Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied as an identification procedure in clinical microbiology and has been widely used in routine laboratory practice because of its economical and diagnostic benefits. The range of applications of MALDI-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology. The purpose of this review is to summarize the contribution of the studies already performed with MALDI-TOF MS concerning antibiotic resistance and to analyze future perspectives in this field. We believe that current research should continue in four main directions, including the detection of antibiotic modifications by degrading enzymes, the detection of resistance mechanism determinants through proteomic studies of multiresistant bacteria, and the analysis of modifications of target sites, such as ribosomal methylation. The quantification of antibiotics is suggested as a new approach to study influx and efflux in bacterial cells. The results of the presented studies demonstrate that MALDI-TOF MS is a relevant tool for the detection of antibiotic resistance and opens new avenues for both clinical and experimental microbiology.
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.
With wide applications of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), statistical comparison of serum peptide profiles and management of patients information play an important role in clinical studies, such as early diagnosis, personalized medicine and biomarker discovery. However, current available software tools mainly focused on data analysis rather than providing a flexible platform for both the management of patients information and mass spectrometry (MS) data analysis.
Here we presented a plug-in-based software, BioSunMS, for both the management of patients information and serum peptide profiles-based statistical analysis. By integrating all functions into a user-friendly desktop application, BioSunMS provided a comprehensive solution for clinical researchers without any knowledge in programming, as well as a plug-in architecture platform with the possibility for developers to add or modify functions without need to recompile the entire application.
BioSunMS provides a plug-in-based solution for managing, analyzing, and sharing high volumes of MALDI-TOF or SELDI-TOF MS data. The software is freely distributed under GNU General Public License (GPL) and can be downloaded from http://sourceforge.net/projects/biosunms/.
Jonah crab Cancer borealis is an excellent model organism long served for many areas of physiology, including the study of endocrinology and neurobiology. Characterizing the neuropeptides present in its nervous system provides the first critical step toward understanding the physiological roles of these complex molecules. Multiple mass spectral techniques were used to comprehensively characterize the neuropeptidome in C. borealis, including matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI FTMS), MALDI time of flight (TOF)/TOF MS and nanoflow liquid chromatography coupled to electrospray ionization quadrupole time of flight tandem mass spectrometry (nanoLC ESI Q TOF MS/MS). In order to enhance the detection signals and expand the dynamic range, direct tissue analysis, tissue extraction, capillary electrophoresis (CE) and off-line HPLC separation have also been employed. In total, 142 peptides were identified, including 85 previously known C. borealis peptides, 22 peptides characterized previously from other decapods, but new to this species, and 35 new peptides de novo sequenced for the first time in this study. Seventeen neuropeptide families were revealed including RFamide, allatostatin (A and B type), RYamide, orcokinin, orcomyotropin, proctolin, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone precursor-related peptide (CPRP), crustacean hyperglycemic hormone (CHH), corazonin, pigment-dispersing hormone (PDH), tachykinin, pyrokinin, SIFamide, red pigment concentrating hormone (RPCH) and HISGLYRamide. Collectively, our results greatly increase the number and expand the coverage of known C. borealis neuropeptides, and thus provide a stronger framework for future studies on the physiological roles played by these molecules in this important model organism.
Cancer borealis; matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI FTMS); electrospray ionization quadrupole time of flight mass spectrometry (ESI-Q-TOF MS); neuropeptides; peptide sequencing; peptidomics; thoracic ganglia; stomatogastric ganglia; commissural ganglia; sinus gland; pericardial organ
With the decipherment of the human genome, new questions have moved into the focus of today's research. One key aspect represents the discovery of DNA variations capable to influence gene transcription, RNA splicing, or regulating processes, and their link to pathology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a powerful tool for the qualitative investigation and relative quantification of variations like single nucleotide polymorphisms, DNA methylation, microsatellite instability, or loss of heterozygosity. After its introduction into proteomics, efforts were made to adopt this technique to DNA analysis. Initially intended for peptide/protein analysis, it held several difficulties for application to nucleic acids. Today, MALDI-TOF-MS has reached worldwide acceptance and application in nucleic acid research, with a wide spectrum of methods being available. One of the most versatile approaches relies on primer extension to genotype single alleles, microsatellite repeat lengths or the methylation status of a given cytosine. Optimized methods comprising intelligent primer design and proper nucleotide selection for primer extension enabled multiplexing of reactions, rendering the analysis more economic due to parallel genotyping of several alleles in a single experiment. Laboratories equipped with MALDI-TOF-MS possess a universal technical platform for the analysis of a large variety of different molecules.
