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 PhoenixH, APIH 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

Mass Spectrometry and Tandem Mass Spectrometry Characterization of Protein Patterns, Protein Markers and Whole Proteomes For Pathogenic Bacteria

There have been many recent reviews published on MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight) MS (mass spectrometry) for identification of bacteria particularly with relevance to clinical microbiology. MALDI-TOF MS is now a mature technique for bacterial identification with great promise. The purpose of this review is to put into perspective MALDI-TOF MS and other widely used mass spectrometry methods for characterization of proteins. MALDI-TOF MS is used for rapid determination of a mass pattern of proteins for bacterial characterization; these proteins are generally not identified. Alternatively, after gel separation, MALDI TOF-TOF MSMS (tandem mass spectrometry) or on-line LC-ESI MS-MS (liquid chromatography electrospray tandem mass spectrometry) specific protein markers can be identified and peptide sequence variation among species assessed. Unlike direct MALDI-TOF MS, sample preparation for gel separation/MALDI-TOF-TOF MS and MS-MS remains quite demanding. Specific marker proteins are readily identified. Sample preparation is quite straightforward for LC-MS-MS. Massive amounts of information (whole proteomes) are provided but bioinformatics is complex. Chromatography and electrospray mass spectrometry instrumentation is also not widely used among microbiologists. Thus, there is a need for further development in sample preparation and instrumental development for rapid and simplified analysis. As MS-MS for microbial characterization reaches maturity, it is to be anticipated that further developments in bioinformatics will also become essential. The genome codes for all proteins that might be synthesized under certain growth conditions but only direct protein identification can prove that specific proteins or networks of proteins are actually expressed which might be of relevance in improving our understanding of bacterial pathogenesis

Detection and sequencing of microRNA using MALDI time-of-flight mass spectrometry

The discovery of microRNAs (miRNAs) and their abilities to regulate in vivo protein synthesis have led to growing interests in miRNA research. Due to their functions on regulating cellular processes that are related to diseases such as cancer, miRNAs can potentially become a new class of diagnostic biomarkers and therapeutic agents. Since early 2000, researchers have reported more than 1,000 human miRNAs. To facilitate high-throughput clinical studies, there are needs for more accurate and robust analytical methods to detect and quantitate miRNAs. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used mainly for proteomics research. In the past two decades, MALDI-TOF MS has also been used in genomics research. Specifically, MALDI-TOF MS has been used in the analysis of oligonucleotides, both DNA and RNA. In this study, the use of MALDI-TOF MS to detect specific miRNA was optimized, and the applicability of tandem MALDI-TOF/TOF to perform partial de novo sequencing was evaluated. The MALDI process requires a small organic compound, often known as "matrix", which readily undergoes desorption on contact with UV laser and assists the ionization of analyte. Therefore, an investigative study was designed to compare five commonly used MALDI matrix compounds for oligonucleotide analysis. The selected matrix compounds includes 3-hydroxypicolinic acid (3-HPA), 2',3',4'-trihydroxy-acetophenone (THAP), 6-aza-thiothymidine (6-ATT), 3,4-diaminobenzophenone (DABP) and 3-hydroxycoumarin (3-HC). The goal is to identify which is the best matrix for supporting the MALDI process and the subsequent tandem MALDI-TOF/TOF measurements. The 4700 Applied Biosystems Proteomics Analyzer was initially used to perform linear MALDI-TOF MS analysis of a selected microRNA standard (miR-124a). The initial results of our MS/MS measurements indicated higher signal intensity or ion count of the parent ion of miRNA is required to achieve sequencing. For this reason, we embarked on a series of studies to increase the signal intensity, especially on the effects of various parameters that were related to the desorption of miRNA during the MALDI process. The results indicated 3-HPA matrix has provided the highest signal intensity. Thus, 3-HPA was used to further optimize the MALDI-TOF MS measurements of miRNA. Once the parameters of MALDI-TOF MS were optimized, the use of tandem MALDI-TOF/TOF MS to perform partial de novo sequencing of miRNA was evaluated. Following the acquisition of initial MS/MS spectra of miR-124a, the use of different collision-induced dissociation (CID) pressures and delayed times to induce the fragmentation of miR-124a parent ion were investigated. It was determined that the 4700 Proteomics Analyzer could have a limitation on measuring the singly-charged miR-124a parent ion (7,161 m/z). This could be due to the current design and/or settings on the reflectron within the MALDI-TOF/TOF instrumentation were not suitable for measuring ions with high molecular masses. To overcome this limitation, the doubly-charged miR-124a parent ion (3,582 m/z) was selected as an alternative for performing the tandem MALDI-TOF/TOF MS measurements. Both post-source decay (PSD) and CID in the positive ion mode were used

Assessment of Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Bacterial and Yeast Identification

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of clinical bacterial and yeast isolates. However, data describing the reproducibility of MALDI-TOF MS for microbial identification are scarce. In this study, we show that MALDI-TOF MS-based microbial identification is highly reproducible and can tolerate numerous variables, including differences in testing environments, instruments, operators, reagent lots, and sample positioning patterns. Finally, we reveal that samples of bacterial and yeast isolates prepared for MALDI-TOF MS identification can be repeatedly analyzed without compromising organism identification

Comparison of biomarker based Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and conventional methods in the identification of clinically relevant bacteria and yeast

