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1.  Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods 
Arthritis Research  2000;2(5):407-414.
The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.
Introduction:
The classification of antinuclear antibodies (ANAs) is important for diagnosis and prognosis and for understanding the molecular pathology of autoimmune disease. Many of the proteins that associate with RNA in the ribonucleoprotein (RNP) complexes of the spliceosome have been found to react with some types of ANA [1], including proteins of the heterogeneous nuclear RNP (hnRNP) complex that associate with newly transcribed pre-mRNA. Autoantibodies to the A2, B1, and B2 proteins of hnRNP found in some patients may be markers of several overlap syndromes [2]. However, ANAs with specificity for these proteins as well as for the D protein also appear to occur in many distinct connective-tissue diseases, although epitope specificities may differ [3]. ANAs with specificity for the C component of hnRNP (consisting of the C1 and C2 proteins) have to our knowledge so far been described in only one case [4]. We here describe the approach taken to unambiguously identify the C1/C2 proteins as ANA targets in the sera of some patients.
Aims:
To determine the fine specificity of sera containing an unusual speckled ANA-staining pattern using a combination of 2D gel electrophoresis and MS.
Methods:
Patient sera were screened for ANAs by indirect immunofluorescence microscopy on HEp-2 cells (cultured carcinoma cells). Sera with an unusual, very regular, speckled ANA pattern were tested for reactivity with components of nuclear extracts of HeLa cells that were separated by one-dimensional (1D) or 2D gel electrophoresis or by reversed-phase high-performance liquid chromatography (HPLC). IgG reactivity was assessed by immunoblotting. Reactive protein spots from 2D separations were excised from the gels and subjected to in-gel digestion with trypsin for subsequent peptide mapping, partial peptide sequencing, and protein identification by MS and tandem MS on a hybrid electrospray ionization/quadrupole/time-of-flight (ESI-Q-TOF) mass spectrometer [5,6,7].
Results:
We observed a strong nuclear staining pattern (titer >1280) with the characteristic even-sized coarse speckles and no staining of nucleoli in sera from three patients. On immunoblots of nuclear extracts from HeLa cells, these sera stained two distinct bands, at Mr 42 000 and 41 000. There activity strongly resembled that of the patient originally described by Stanek et al [4]. The antigens were enriched by fractionating the extract using reversed-phase HPLC on a C4 column, and the two reactive spots on 2D separations were excised for identification. The two components appeared to be of approximately the same isoelectric points, although their molecular masses differed by approximately 2000. Peptide-mass mapping was performed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS on the tryptic peptide mixture generated by digestion of the two excised proteins. The database search suggested that the two proteins were C1/C2 hnRNPs (Swissprot accession number P07910). The identity of the proteins was further confirmed by tandem MS using an ESI-Q-TOF instrument. One peptide carrying two positive charges (m/z 580.32 Da), corresponding to a peptide mass of 1158.7 Da, was selected as a precursor ion and partially sequenced by collisional fragmentation. The fragmented peptide was found to represent the tryptic fragment VDSLLENLEK, ie amino acids 207-216 (C2 protein numbering). Four other peptides were partially sequenced and all of them matched the human C1/C2 hnRNP sequence. The theoretical masses of C1 and C2 are 32.0 and 33.3 kDa, respectively. The difference between the two sequences is a 13-amino-acid insert in C2 between positions 107 and 108 of C1. The presence of a specific tryptic fragment in the MALDI-TOF peptide-mass map from the higher-molecular-mass spot containing a 13-amino-acid insert that was not present in the lower-molecular-mass spot, further demonstrated that the two components represented the two isoforms of the C class of hnRNPs.