MALDI-TOF; Mass spectrometry; Single nucleotide polymorphism; Methylation; Microsatellite instability; Genotyping
The purpose of this study was to use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in osteoarthritis research. Our aim was to find differentially expressed disease-related and condition-specific peptide in synovial fluid in the knee joint of patients suffering from osteoarthritis (OA), and to develop and validate the peptide classification model for OA diagnosis.
Based on the American College of Rheumatology criteria, 30 OA cases and ten healthy donors were enrolled and underwent analysis. Magnetic beads-based weak cation exchange chromatography (MB-WCX) was performed for sample processing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was conducted for peptide profile. ClinProt software 2.2 was used for data analysis and a genetic algorithm was created for class prediction.
Two peptide peaks were found which may be characterised as the potential diagnostic markers for OA. Two other significantly different peptide peaks were found in OA patients at a medium stage compared to the early and late stages. A genetic algorithm (GA) was used to establish differential diagnosis models of OA. As a result, the algorithm models marked 100% of OA, and of 97.92% of medium-stage OA.
This study demonstrated that use of proteomics methods to identify potential biomarkers of OA is possible, and the identified potential biomarkers may be potential markers for diagnosis and monitoring the progression of OA.
Breast cancer is one of the most common types of invasive cancer in females worldwide. Despite major advances in early cancer detection and emerging therapeutic strategies, further improvement has to be achieved for precise diagnosis to reduce the chance of metastasis and relapses. Recent proteomic technologies have offered a promising opportunity for the identification of new breast cancer biomarkers. Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and the derived surface-enhanced laser desorption/ionization mass spectrometry (SELDI-TOF MS) enable the development of high-throughput proteome analysis based on comprehensive reliable biomarkers. In this review, we examined proteomic technologies and their applications, and provided focus on the proteomics-based profiling analyses of tumor tissues/cells in order to identify and confirm novel biomarkers of breast cancer.
breast cancer; proteomics; biomarkers; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality.
This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed.
MALDI-TOF MS; food microbiology; species identification; bacteria; food isolates
Recent advancement in mass spectrometry leads us to a new era of proteomic analysis. Human saliva can be easily collected; however, the complexity of the salivary proteome in the past prevented the use of saliva for proteomic analysis. Here we review the development of proteomic analyses for human saliva and focus on the use of a new mass spectrometric technology known as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). SELDI-TOF, a modification of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF), combines the precision of mass spectrometry and the high-through-put nature of protein arrays known as Protein Chips. This technology shows a promising future for salivary proteomic analysis in monitoring treatments and diseases, as well as novel biomarker discovery.
Proteomics; mass spectrometry; biomarker; biomarker discovery; proteomic profiling; protein expression; SELDI; SELDI-TOF; protein chips.
Whole-cell matrix–assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been successfully applied for bacterial identification and typing of many pathogens. The fast and reliable qualities of MALDI-TOF MS make it suitable for clinical diagnostics. MALDI-TOF MS for the identification and cluster analysis of Streptococcus pyogenes, however, has not been reported. The goal of our study was to evaluate this approach for the rapid identification and typing of S. pyogenes.
65 S. pyogenes isolates were obtained from the hospital. The samples were prepared and MALDI-TOF MS measurements were conducted as previously reported. Identification of unknown spectra was performed via a pattern recognition algorithm with a reference spectra and a dendrogram was constructed using the statistical toolbox in Matlab 7.1 integrated in the MALDI Biotyper 2.0 software.
For identification, 61 of 65 S. pyogenes isolates could be identified correctly by MALDI-TOF MS with BioType 2.0 when compared to biochemical identification (API Strep), with an accuracy of 93.85%. In clustering analysis, 44 of 65 isolates were in accordance with those established by M typing, with a matching rate of 67.69%. When only the M type prevalence in China was considered, 41 of 45 isolates were in agreement with M typing, with a matching rate of 91.1%.
It was here shown that MALDI-TOF MS with Soft Biotype 2.0 and its database could facilitate rapid identification of S. pyogenes. It may present an attractive alternative to traditional biochemical methods of identification. However, for classification, more isolates and advances in the MALDI-TOF MS technology are needed to improve accuracy.
