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。     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice

Background

Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed.

Methodology

We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10⁵ colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours.

Conclusions

Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.     

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.     

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.     

Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

To evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as a tool for rapid identification of common clinical bacterial isolates, we analyzed 25 carefully selected isolates of pathogenic Escherichia coli (E. coli) and additional Enterobacteriaceae members. Organisms were prepared according to clinical microbiological protocols and analyzed with minimal additional processing. Spectra were reproducible from preparation to preparation and comprised 40-100 peaks primarily representing intracellular proteins with masses up to 25 kDa. Spectra of 14 genetically diverse bacteremic isolates of E. coli were compared with isolates representing other genera within the Enterobacteriaceae family. Using a new spectrum comparison algorithm, E. coli isolates were closely related to each other and were readily distinguishable from other Enterobacteriaceae, including Salmonella and Shigella. Presently, the methodology permits the analysis of 40 unknown isolates per hour per instrument. These results suggest that MALDI-ToF MS offers a rapid and reliable approach for performing phyloproteomics i.e., identification of unknown bacterial isolates based on similarities within protein biomarker databases.     

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.     

Global analysis of circulating immune cells by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Background

MALDI-TOF mass spectrometry is currently used in microbiological diagnosis to characterize bacterial populations. Our aim was to determine whether this technique could be applied to intact eukaryotic cells, and in particular, to cells involved in the immune response.

Methodology/Principal Findings

A comparison of frozen monocytes, T lymphocytes and polymorphonuclear leukocytes revealed specific peak profiles. We also found that twenty cell types had specific profiles, permitting the establishment of a cell database. The circulating immune cells, namely monocytes, T lymphocytes and polymorphonuclear cells, were distinct from tissue immune cells such as monocyte-derived macrophages and dendritic cells. In addition, MALDI-TOF mass spectrometry was valuable to easily identify the signatures of monocytes and T lymphocytes in peripheral mononuclear cells.

Conclusions/Significance

This method was rapid and easy to perform, and unlike flow cytometry, it did not require any additional components such as specific antibodies. The MALDI-TOF mass spectrometry approach could be extended to analyze the cell composition of tissues and the activation state of immune cells.     

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.     

Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Nucleoside diphosphate kinase (NDPK, Nm23) has been implicated as a multifunctional protein. However, the regulatory mechanism of NDPK is poorly understood. We have examined the modification of NDPK in oxidative stresses. We found that oxidative stresses including diamide and H(2)O(2) treatment cause disulfide cross-linking of NDPK inside cells. This cross-linking was reversible in response to mild oxidative stress, and irreversible to strong stress. This suggests that disulfide cross-linked NDPK may be a possible mechanism in the modification of cellular regulation. To confirm this idea, oxidative modification of NDPK has been performed in vitro using purified human NDPK H(2)O(2) inactivated the nucleoside diphosphate (NDP) kinase activity of NDPK by producing intermolecular disulfide bonds. Disulfide cross-linking of NDPK also dissociated the native hexameric structure into a dimeric form. The oxidation sites were identified by the analysis of tryptic peptides of oxidized NDPK, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Intermolecular cross-linking between Cys109-Cys109, which is highly possible based on the X-ray crystal structure of NDPK-A, and oxidations of four methionine residues were identified in H(2)O(2)-treated NDPK. This cross-linkng was confirmed using mutant C109A (NDPK-A(C109A)) which had similar enzymatic activity as a wild NDPK-A. Mutant NDPK-A(C109A) was not cross-linked and was not easily denatured by the oxidant. Therefore, enzymatic activity and the quaternary structure of NDPK appear to be regulated by cross-linking with oxidant. These findings suggest one of the regulatory mechanisms of NDPK in various cellular processes.     

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.     

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.     

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.     

Improving sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.     

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.     

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of bacteria cultured in liquid media

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for many years to rapidly identify whole bacteria. However, no consistent methodology exists for the rapid identification of bacteria cultured in liquid media. Thus, in this study we explored the use of MALDI-TOF MS analysis for rapid identification of cells cultured in liquid media. We determined that 2,5-dihydroxybenzoic acid (50 mg mL^?1, 50% acetonitrile, 0.1% trifluoroacetic acid) was the best matrix solution for MALDI-TOF MS for this type of study. Moreover, the tested strains were successfully differentiated by principal component analysis, and the main characteristics of the mass peaks for each species were found in mixed culture samples. In addition, we found that the minimum number of cells for detection was 1.8×10³. In conclusion, our findings suggest that MS-based techniques can be developed as an auxiliary method for rapidly and accurately identifying bacteria cultured in liquid media.     

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.