass spectra measured from the DHB solution were identified, which can be used for internal calibration of the mass axis. Copyright © 2014 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.     

Hydrazide and hydrazine reagents as reactive matrices for MALDI‐MS to detect gaseous aldehydes

The reagents 19 hydrazide and 14 hydrazine were examined to function as reactive matrices for matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to detect gaseous aldehydes. Among them, two hydrazide (2‐hydroxybenzohydrazide and 3‐hydroxy‐2‐naphthoic acid hydrazide) and two hydrazine reagents [2‐hydrazinoquinoline and 2,4‐dinitrophenylhydrazine (DNPH)] were found to react efficiently with carbonyl groups of gaseous aldehydes (formaldehyde, acetaldehyde and propionaldehyde); these are the main factors for sick building syndrome and operate as reactive matrices for MALDI‐MS. Results from accurate mass measurements by JMS‐S3000 Spiral‐TOF suggested that protonated ion peaks corresponding to [M + H]⁺ from the resulting derivatives were observed in all cases with the gaseous aldehydes in an incubation, time‐dependent manner. The two hydrazide and two hydrazine reagents all possessed absorbances at 337 nm (wavelength of MALDI nitrogen laser), with, significant electrical conductivity of the matrix crystal and functional groups, such as hydroxy group and amino group, being important for desorption/ionization efficiency in MALDI‐MS. To our knowledge, this is the first report that gaseous molecules could be derivatized and detected directly in a single step by MALDI‐MS using novel reactive matrices that were derivatizing agents with the ability to enhance desorption/ionization efficiency. Copyright © 2014 John Wiley & Sons, Ltd.     

Improved MALDI‐MS method for the highly sensitive and reproducible detection of biomarker peaks for the proteotyping of Salmonella serotypes

The rapid identification and classification of pathogenic microorganisms, including Salmonella enterica, is important for the surveillance and prevention of foodborne diseases. Matrix‐assisted laser desorptionionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) has been shown to be an effective tool for the rapid identification of microorganisms. In a previous report, a mass database consisting of 12 biomarker proteins, S8, L15, L17, L21, L25, S7, superoxide dismutase (SodA), peptidylprolyl cis‐trans isomerase C, Gns, YibT, YaiA, and YciF, was introduced for the serotyping of S. enterica via MALDI‐MS (Applied Microbiology and Biotechnology, 2017, 101, 8557‐8569). However, the reproducibility of peak detection of biomarkers such as SodA at mz 23 000 was poor. We report here an optimized MALDI‐MS method for detecting these biomarkers with high sensitivity and reproducibility. The issue was solved by controlling the bacterial concentration at 1 × 10 to 1 × 10² MFU (3 × 10⁶ to 3 × 10⁷ CFUμL, as calculated from the MFU), using the colony suspension supernatant obtained by centrifugation, and using matrix additives such as methylenediphosphonic acid and N‐decyl‐β‐D‐maltopyranoside. We propose that the method including the above steps is one of the best for detecting biomarkers with high sensitivity and reproducibility.     

Applications of MALDI‐TOF MS to large‐scale human mtDNA population‐based studies

Analysis of the mitochondrial DNA variation in populations is commonly carried out in many fields of biomedical research. We propose the analysis of mitochondrial DNA coding region SNP (mtSNP) variation to a high level of phylogenetic resolution based on MALDI‐TOF MS. The African phylogeny has been chosen to test the applicability of the technique but any other part of the worldwide phylogeny (or any other mtSNP panel) could be equally suitable for MALDI‐TOF MS genotyping. SNP selection thus aimed to fully cover all the mtSNPs defining major and minor branches of the known African tree, including, macro‐haplogroup L, and haplogroups M1, and U6. A total of 230 mtSNPs were finally selected. We used tests samples collected from two different African locations, namely, Mozambique and Chad Basin. Different internal genotyping controls and other indirect approaches (e.g. phylogenetic checking coupled with automatic sequencing) were used in order to evaluate the reproducibility of the technique, which resulted to be 100% using samples previously subjected to whole genome amplification. The advantages of the MALDI‐TOF MS are also discussed in comparison with other popular methods such as minisequencing, highlighting its high‐throughput nature, which is particularly suitable for case–control medical studies, forensic databasing or population and anthropological studies.     

A MALDI‐TOF MS study of lanthanide(III)‐cored poly(phenylenevinylene) dendrimers

An extensive study by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry (MS) of some first‐generation and second‐generation lanthanide(III)‐cored poly(phenylenevinylene) dendrimers is described. The complexes were obtained by self‐assembly of suitably functionalized carboxylate dendrons around the lanthanide ion (La³⁺, Er³⁺). Fourier transform infrared (FT‐IR) spectroscopy gave reasonable evidence for the proposed structures. However, MS was used to ascertain unequivocally the complex formation. The most reliable results were found in the negative reflector mode, using 2‐[(2E)‐3‐(4‐tert‐butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile (DCTB) as matrix. Well‐defined and highly resolved base peaks corresponding to negative ions of [Gn₄La]^? and [Gn₄Er]^? were found in all cases, with an excellent match between the theoretical and observed isotope distributions. However, the 3 : 1 stoichiometry used in the synthesis guarantees an empirical formula Gn₃Ln for the complexes. Copyright © 2008 John Wiley & Sons, Ltd.     

Detergent‐assisted sample preparation for MALDI‐MS: Investigation of octylglucoside and docecylmaltoside for matrix crystallization,on‐plate digestion,and trypsin activity

We show an easy and fast method for improved detection of lipophilic peptides with MALDI‐MS utilizing the nonionic detergents n‐octylglucoside and n‐dodecylmaltoside (laurylmaltoside). Investigations comprised on‐plate digestion of proteins with trypsin, detergent effects on the protease trypsin, and the changes in MALDI matrix crystallization. Investigations also exhibited a higher tryptic activity in trypsin activity assay of 139% when using laurylmaltoside as supplement. Crystallization changed toward a more homogeneous crystal distribution and especially trypsinized insulin spectra recorded with MALDI‐MS showed improved detectability of lipophilic peptides.     

Molecular weight analysis of water‐soluble poly(phenylene ethynylene)s using MALDI‐TOF MS

Molecular weights of seven poly(phenylene ethynylene)‐based water‐soluble conjugated polyelectrolytes (CPEs) obtained through Sonogashira coupling are determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). A standard sample preparation protocol is developed to characterize the seven CPEs using 2,5‐dihydroxybenzoic acid as the matrix (M) and AgTFA as the cationization reagent (CR). High‐quality MALDI mass spectra are obtained at volume mixing ratios (CPE/M/CR) of 5/5/1 for anionic polymers (P1–P4) and 5/50/1 for cationic polymers (P5–P7). Molecular weight, molecular weight distribution, and end‐group information are analyzed. The effects of molecular weight of CPEs on optical and quenching properties are also studied. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2537–2543