Graphene was utilized for the first time as matrix for the analysis of low-molecular weight compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Polar compounds including amino acids, polyamines, anticancer drugs and nucleosides could be successfully analyzed. Additionally, nonpolar compounds including steroids could be detected with high resolution and sensitivity. Compared with conventional matrix, graphene exhibited high desorption/ionization efficiency for nonpolar compounds. The graphene matrix functions as substrate to trap analytes, and it transfers energy to the analytes upon laser irradiation, which allowed for the analytes to be readily desorbed/ionized and interference of intrinsic matrix ions to be eliminated. The use of graphene as matrix avoided the fragmentation of analytes and provided good reproducibility and high salt tolerance, underscoring the potential application of graphene as matrix for MALDI-MS analysis of practical samples in complex sample matrices. We also demonstrated that the use of graphene as adsorbent for the solid-phase extraction of squalene could improve greatly the detection limit. This work not only opens a new field for applications of graphene, but also offers a new technique for high-speed analysis of low-molecular weight compounds in areas such as metabolism research and natural products characterization.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS) allows rapid and reliable identification of microorganisms,
particularly clinically important pathogens.
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
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).
MALDI-TOF MS represents a rapid, reliable and cost-effective identification
technique for clinically relevant bacteria.
As osteoarthritis (OA) is a highly heterogeneous disease in terms of progression, establishment of prognostic biomarkers would be highly beneficial for treatment. The present study was performed to identify novel biomarkers capable of predicting the progression of knee OA.
A total of 69 plasma samples (OA patients undergoing radiographic progression, n = 25; nonprogression, n = 33; healthy donors, n = 11) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and ion peaks of interest were identified by liquid chromatography and matrix-assisted laser desorption/ionization (MALDI)-TOF MS. The identities of these proteins were further validated by immunoprecipitation combined with SELDI-TOF MS analysis.
SELDI-TOF MS analysis indicated that the intensities of 3 ion peaks differed significantly between progressors and nonprogressors. Subsequent analyses indicated that these peaks corresponded to apolipoprotein C-I, C-III, and an N-terminal truncated form of transthyretin, respectively. The identities of these proteins were confirmed by the loss of ion peaks in SELDI-TOF MS spectra by immunoprecipitation using specific antibodies for the respective proteins.
Three potential biomarkers were identified whose serum levels differed significantly between OA progressors and nonprogressors. These biomarkers are expected to be prognostic biomarkers for knee OA and to facilitate the development of novel disease-modifying treatments for OA.
osteoarthritis; biomarker; proteomics; surface-enhanced laser desorption/ionization (SELDI)
Individual characteristics of pathophysiology and course of depressive episodes are at present not considered in diagnostics. There are no biological markers available that can assist in categorizing subtypes of depression and detecting molecular variances related to disease-causing mechanisms between depressed patients. Identification of such differences is important to create patient subgroups, which will benefit from medications that specifically target the pathophysiology underlying their clinical condition. To detect characteristic biological markers for major depression, we analyzed the cerebrospinal fluid (CSF) proteome of depressed vs control persons, using two-dimensional polyacrylamide gel electrophoresis and time-of-flight (TOF) mass spectrometry peptide profiling. Proteins of interest were identified by matrix-assisted laser desorption ionization TOF mass spectrometry (MALDI-TOF-MS). Validation of protein markers was performed by immunoblotting. We found 11 proteins and 144 peptide features that differed significantly between CSF from depressed patients and controls. In addition, we detected differences in the phosphorylation pattern of several CSF proteins. A subset of the differentially expressed proteins implicated in brain metabolism or central nervous system disease was validated by immunoblotting. The identified proteins are involved in neuroprotection and neuronal development, sleep regulation, and amyloid plaque deposition in the aging brain. This is one of the first hypothesis-free studies that identify characteristic protein expression differences in CSF of depressed patients. Proteomic approaches represent a powerful tool for the identification of disease markers for subgroups of patients with major depression.
major depression; cerebrospinal fluid biomarkers; phosphoproteins; PEDF; cystatin C; PGDS; molecular & cellular neurobiology; depression; unipolar/bipolar; mood/anxiety/stress disorders; psychiatry & Behavioral sciences; proteomics; cerebrospinal fluid
The advent of contemporary proteomic technologies has ushered in definite advances to the field of auditory research and has provided the potential for a dramatic increase in applications in the near future. Two dimensional-differential gel electrophoresis (2D-DIGE) followed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), antibody microarrays and tandem mass spectrometry have evolved as the major tools. Each of these techniques has unique features with distinct advantages. This review attempts to highlight the common as well as diverse characteristics of these methods and their suitability and application to different experimental conditions employed to investigate the auditory system. In addition a glimpse of the valuable scientific information that has been gained in the hearing field using a proteomic approach is given. Finally, a brief view of the directions that auditory proteomics research is headed for has been discussed.