The patient whose case prompted us to investigate the specificities of these antibodies was a 72-year-old man who had arthralgias and oligoarthritis but did not fulfill the criteria for rheumatoid arthritis and did not have dermatological complaints. The reactivity of various patient groups to the C1/C2 hnRNP autoantigens was subsequently tested by immunoblotting of HeLa-cell nuclear extracts. Of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis, none had IgG antibodies reacting with the two bands. Of sera from 139 consecutive patients who had moderately to strongly positive speckled ANA patterns shown by indirect immunofluorescence on HEp-2 cells, only two reacted with the C1/C2 hnRNP bands in immunoblotting. One of these was from a young woman (22 years old) whose complaints of muscle tenderness were not explained by objective findings or abnormal laboratory test results. The third patient that we identified through ANA screening followed by immunoblotting was a 54-year-old male who was being treated with methotrexate for long-standing polymyositis in addition to psoriasis and possible osteoporosis.
Discussion:
The results confirm the existence of anti-C1/C2 antibodies in some patients with speckled ANAs. The antigens were identified through the use of biochemical methods using high-resolution separation techniques combined with mass-spectrometry peptide mapping and database searches. As a general approach, this is a powerful way to identify new antigens using small amounts of material without the need for conventional protein sequencing. The approach does require, however, that the proteins can be found in databases, that they are not extensively post-translationally modified, that they can be digested enzymatically, and that they can be isolated in appropriately pure form by the separation technique used.
It is not known at present if the C1/C2 antibodies may have pathogenic relevance and/or relate to specific diagnoses or subsets within the group of connective-tissue diseases. It does appear that the reactivity is quite rare among ANA-positive patients, and therefore many patients will have to be examined to determine these issues. The fact that the antibodies to the C1/C2 hnRNPs are revealed by indirect immunofluorescence would indicate that the epitopes are accessible in intact, fixed HEp-2 cells and thus probably reside outside the nucleic-acid-binding domains that would be expected to be covered by RNA.
PMCID: PMC17817  PMID: 11056675
antinuclear antibodies; autoantibodies; heterogeneous nuclear ribonucleoproteins C1/C2; mass spectrometry
2.  Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry for Detection of Antibiotic Resistance Mechanisms: from Research to Routine Diagnosis 
Clinical Microbiology Reviews  2013;26(1):103-114.
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.
doi:10.1128/CMR.00058-12
PMCID: PMC3553667  PMID: 23297261
3.  Relative Quantitation of Neuropeptides Over a Thousand-fold Concentration Range 
Neuropeptides are essential cell-to-cell signaling molecules that influence diverse regulatory and behavioral functions within biological systems. Differing in their amino acid sequences and posttranslational modifications, hundreds of neuropeptides are produced via a series of enzymatic processing steps, and their levels vary with location, time, and physiological condition. Due to their wide range of endogenous concentrations and inherent chemical complexity, using mass spectrometry (MS) to accurately quantify changes in peptide levels can be challenging. Here we evaluate three different MS systems for their ability to accurately measure neuropeptide levels: capillary liquid chromatography-electrospray ionization-ion trap (CapLC-ESI-IT) MS, ultraperformance liquid chromatography- electrospray ionization-quadrupole-time-of-flight (UPLC-LC-ESI-Q-TOF) MS, and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS. Specifically, eight sample mixtures composed of five neuropeptide standards, with four technical replicates of each, were labeled with H4/D4-succinic anhydride, followed by relative peptide quantitation using the three MS platforms. For these samples, the CapLC-ESI-IT MS platform offered the most robust ability to accurately quantify peptides over a concentration range of 1200-fold, although it required larger sample sizes than the other two platforms. Both the UPLC-ESI-Q-TOF MS and the MALDI-TOF MS systems had lower limits of quantification, with the MALDI-TOF having the lowest. By implementing several data acquisition schemes and optimizing the data analysis approaches, we were able to accurately quantify peptides over a three orders of magnitude concentration range using either the UPLC or MALDI-TOF platforms. Overall these results increase our understanding of both the capabilities and limits of using MS-based approaches to measure peptides.
doi:10.1007/s13361-012-0481-0
PMCID: PMC3515743  PMID: 22993045
4.  MALDI-TOF Mass Spectrometry Detection of Pathogens in Vectors: The Borrelia crocidurae/Ornithodoros sonrai Paradigm 
Background
In Africa, relapsing fever borreliae are neglected vector-borne pathogens that cause mild to deadly septicemia and miscarriage. Screening vectors for the presence of borreliae currently requires technically demanding, time- and resource-consuming molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has recently emerged as a tool for the rapid identification of vectors and the identification of cultured borreliae. We investigated whether MALDI-TOF-MS could detect relapsing fever borreliae directly in ticks.
Methodology/Principal Findings
As a first step, a Borrelia MALDI-TOF-MS database was created to house the newly determined Mean Spectrum Projections for four Lyme disease group and ten relapsing fever group reference borreliae. MALDI-TOF-MS yielded a unique protein profile for each of the 14 tested Borrelia species, with 100% reproducibility over 12 repeats. In a second proof-of-concept step, the Borrelia database and a custom software program that subtracts the uninfected O. sonrai profile were used to detect Borrelia crocidurae in 20 Ornithodoros sonrai ticks, including eight ticks that tested positive for B. crocidurae by PCR-sequencing. A B. crocidurae-specific pattern consisting of 3405, 5071, 5898, 7041, 8580 and 9757-m/z peaks was found in all B. crocidurae-infected ticks and not found in any of the un-infected ticks. In a final blind validation step, MALDI-TOF-MS exhibited 88.9% sensitivity and 93.75% specificity for the detection of B. crocidurae in 50 O. sonrai ticks, including 18 that tested positive for B. crocidurae by PCR-sequencing. MALDI-TOF-MS took 45 minutes to be completed.
Conclusions/Significance
After the development of an appropriate database, MALDI-TOF-MS can be used to identify tick species and the presence of relapsing fever borreliae in a single assay. This work paves the way for the use of MALDI-TOF-MS for the dual identification of vectors and vectorized pathogens.
Author Summary
In Africa, relapsing fever borreliae are neglected vector-borne infections that cause mild to deadly septicemia and miscarriage. The causative relapsing fever borreliae are transmitted by the bite of soft ticks, except for Borrelia recurrentis which is transmitted by body lice. Screening vectors for these relapsing fever borreliae currently relies on time- and resource-consuming methods such as polymerase chain reaction-based method. Here, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the rapid detection of borreliae in ticks. As a first step, we created a Borrelia MALDI-TOF-MS database and we detected B. crocidurae in Ornithodoros sonrai ticks. As a blind validation step, the 45-minute MALDI-TOF-MS exhibited a 88.9% sensitivity and a 93.75% specificity for the detection of B. crocidurae in 50 O. sonrai ticks including 18 ticks detected positive for B. crocidurae by PCR-sequencing. These findings provide the proof-of-concept that MALDI-TOF-MS can be used to identify tick species and the presence of relapsing fever borreliae. This technique could be translated for field applications.
doi:10.1371/journal.pntd.0002984
PMCID: PMC4109908  PMID: 25058611
5.  Detection of Rickettsia spp in Ticks by MALDI-TOF MS 
PLoS Neglected Tropical Diseases  2015;9(2):e0003473.
Background
Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases.
Methodology/Principal Findings
The objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels.
Conclusions/Significance
Taken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns.
Author Summary
Tick-borne rickettsioses include mild to life-threatening diseases in humans worldwide. When removing an attached tick from the human body, patients and physicians may have two questions: 1) is the tick a known vector of a human infectious disease, and 2) is the tick infected by a pathogenic agent that could have been transmitted during the attachment period? The morphological identification of Ticks is difficult, and requires expertise and specific documentation. The use of Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged as an effective, rapid and inexpensive tool to identify arthropods including Ticks. Here, we show the utility of MALDI-TOF MS for the dual identification of tick species and the rapid detection of Rickettsia spp in Ticks. Such results can be used to guide decisions related to specific patient monitoring or the administration of preventive treatment. Additionally, the low consumable costs, the minimum time required for sample preparation and the rapid availability of the results of MALDI-TOF MS could be useful for epidemiological studies and tick-borne disease monitoring via the dual identification of vectors and the pathogens they carry in one step. These results present new opportunities for the management of other vector-borne diseases that are of importance to public health.
doi:10.1371/journal.pntd.0003473
PMCID: PMC4319929  PMID: 25659152
6.  Development and Validation of an In-House Database for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Yeast Identification Using a Fast Protein Extraction Procedure 
Journal of Clinical Microbiology  2014;52(5):1453-1458.
In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of ≥2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies.
doi:10.1128/JCM.03355-13
PMCID: PMC3993681  PMID: 24554755
7.  Prospective Evaluation of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System in a Hospital Clinical Microbiology Laboratory for Identification of Bacteria and Yeasts: a Bench-by-Bench Study for Assessing the Impact on Time to Identification and Cost-Effectiveness 
Journal of Clinical Microbiology  2012;50(10):3301-3308.
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.
doi:10.1128/JCM.01405-12
PMCID: PMC3457442  PMID: 22855510
8.  Analysis of RNA cleavage by MALDI-TOF mass spectrometry 
Nucleic Acids Research  2012;41(1):e2.
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.
doi:10.1093/nar/gks811
PMCID: PMC3592410  PMID: 22941655
9.  Identification and Cluster Analysis of Streptococcus pyogenes by MALDI-TOF Mass Spectrometry 
PLoS ONE  2012;7(11):e47152.
Background
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.
Methods
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.
Results
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%.
Conclusions
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.
doi:10.1371/journal.pone.0047152
PMCID: PMC3492366  PMID: 23144803
10.  Direct Identification of Urinary Tract Pathogens from Urine Samples by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿  
Journal of Clinical Microbiology  2010;48(6):2110-2115.
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.
doi:10.1128/JCM.02215-09
PMCID: PMC2884468  PMID: 20392910
11.  Species determination of Culicoides biting midges via peptide profiling using matrix-assisted laser desorption ionization mass spectrometry 
Parasites & Vectors  2014;7(1):392.
Background
Culicoides biting midges are vectors of bluetongue and Schmallenberg viruses that inflict large-scale disease epidemics in ruminant livestock in Europe. Methods based on morphological characteristics and sequencing of genetic markers are most commonly employed to differentiate Culicoides to species level. Proteomic methods, however, are also increasingly being used as an alternative method of identification. These techniques have the potential to be rapid and may also offer advantages over DNA-based techniques. The aim of this proof-of-principle study was to develop a simple MALDI-MS based method to differentiate Culicoides from different species by peptide patterns with the additional option of identifying discriminating peptides.
Methods
Proteins extracted from 7 Culicoides species were digested and resulting peptides purified. Peptide mass fingerprint (PMF) spectra were recorded using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peak patterns analysed in R using the MALDIquant R package. Additionally, offline liquid chromatography (LC) MALDI-TOF tandem mass spectrometry (MS/MS) was applied to determine the identity of peptide peaks in one exemplary MALDI spectrum obtained using an unfractionated extract.
Results
We showed that the majority of Culicoides species yielded reproducible mass spectra with peak patterns that were suitable for classification. The dendrogram obtained by MS showed tentative similarities to a dendrogram generated from cytochrome oxidase I (COX1) sequences. Using offline LC-MALDI-TOF-MS/MS we determined the identity of 28 peptide peaks observed in one MALDI spectrum in a mass range from 1.1 to 3.1 kDa. All identified peptides were identical to other dipteran species and derived from one of five highly abundant proteins due to an absence of available Culicoides data.
Conclusion
Shotgun mass mapping by MALDI-TOF-MS has been shown to be compatible with morphological and genetic identification of specimens. Furthermore, the method performs at least as well as an alternative approach based on MS spectra of intact proteins, thus establishing the procedure as a method in its own right, with the additional option of concurrently using the same samples in other MS-based applications for protein identifications. The future availability of genomic information for different Culicoides species may enable a more stringent peptide detection based on Culicoides-specific sequence information.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-3305-7-392) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-3305-7-392
PMCID: PMC4158057  PMID: 25152308
Culicoides; Species typing; MALDI-TOF-MS; Shotgun mass mapping; MALDIquant
12.  Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology 
Clinical Microbiology Reviews  2013;26(3):547-603.
