Rapid discrimination of environmental Vibrio by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The aim of this study was to discriminate 30 Vibrio strains isolated from two wastewater treatment plants from Agadir, Morocco by two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Out of the 30 strains of Vibrio examined in this study, 5 isolates could not be typed by PFGE and consistently appeared as a smear on the gel. In general, high genetic biodiversity among the Vibrio strains was found regardless to the isolation source. The results of MALDI TOF analysis show a high congruence of strain grouping demonstrating the accuracy and reliability of MALDI-TOF MS.     

Multiplex single-nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A robust high-throughput single-nucleotide polymorphism (SNP) genotyping method is reported, which applies allele-specific extension to achieve allelic discrimination and uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to measure the natural molecular weight difference of oligonucleotides for determination of the base in a single-nucleotide polymorphic location. Tenfold PCR is performed successfully by carefully designing the primers and adjusting the conditions of PCR. In addition, two ways used for PCR product purification are compared and the matrix used in mass spectrometry for high-throughput oligonucleotide analysis is evaluated. The result here shows that the method is very effective and suitable for high-throughput genotyping of SNPs.     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3′ and 5′ ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as described previously. On the basis of ¹⁸O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair.     

Determination of prolidase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Proline-containing peptides of the X-proline type are cleaved by the dipeptidase prolidase. The classical method of prolidase assay relied on the colorimetric estimation of the liberated proline with ninhydrin using acidic media and heat. This method, however, gave inconsistent results due to the nonspecificity of the ninhydrin color reaction. We report here a method for the detection of the liberated proline using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Human sera were incubated with a mixture containing the dipeptide glycyl-proline in Tris-HCl supplemented with manganese at 37 degrees C for 24h. The samples were precipitated with trifluoroacetic acid and centrifuged. An aliquot of the supernatant was mixed with an equal volume of ferulic acid solution. An aliquot from this mixture was spotted on a stainless steel mass spectrometry grid and analyzed using MALDI-TOF mass spectrometry. The activity of the enzyme was determined by the complete disappearance of the glycyl-proline peak with the concomitant appearance of the proline peak and can be expressed in terms of the ratio of the area beneath the proline to the area beneath the glycyl-proline peak. Subjects homozygous for prolidase deficiency had a ratio ranging from 0.006 to 0.04 while obligatory heterozygotes had a ratio ranging from around 1.1 to 2.4. Normal subjects had ratios ranging from 9 to 239. Using this method we have unambiguously identified subjects with homozygous or heterozygous prolidase deficiency. In addition to the advantage of rapid sample preparation time, this method is highly specific, reproducible, and sensitive.     

基质辅助激光解吸电离飞行时间质谱在微生物鉴定中的研究进展

与传统的微生物鉴定技术相比,基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文,总结最新的研究进展,发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势,加快了微生物鉴定的进程,同时探索该技术在新领域的最新进展和面临的挑战,以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。     

Quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.     

Mass determination of major plasma proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) serves as a rapid and accurate means to determine masses of proteins independent of their shapes or interactions with other molecules. It provides one of the most fundamental characterizations of major plasma proteins. Purified proteins in saline or serum specimens were prepared for analysis by dilution, mixing with a solution of sinapinic acid, and drying on a target plate. Specimens were analyzed in a linear TOF mode with external calibration. Analyses of 24 purified plasma proteins showed predominance of singly charged ions with lesser amounts of dimer and doubly charged monomer, and provided measured masses for these proteins. A number of proteins, including albumin, transferrin, apolipoproteins A-I, A-II, C-I, C-II, and C-III, and prealbumin, could be analyzed directly in serum with appropriate dilution. Measured values for masses of major plasma proteins will assist in analysis of serum and plasma. It is possible to analyze a number of components by MALDI-TOF/MS directly in diluted serum. Extremely simple sample preparation techniques may be useful in analyzing structural variation of several major plasma proteins, particularly those with masses <30 kDa, including a number of apolipoproteins and markers of nutritional status or acute phase responses.     