proteomics; DIGE; mass spectrometry; antibody microarray; pathway analysis; hearing
The American lobster Homarus americanus is a decapod crustacean with both high economic and scientific importance. To facilitate physiological investigations of peptide transmitter/hormone function in this species, we have used matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF-MS/MS) to elucidate the peptidome present in its nervous system and neuroendocrine organs. In total, 84 peptides were identified, including 27 previously known H. americanus peptides (e.g. VYRKPPFNGSIFamide [Val1-SIFamide]), 23 peptides characterized previously from other decapods, but new to the American lobster (e.g. pQTFQYSRGWTNamide [Arg7-corazonin]), and 34 new peptides de novo sequenced/detected for the first time in this study. Of particular note are a novel B-type allatostatin (TNWNKFQGSWamide) and several novel FMRFamide-related peptides, including an unsulfated analog of sulfakinin (GGGEYDDYGHLRFamide), two myosuppressins (QDLDHVFLRFamide and pQDLDHVFLRFamide), and a collection of short neuropeptide F isoforms (e.g. DTSTPALRLRFamide, and FEPSLRLRFamide). Our data also include the first detection of multiple tachykinin-related peptides in a non-brachyuran decapod, as well as the identification of potential individual-specific variants of orcokinin and orcomyotropin-related peptide. Taken collectively, our results not only expand greatly the number of known H. americanus neuropeptides, but also provide a framework for future studies on the physiological roles played by these molecules in this commercially and scientifically important species.
Homarus americanus; matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS); electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS); matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); neuropeptide; neurohormone; peptide sequencing; supraoesophageal ganglion; suboesophageal ganglion; thoracic ganglia; abdominal ganglia; eyestalk ganglia (ESG); stomatogastric ganglion (STG); sinus gland (SG); pericardial organ (PO); ventral nerve cord (VNC)
The differentiation of species within the Streptococcus mitis group has posed a problem in the routine diagnostic microbiology laboratory for some time. It also constitutes a major weakness of recently introduced matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) fingerprinting systems. As the phylogenetic resolution of the spectral similarity measures employed by these systems is insufficient to reliably distinguish between the most closely related members of the group, the major pathogen Streptococcus pneumoniae is frequently misidentified. In this study, a comparative analysis of MALDI-TOF spectra of several species from the S. mitis group has been performed in order to identify single peaks that could be used to improve mass spectrometry-based identification of the respective species. A characteristic peak profile could be identified that unambiguously distinguished the 14 S. pneumoniae isolates studied from 33 nonpneumococcal isolates of the S. mitis group. In addition, specific peak combinations could be assigned to other members of the group. The findings of this study suggest that it is possible to distinguish different species of the S. mitis group by close analysis of their mass peak profiles.
A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2′,3′-cyclic phosphate, 3′-phosphate, 3′-phosphoglycolate, 5′- hydroxyl and 5′- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2′-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.
Matrix-Assisted Laser Desorption/Ionisation (MALDI) mass spectrometry imaging (MSI) uses the power of high mass resolution time of flight (ToF) mass spectrometry coupled to the raster of lasers shots across the cut surface of tissues to provide new insights into the spatial distribution of biomolecules within biological tissues. The history of this technique in animals and plants is considered and the potential for analysis of proteins by this technique in plants is discussed. Protein biomarker identification from MALDI-MSI is a challenge and a number of different approaches to address this bottleneck are discussed. The technical considerations needed for MALDI-MSI are reviewed and these are presented alongside examples from our own work and a protocol for MALDI-MSI of proteins in plant samples.
MALDI MS imaging; MSI; mass spectrometry imaging; plant; plant imaging soybean; protein; protein imaging
Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology has been applied to the exploration of biomarkers for early cancer diagnosis, but more effort is required to identify a single sensitive and specific biomarker. For early diagnosis, a proteomic profile is the gold standard, but inconvenient for clinical use since the profile peaks are quantitative. It would therefore be helpful to find a minimized profile, comprising fewer peaks than the original using an existing algorithm and compare it with other traditional statistical methods.
In the present study, principal component analysis (PCA) in the ClinProt-Tools of MALDI-TOF MS was used to establish a mini-optimized proteomic profile from gastric cancer patients and healthy controls, and the result was compared with t-test and Flexanalysis software.