SUMMARY
Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the “nuts and bolts” of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.
doi:10.1128/CMR.00072-12
PMCID: PMC3719498  PMID: 23824373
13.  BioSunMS: a plug-in-based software for the management of patients information and the analysis of peptide profiles from mass spectrometry 
Background
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.
Results
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.
Conclusion
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/.
doi:10.1186/1472-6947-9-13
PMCID: PMC2654546  PMID: 19220920
14.  The C-Terminal Fragment of Prostate-Specific Antigen, a 2331 Da Peptide, as a New Urinary Pathognomonic Biomarker Candidate for Diagnosing Prostate Cancer 
PLoS ONE  2014;9(9):e107234.
Background and Objectives
Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, although PSA is an excellent organ-specific marker, it is not a cancer-specific marker. Therefore, the aim of this study was to discover new biomarkers for the diagnosis of PCa.
Materials and Methods
We focused on urine samples voided following prostate massage (digital rectal examination [DRE]) and conducted a peptidomic analysis of these samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MSn). Urinary biomaterials were concentrated and desalted using CM-Sepharose prior to the following analyses being performed by MALDI-TOF/MSn: 1) differential analyses of mass spectra; 2) determination of amino acid sequences; and 3) quantitative analyses using a stable isotope-labeled internal standard.
Results
Multivariate analysis of the MALDI-TOF/MS mass spectra of urinary extracts revealed a 2331 Da peptide in urine samples following DRE. This peptide was identified as a C-terminal PSA fragment composed of 19 amino acid residues. Moreover, quantitative analysis of the relationship between isotope-labeled synthetic and intact peptides using MALDI-TOF/MS revealed that this peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects.
Conclusion
The results of the present study indicate that the 2331 Da peptide fragment of PSA may become a new pathognomonic biomarker for the diagnosis of PCa. A further large-scale investigation is currently underway to assess the possibility of using this peptide in the early detection of PCa.
doi:10.1371/journal.pone.0107234
PMCID: PMC4169392  PMID: 25233230
15.  Interlaboratory Comparison of Sample Preparation Methods, Database Expansions, and Cutoff Values for Identification of Yeasts by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using a Yeast Test Panel 
Journal of Clinical Microbiology  2014;52(8):3023-3029.
An interlaboratory study using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n = 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases. Logistic regression analysis was performed to analyze the association between the number of spectra in the database and the percentage of strains that were correctly identified. A total of 5,460 MALDI-TOF MS results were obtained. Using all results, the area under the ROC curve was 0.95 (95% confidence interval [CI], 0.94 to 0.96). With a sensitivity of 0.84 and a specificity of 0.97, a cutoff value of 1.7 was considered optimal. The overall percentage of correct identifications (formic acid-ethanol extraction method, score ≥ 1.7) was 61.5% when the commercial Bruker Daltonics database (BDAL) was used, and it increased to 86.8% by using an extended BDAL supplemented with a Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre in-house database (BDAL+CBS in-house). A greater number of main spectra (MSP) in the database was associated with a higher percentage of correct identifications (odds ratio [OR], 1.10; 95% CI, 1.05 to 1.15; P < 0.01). The results from the direct transfer method ranged from 0% to 82.9% correct identifications, with the results of the top four centers ranging from 71.4% to 82.9% correct identifications. This study supports the use of a cutoff value of 1.7 for the identification of yeasts using MALDI-TOF MS. The inclusion of enough isolates of the same species in the database can enhance the proportion of correctly identified strains. Further optimization of the preparation methods, especially of the direct transfer method, may contribute to improved diagnosis of yeast-related infections.
doi:10.1128/JCM.00563-14
PMCID: PMC4136148  PMID: 24920782
16.  Rapid Identification of Vibrio parahaemolyticus by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿ † 
Applied and Environmental Microbiology  2009;75(21):6745-6756.
Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.
doi:10.1128/AEM.01171-09
PMCID: PMC2772414  PMID: 19749061
17.  Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Bacterial Strains Routinely Isolated in a Clinical Microbiology Laboratory ▿  
Journal of Clinical Microbiology  2010;48(5):1549-1554.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.
doi:10.1128/JCM.01794-09
PMCID: PMC2863943  PMID: 20220166
18.  Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling 
PLoS ONE  2013;8(2):e57486.
Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.
doi:10.1371/journal.pone.0057486
PMCID: PMC3585343  PMID: 23469000
19.  Identification of Lactobacillus from the Saliva of Adult Patients with Caries Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry 
PLoS ONE  2014;9(8):e106185.
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. Bacteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification.
doi:10.1371/journal.pone.0106185
PMCID: PMC4148440  PMID: 25166027
20.  Protease- and acid-catalyzed labeling workflows employing 18O-enriched water 
Short abstract
Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.
Long abstract
Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1–3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g., iTRAQ, ICAT) and metabolic (e.g., SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10–11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such “double-labeling” enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.
doi:10.3791/3891
PMCID: PMC3605716  PMID: 23462971
MALDI-TOF mass spectrometry; proteomics; proteolysis; quantification; stable isotope labeling
21.  Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿ †  
Applied and Environmental Microbiology  2008;74(17):5402-5407.
Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.
doi:10.1128/AEM.02689-07
PMCID: PMC2546641  PMID: 18606788
22.  Rapid identification of oral Actinomyces species cultivated from subgingival biofilm by MALDI-TOF-MS 
Journal of Oral Microbiology  2015;7:10.3402/jom.v7.26110.
Background
Actinomyces are a common part of the residential flora of the human intestinal tract, genitourinary system and skin. Isolation and identification of Actinomyces by conventional methods is often difficult and time consuming. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a rapid and simple method to identify bacteria.
Objective
The present study evaluated a new in-house algorithm using MALDI-TOF-MS for rapid identification of different species of oral Actinomyces cultivated from subgingival biofilm.
Design
Eleven reference strains and 674 clinical strains were used in this study. All the strains were preliminarily identified using biochemical methods and then subjected to MALDI-TOF-MS analysis using both similarity-based analysis and classification methods (support vector machine [SVM]). The genotype of the reference strains and of 232 clinical strains was identified by sequence analysis of the 16S ribosomal RNA (rRNA).
Results
The sequence analysis of the 16S rRNA gene of all references strains confirmed their previous identification. The MALDI-TOF-MS spectra obtained from the reference strains and the other clinical strains undoubtedly identified as Actinomyces by 16S rRNA sequencing were used to create the mass spectra reference database. Already a visual inspection of the mass spectra of different species reveals both similarities and differences. However, the differences between them are not large enough to allow a reliable differentiation by similarity analysis. Therefore, classification methods were applied as an alternative approach for differentiation and identification of Actinomyces at the species level. A cross-validation of the reference database representing 14 Actinomyces species yielded correct results for all species which were represented by more than two strains in the database.
Conclusions
Our results suggest that a combination of MALDI-TOF-MS with powerful classification algorithms, such as SVMs, provide a useful tool for the differentiation and identification of oral Actinomyces.
doi:10.3402/jom.v7.26110
PMCID: PMC4297926  PMID: 25597306
MALDI-TOF-MS; oral Actinomyces; support vector machine
23.  Technologies in the Whole-Genome Age: MALDI-TOF-Based Genotyping 
Summary
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.
doi:10.1159/000225089
PMCID: PMC2941830  PMID: 21049076
MALDI-TOF; Mass spectrometry; Single nucleotide polymorphism; Methylation; Microsatellite instability; Genotyping
24.  Expanding the Crustacean Neuropeptidome using a Multi-Faceted Mass Spectrometric Approach 
Journal of proteome research  2009;8(5):2426-2437.
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.
doi:10.1021/pr801047v
PMCID: PMC2692460  PMID: 19222238
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
25.  Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS 
BMC Microbiology  2011;11:267.
Background
The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays.
Results
In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences.
Conclusions
MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.
doi:10.1186/1471-2180-11-267
PMCID: PMC3314589  PMID: 22192890

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