Structural analysis of cartilage proteoglycans and glycoproteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cartilage extracellular matrix molecules synthesized and maintained by chondrocytes form a strong, elastic tissue functioning to cushion and protect the subchondral bone. Osteoarthritis is characterized by degradation of cartilage extracellular matrix molecules resulting in fibrillation, irreversible erosion, and eventual failure of the tissue. With recent interest in the degradation of cartilage extracellular matrix molecules, a need for more detailed structural information exists. Posttranslational modifications are believed to play a role in determining the susceptibility of these molecules to proteolytic degradation during the development of osteoarthritis. The purpose of this paper is to show how the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to extracellular matrix protein and proteoglycan structure will help elucidate problems in extracellular matrix biochemistry. Methodological issues relating to the high molecular weight, polydispersity, and high degree of posttranslational modification of these molecules are discussed. MALDI-TOF mass spectrometry provides an improved level of detail for extracellular matrix protein and proteoglycan structure and is useful in addressing issues surrounding the causes of degradation during osteoarthritis.     

Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Background

MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory.

Materials and Methods

First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification.

Results

The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides.

Conclusions

This work''s seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.     

Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry was employed to analyze DNA methylation carried out by the Escherichia coli dam DNA methyltransferase using oligonucleotide substrates with molecular masses of 5000-10,000 Da per strand. The mass spectrometry assay offers several advantages: (i) it directly shows the methylation as the increase in the mass of the substrate DNA, (ii) it is nonradioactive, (iii) it is quantitative, and (iv) it can be automated for high-throughput applications. Since unmethylated and methylated DNA are detected, the ratio of methylation can be determined directly and accurately. Furthermore, the assay allows detection individually of the methylation of several substrates in competition, offering an ideal setup to analyze the specificity of DNA interacting with enzymes. We could not identify methylation at any noncanonical site, indicating that the dam MTase is a very specific enzyme. Finally, MALDI-TOF mass spectrometry permitted assessment of the number of methyl groups incorporated into each DNA strand, thereby, allowing study of mechanistic details such as the processivity of the methylation reaction. We provide evidence that the dam MTase modifies DNA in a processive reaction, confirming earlier findings.     

Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces 'spot to spot' variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi-quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC-photodiode array analysis. These data show that MALDI/TOF-MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.     

Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry

Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.     

Phosphopeptide sequencing by in-source decay spectrum in delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Protein phosphorylation underlies numerous cellular signaling processes. Since a reliable prediction of phosphorylation site(s) based on a consensus amino acid sequence is rather difficult to date, determination of phosphorylation site(s) in phosphoproteins is a crucial step toward the understanding of their function at the molecular level. A conventional protocol for the determination of phosphorylation sites utilizes radioactive labeling of a phosphoprotein by (32)P and purification of digested peptides carrying radioactivity, followed by Edman degradation. This method is not only tedious, but also indirect because the evidence will be based on disappearance of a phenylthiohydantoin signal from the degradation cycle where the (32)P radioactivity is eluted. Several methodologies have been developed to determine the phosphorylation sites directly by using mass spectrometry. These include collision-induced dissociation (CID) and post-source decay (PSD), both of which tend to produce fragment ions less efficiently as the number of residues exceeds 20. Moreover, in both decay processes, there is a tendency for the phosphate group to be removed during the breakdown of the main peptide chain. We report a method that allows direct observation of phosphorylated peptide fragments of phosphopeptides exceeding 20 residues by using an in-source decay fragmentation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, yielding results which are difficult or impossible to obtain by existing methods using CID or PSD.     

Correlation between phosphorylation ratios by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and radioactivities by radioactive assay

To investigate the correlation between the counts per minute (CPM) by radioactivity assay and the phosphorylation ratio by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we prepared 136 peptide substrates. The correlation coefficient of phosphorylation ratios to CPM was 0.77 for all samples. However, the more the numbers of positively charged amino acids increased, the more the correlation coefficient increased. Although positively charged amino acids can have an effect on the correlation results, MALDI-TOF MS analysis is a useful means for monitoring phosphorylated peptide and protein kinase activity instead of radioactivity assays.     

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI–TOF and MALDI–qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250 ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H]⁺ ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI–MS; however, these fungi may be successfully analyzed by MALDI–TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI–TOF MS.