Eight peaks were selected as the mini-optimized proteomic profile to help differentiate between gastric cancer patients and healthy controls. The peaks at m/z 4212 were regarded as the most important peak by the PCA algorithm. The peaks at m/z 1866 and 2863 were identified as deriving from complement component C3 and apolipoprotein A1, respectively.
PCA enabled us to identify a mini-optimized profile consisting of significantly differentiating peaks and offered the clue for further research.
MALDI-TOF MS; mini-profile; PCA; proteomics
Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 × g) to remove leukocytes and then at high revolutions (15,500 × g) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >105 CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was Escherichia coli (173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%). Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved.
The increased interests on using small non-coding RNA such as microRNA as targets for drug development or diagnosis of different diseases have raised the demand for more accurate analysis of RNA. The most common method for nucleic acid analysis is sequencing. Unlabeled RNA sequencing ladders can be directly measured by using mass spectrometry (MS), and RNA sequence is determined by the mass difference between successive peaks in the mass spectrum. Among various MS techniques, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) requires relatively small sample size, and provides high sample throughput as well as the largest mass range. In a standard MALDI sample preparation method, a sample of interest is mixed with an excess amount of MALDI matrix. In this study, by taking advantage of the acidity of MALDI matrix, a unique yet simple approach to generate RNA sequencing ladders is developed. By using high resolution MALDI SpiralTOF MS, monoisotopic mass of each RNA fragment generated from selected microRNAs was measured. To deduce the RNA sequence, the nucleotide composition of each individual RNA fragment was calculated from its measured monoisotopic mass. The RNA sequence was then determined by the difference between the nucleotide compositions. The advantage of this approach is the nucleotide composition of each RNA fragment can be crosschecked with the nucleotide composition of an adjacent fragment within the same sequencing ladder. The crosschecking of a specific nucleotide composition can also be repeated with those obtain from other RNA fragments within the same sequencing ladder, thus allowing the RNA sequence to be confirmed multiple times. This sequencing method (patent pending) can also achieve double read of RNA sequences. In a blinded study, the RNA sequences of three different human microRNAs that were obtained by using this method matched 100% with the known sequences, and 100% sequence coverage was achieved.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) of intact bacteria yields a reproducible spectrum depending upon growth conditions, strain, or species. Using whole viable bacteria we describe here the application of MALDI-TOF-MS to the identification of coagulase-negative staphylococci (CoNS). Our aim was, once a bacterium has been recognized as Micrococcaceae, to identify peaks in the spectrum that can be used to identify the species or subspecies. MALDI-TOF-MS was performed using bacteria obtained from one isolated colony. One reference strain for each of the 23 clinically relevant species or subspecies of Micrococcaceae was selected. For each reference strain, the MALDI-TOF-MS profile of 10 colonies obtained from 10 different passages was analyzed. For each strain, only peaks that were conserved in the spectra of all 10 isolated colonies and with a relative intensity above 0.1 were retained, thus leading to a set of 3 to 14 selected peaks per strain. The MALDI-TOF-MS profile of 196 tested strains was then compared with that of the set of selected peaks of each of the 23 reference strains. In all cases the best hit was with the set of peaks of the reference strain belonging to the same species as that of the tested strain, thus demonstrating that the 23 sets of selected peaks can be used as a database for the rapid species identification of CoNS. Similar results were obtained using four different growth conditions. Extending this strategy to other groups of relevant pathogenic bacteria will allow rapid bacterial identification.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging mass spectrometry, also called MALDI-imaging, is a label-free bioanalytical technique used for spatially-resolved chemical analysis of a sample. Usually, MALDI-imaging is exploited for analysis of a specially prepared tissue section thaw mounted onto glass slide. A tremendous development of the MALDI-imaging technique has been observed during the last decade. Currently, it is one of the most promising innovative measurement techniques in biochemistry and a powerful and versatile tool for spatially-resolved chemical analysis of diverse sample types ranging from biological and plant tissues to bio and polymer thin films. In this paper, we outline computational methods for analyzing MALDI-imaging data with the emphasis on multivariate statistical methods, discuss their pros and cons, and give recommendations on their application. The methods of unsupervised data mining as well as supervised classification methods for biomarker discovery are elucidated. We also present a high-throughput computational pipeline for interpretation of MALDI-imaging data using spatial segmentation. Finally, we discuss current challenges associated with the statistical analysis of MALDI-imaging data.