Background: MALDI-TOF MS is an analytical method that has recently become integral in the identification of microorganisms in clinical laboratories. It relies on databases that majorly employ pattern recognition or fingerprinting. Biomarker based databases have also been developed and there is optimism that these may be superior to pattern recognition based databases. This study compared the performance of ribosomal biomarker based MALDI-TOF MS and conventional methods in the identification of selected bacteria and yeast. Methods: The study was a cross sectional study identifying clinically relevant bacteria and yeast isolated from varied clinical specimens submitted to a clinical laboratory. The identification of bacteria using conventional Vitek 2™ automated system, serotyping and MALDI-TOF MS was performed as per standard operating procedures. Comparison of sensitivities were then carried out using Pearson Chi-Square test and p-value of Results: Of the 383 isolates MALDI-TOF MS and conventional methods identified 97.6 and 95.7% (p = 0.231) to the genus level and 97.4 and 88.0% (p = 0.000) to the species level respectively. Biomarker based MALDI-TOF MS was significantly superior to Vitek 2™ in the identification of Gram negative bacteria and Gram positive bacteria to the species level. For the Gram positive bacteria, significant difference was observed in the identification of Coagulase negative Staphylococci (p = 0.000) and Enterococcus (p = 0.008). Significant difference was also observed between serotyping and MALDI-TOF MS (p = 0.005) and this was attributed to the lack of identification of Shigella species by MALDI-TOF MS. There was no significant difference observed in the identification of yeast however some species of Candida were unidentified by MALDI-TOF MS. Conclusion: Biomarker based MALDI-TOF MS had good performance in a clinical laboratory setting with high sensitivities in the identification of clinically relevant microorganisms

Subspecies typing of Streptococcus agalactiae based on ribosomal subunit protein mass variation by MALDI-TOF MS

Background: A ribosomal subunit protein (rsp)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was developed for fast subspecies-level typing of Streptococcus agalactiae (Group B Streptococcus, GBS), a major cause of neonatal sepsis and meningitis. Methods: A total of 796 GBS whole genome sequences, covering the genetic diversity of the global GBS population, were used to in silico predict molecular mass variability of 28 rsp and to identify unique rsp mass combinations, termed “rsp-profiles”. The in silico established GBS typing scheme was validated by MALDI-TOF MS analysis of GBS isolates at two independent research sites in Europe and South East Asia. Results: We identified in silico 62 rsp-profiles, with the majority (>80%) of the 796 GBS isolates displaying one of the six rsp-profiles 1-6. These dominant rsp-profiles classify GBS strains in high concordance with the core-genome based phylogenetic clustering. Validation of our approach by in-house MALDI-TOF MS analysis of 248 GBS isolates and external analysis of 8 GBS isolates showed that across different laboratories and MALDI-TOF MS platforms, the 28 rsp were detected reliably in the mass spectra, allowing assignment of clinical isolates to rsp-profiles at high sensitivity (99%) and specificity (97%). Our approach distinguishes the major phylogenetic GBS genotypes, identifies hyper-virulent strains, predicts the probable capsular serotype and surface protein variants and distinguishes between GBS genotypes of human and animal origin. Conclusion: We combine the information depth of whole genome sequences with the highly cost efficient, rapid and robust MALDI-TOF MS approach facilitating high-throughput, inter-laboratory, large-scale GBS epidemiological and clinical studies based on pre-defined rsp-profiles

Why Not Mold? A Meta-Narrative Review of MALDI-TOF MS

Hospitals have recorded immunocompromised patients to be highly susceptible to hospital acquired infections caused by yeasts and molds. Successfully identifying such pathogens is important for the rapid diagnosis and treatment of these infections, marking the significance of this study. Since 2010, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has made mold identification more efficient but its usage in clinical laboratories is widely restricted. With an aim to understand this paradox, this meta-narrative review investigates the limited use of MALDI-TOF MS in mold identification, including factors affecting the accuracy and preparation of specimens. PubMed Central Database was accessed through the MD Anderson Research Library for this critical literature review and included articles from 2019 to 2024. Search parameters included keywords ‘MALDI-TOF MS’, ‘mold’, and ‘fungus’ —with ‘nosocomial’ added as an advanced search filter. This review emphasized findings of publications from 2019 and 2020, which both concur the lack of a comprehensive and standardized database for mold species was a major limitation of MALDI-TOF MS. Consequently, such limitations characterize MALDI-TOF MS as a verification tool for primary identification methods like microscopic analysis. This gap reveals potential for expanding the use of MALDI-TOF MS for mold identification by developing a complete database for mold species or implementing rigorous pre-analytical controls and utilizing software databases alongside MALDI-TOF analysis. Future research could investigate software, agencies, and parameters needed to create a universal database for molds or review more publications to determine whether MALDI-TOF MS can and should replace microscopic analysis.https://openworks.mdanderson.org/rmps24/1010/thumbnail.jp

MALDI-TOF Mass Spectrometry Discriminates Known Species and Marine Environmental Isolates of Pseudoalteromonas

The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies

Liquid MALDI MS analysis of complex peptide and proteome samples

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, i.e. the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints which led to a higher confidence in protein identification compared to solid (crystalline) MALDI samples. However, post-source decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples as the MS/MS ion search score was lower than for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable