Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically relevant bacteria

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens.

Methodology/Principal Findings

We compared the identification efficiency of MALDI-TOF MS with that of Phoenix®, API® and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7% of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63% of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn''s kappa values: 0.9 to 0.93).

Conclusions/Significance

MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria.     

Characterization of propionic acid bacteria using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry

It was shown that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry could be used for the diagnostic characterization of propionic acid bacteria (PABs). The spectra of proteins (whole PAB cells) with a molecular mass of 3000 to 11 000 were obtained and analyzed using three matrices: sinapinic (SA), 2,5-dihydroxibenzoic (DHB), and α-cyano-4-hydroxycinnamic acid (HCCA). The MALDI spectra of PAB revealed the protein peaks characteristic of (1) the genus Propionibacterium (3496, 5386, 5605, 10 470), (2) the groups of species sharing the common composition of their cell walls and fatty acids, and (3) a species (four species were investigated). Exemplified by the P. shermanii strains (the collection and mutant ones) producing and not producing vitamin B₁₂, the possibility of using MALDI profiles for strain differentiation was confirmed. The MALDI profiles of the propionic acid cocci of the genus Luteococcus differ substantially from the profiles of PAB strains of the genus Propionibacterium, which is an additional proof of the validity of whole-cell MALDI spectra for generic differentiation of bacteria. Our investigation shows that the bacterial groups determined using the MALDI profiles correlate with the phylogenetic 16S rRNA gene groups, thus demonstrating the high resolution of this method for the differentiation of intraspecific differences (subspecies, strains).     

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.     

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.     

Use of surface-enhanced laser desorption/ionization -time of flight to explore bacterial proteomes

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is a recent technology that allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. We compared three simple procedures for protein extraction from bacteria for compatibility with the ProteinChip Array; we also determined the amount of protein required for each analysis. The protocol for the SELDI analysis was evaluated by generating protein expression profiles of a Streptococcus pneumoniae strain grown in different conditions and those of different strains of the same species. The protocol also was successfully applied to a wide range of Gram positive and negative bacteria. The results of this study suggest the appropriateness of this technology for microorganism protein profiling as complementary or alternative to two-dimensional gel electrophoresis.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of yeasts is contingent on robust reference spectra

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods.

Methods

MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster.

Principal Findings

Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results.

Conclusions

MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.     

Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin

Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.     

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.     

Matrix-assisted laser desorption/ionization mass spectrometry of immobilized duplex DNA probes.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry was used to analyze short DNA duplex probes with one strand immobilized on solid supports (straptavidin-coated magnetic beads or controlled pore glass beads). Only the non-immobilized strand could be detected. Partial denaturation was found when the duplex probes were mixed with 3-hydroxypicolinic acid, ammonium citrate matrix. The strategy has several applications, such as fast DNA sequence analysis and DNA diagnostics.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of glycosphingolipids including gangliosides.

Long chain base compositions of gangliosides containing mainly stearic acid could be determined without any chemical modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS). The analytical results for the long chain base compositions of various samples of GM1 from the brain tissues of patients with different diseases at different ages confirmed that the proportion of d20:1 (icosasphingosine) and d20 (icosa-sphinganine) of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50%; this value was evidently higher than the proportion of d20:1 and d20 of GM1 in various adult mammalian brains. A long chain base composition of GM1 from the brain tissue of a patient with infantile type of GM1-gangliosidosis at 4y2m was abnormal and so was in two sibling patients with Spielmeyer-Vogt type of juvenile amaurotic idiocy at 19y and 21y in spite of that in the latter there was no accumulation of GM1 in the brain tissue. On the other hand, a patient with adult type of GM1 gangliosidosis at 66y showed a local accumulation of GM1 in the putamen and caudate nucleus, but its long chain base composition was found to be normal. It was of interest that the white matter of Eker rat with hereditary renal carcinoma contained a large amount of plasmalocerebroside as compared with the amount of cerebroside and sphingomyelin. The individual molecular species of plasmalocerebroside were identified by DE MALDI-TOF MS.     

MALDI-TOF MS技术在临床病原真菌鉴定中的应用进展

基质辅助激光解吸电离飞行时间质谱技术（MALDI-TOF-MS）目前是一种快速而可靠的微生物鉴定方法.随着可鉴定真菌谱的完善,MALDI-TOF MS技术已逐步应用于临床常见致病酵母菌、酵母样真菌和丝状菌的鉴定中,本文将就此做一综述.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.     

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight (UV-MALDI-TOF) mass spectra of N-acylated and N,O-acylated glycosylamines

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.     

Improving the sensitivity of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry by using polyethylene glycol modified polyurethane MALDI target

Previous studies in our group have shown that the analyte signal in a matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) experiment is strongly influenced by the binding interactions between the target surface and the analyte. Specifically, the analyte signal increases with decreases in surface binding affinity, which has been attributed to more unbound analyte being available for incorporation within the MALDI matrix. In this work, polyethylene glycol (PEG) was chemically grafted onto a polyurethane (PU) film to produce a MALDI target having reduced surface-protein binding affinity, and the effect of this modification on protein MALDI ion signals was investigated. The proteins myoglobin, lysozyme, and albumin were used to evaluate the PEG PU modified target as compared with a PU target and a commercial stainless steel target. It is shown that there are enhancements in the protein MALDI ion signals on the PEG PU modified target and that the limit of detection for these proteins is decreased by a factor of 2 to 6 in comparison with the unmodified PU and the commercial stainless steel targets.     

Matrix-assisted laser desorption/ionization mass spectrometry of neutral and acidic oligosaccharides with collision-induced dissociation

Using ribonuclease B and human alpha 1-acid glycoprotein (AGP) as model glycoproteins, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) is validated here as an effective tool for oligosaccharide sequencing. The spectra acquired for high-mannose and complex oligosaccharide structures show characteristic fragments resulting from cleavages of the glycosidic bonds and a few cross-ring cleavages. Esterification of the sialic acid residues is essential in stabilizing the acidic N-linked oligosaccharides. An important analytical feature observed in all acquired spectra is the occurrence of cleavages on the same antenna up to the branching point, as deduced from the absence of fragmentation due to the simultaneous cleavages on two or more antennas.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identifying the composition of labeled proteins

Labeled proteins are extensively used in molecular biology and environmental science. The determination of the composition and label ratio is very important for monitoring the efficiency of their separation and purification. In this paper a novel method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry was developed for this purpose. The results obtained for three commercial labeled proteins showed that they are mixtures of different conjugates. In some cases, the label ratio obtained by UV spectrometry and MALDI mass spectrometry was strikingly different. For fluorescent labels such as fluorescein isothiocyanate, MALDI mass spectrometry determines the number of covalently bound labels, whereas UV absorption yields both bound and adsorbed labels. For biotinylated proteins, label ratios obtained by the 4-hydroxyazabenzene-2'-carboxylic acid (HABA)-avidin method were found to be much smaller those determined by MALDI mass spectrometry. The HABA-avidin method may therefore not be suitable for the determination of biotin label ratios.     

Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.     

Comparison of vacuum matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI (AP-MALDI) tandem mass spectrometry of 2-dimensional separated and trypsin-digested glomerular proteins for database search derived identification

Mass spectrometric based sequencing of enzymatic generated peptides is widely used to obtain specific sequence tags allowing the unambiguous identification of proteins. In the present study, two types of desorption/ionization techniques combined with different modes of ion dissociation, namely vacuum matrix-assisted laser desorption/ionization (vMALDI) high energy collision induced dissociation (CID) and post-source decay (PSD) as well as atmospheric pressure (AP)-MALDI low energy CID, were applied for the fragmentation of singly protonated peptide ions, which were derived from two-dimensional separated, silver-stained and trypsin-digested hydrophilic as well as hydrophobic glomerular proteins. Thereby, defined properties of the individual fragmentation pattern generated by the specified modes could be observed. Furthermore, the compatibility of the varying PSD and CID (MS/MS) data with database search derived identification using two public accessible search algorithms has been evaluated. The peptide sequence tag information obtained by PSD and high energy CID enabled in the majority of cases an unambiguous identification. In contrast, part of the data obtained by low energy CID were not assignable using similar search parameters and therefore no clear results were obtainable. The knowledge of the properties of available MALDI-based fragmentation techniques presents an important factor for data interpretation using public accessible search algorithms and moreover for the identification of two-dimensional gel separated proteins.