[Determination of the derivatization reactivity between α/ß-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry].

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two ß-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the ß-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the ß-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

Discovery of mass spectral peak markers and protein biomarkers in fish muscle exudates for rapid and precise recognition of fish species via magnetic beads (MBs) and mass spectrometry.

In this study, muscle exudates from five fishes belonging to the family Sciaenidae, in the order Perciformes, were analyzed as models for the discovery of biomarkers by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). MagSi-weak cation exchange magnetic beads (WCX-MBs) were utilized for the enrichment of proteins from fish exudate samples, allowing protein biomarkers to be identified and subsequently used for fish species differentiation. Buffers with pH ranging from 4.0 to 9.0 can provide an environment for proteins in fish muscle exudate to bind to the WCX-MBs. The optimal enrichment based on WCX-MBs can be achieved when the exudate samples are diluted 100folds. More species-specific biomarkers in mass spectra can be identified when using WCX-MBs. The number of ions that can be considered as peak markers and can differentiate the analyzed fishes increases from 38 to 121 when using WCX-MBs to isolate peptides/protein in fish muscle exudate. Particularly, eight peak markers in mass spectra were assigned to be specific to Nibea albiflora (NA), three peak markers specific to Larimichthys crocea (LC), two peak markers specific to Miichthys miiuy (MM), seven peak markers specific to Collichthys lucidus (CL), and six peak markers specific to Larimichthys polyactis (LP). Furthermore, five proteins were identified based on the characterization of tryptic peptides and their potential to be biomarkers, of which four proteins specific to CL and one specific to LC were identified. The single-blind samples analysis demonstrated that these species-specific peak markers and protein biomarkers can be successfully utilized for corresponding fish recognition. The utilization of WCX-MBs can improve the discovery of fish species-specific biomarkers in fish muscle exudate samples. The present protocol holds potential of being a rapid and accurate identification tool for recognition of fish species.

[Preparation of magnetic carbon nitride composite toward phosphopeptide enrichment].

Protein phosphorylation plays an important role in cellular signaling and disease development. Advances in mass spectrometry-based proteomics have enabled qualitative and quantitative phosphorylation studies as well as in-depth biological explorations for biomarker discovery and signaling pathway analysis. However, the dynamic changes that occur during phosphorylation and the low abundance of target analytes render direct analysis difficult because mass spectral detection offers no selectivity, unlike immunoassays such as Western blot and enzyme-linked immunosorbent assay (ELISA). The present study aimed to solve one of the key problems in the specific and efficient isolation of phosphorylated peptides. A method based on a magnetic carbon nitride composite coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the enrichment and analysis of phosphopeptides with low abundance in complex samples. Magnetic carbon nitride composite was synthesized and characterized by electron microscopy, infrared spectroscopy, and X-ray diffractometry. The composite showed a well-distributed two-dimensional layered structure and functional groups with excellent paramagnetic performance. Two classical phosphoproteins, namely, α- and ß-caseins, were selected as model phosphorylated samples to assess the performance of the proposed enrichment technique. The magnetic carbon nitride composite exhibited high selectivity and sensitivity for phosphopeptide enrichment. The limit of detection was determined by MALDI-TOF-MS analysis to be 0.1 fmol. The selectivity of the method was investigated using the digest mixtures of α-casein, ß-casein, and bovine serum albumin (BSA) with different mass ratios (1∶1∶1000, 1∶1∶2000, and 1∶1∶5000). Direct analysis of the samples revealed the dominance of spectral signals from the abundant peptides in BSA. After enrichment with the magnetic carbon nitride composite, the high concentration of background proteins was washed away and only the signals of the phosphopeptides were captured. The signals from the casein proteins were clearly observed with little background noise, indicating the high selectivity of the composite material. The robustness of the method was tested by assessing the reusability of the same batch of magnetic carbon nitride materials over 20 cycles of enrichment. The composite showed nearly the same enrichment ability even after several cycles of reuse, demonstrating its potential applicability for a large number of clinical samples. Finally, the method was applied to the analysis of phosphopeptides from several commonly used phosphoprotein-containing samples, including skimmed milk digest, human serum, and human saliva; these samples are significant in the analysis of food quality, disease biomarkers, and liquid biopsies for cancer. Without enrichment, no phosphopeptide was detected because of the high abundance of nonphosphopeptide materials dominating the spectral signals obtained. After pretreatment with the developed magnetic carbon nitride composite, most of the phosphosites were identified with high selectivity and sensitivity via MALDI-TOF-MS. These results revealed the practicality of the developed approach for clinical applications. In addition, our method may potentially be employed for phosphoproteomics with real complex biological samples.

Molecular epidemiology and molecular typing methods of Acinetobacter baumannii: An updated review.

The aim of this study was to go through the molecular methods used for typing of carbapenem-resistant Acientobacter baumannii (CRAB) isolates for investigating the molecular epidemiology all over the world. Multiple typing techniques are required to understand the source and nature of outbreaks caused by Acientobacter baumannii (A. baumannii) and acquired resistance to antimicrobials. Nowadays, there is gradual shift from traditional typing methods to modern molecular methods to study molecular epidemiology and infection control. Molecular typing of A. baumannii strains has been revolutionized significantly in the last 2 decades. A few sequencing-based techniques have been proven as a breakthrough and opened new prospects, which have not been achieved by the traditional methods. In this review, discussed different pre-existing and recently used typing methods to explore the molecular epidemiology of A. baumannii pertaining in context with human infections.

Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.

We tested a technique (FT-IR) to identify four different, common bacteria from 104 children with serious infections and compared it to lab methods for diagnosis. FT-IR was more accurate. We tested if it could identify subtypes of bacteria, which is important in outbreaks. It was able to subtype two species, but not the two other species. However, it is a much faster and cheaper technique than the gold standard. It may be useful in certain outbreaks. We also investigated the trends between genes and the length of hospital stay. This can support further laboratory research. As a fast, low-cost test, FT-IR warrants further testing before it is applied to clinical labs.

Quantum dots (QDs) attached magnetic beads (MBs) for on-chip efficient capture and detection of bacteria in ready-to-eat (RTE) foods.

In this study, we established a versatile and simple magnetic-assisted microfluidic method for fast bacterial detection. Quantum dots (QDs) were loaded onto magnetic beads (MBs) to construct performance enhanced on-chip capture of bacteria. Escherichia coli (E. coli), as a model bacterium was studied. CdSe QDs were deposited onto the surface of Fe3O4 MBs through layer-by-layer self-assembly to enhance the loading of antibodies (Abs). MBs functionalized with anti-E. coli antibody molecules in a micropillar-based microfluidic chip were utilized to capture E. coli, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for characterization of captured bacteria. This method was found capable of specifically isolating E. coli within the range of 1.0 to 1.0 × 109 CFU/mL, having a detection limit (LOD) of 10 CFU/mL. The average similarity score among mass spectra for the bacterial capture obtained in independent experiments is calculated as 0.97 ± 0.01 (n = 3), which shows this work's excellent reproducibility for bacterial capture. Bacterial growth on ready-to-eat (RTE) foods during its time of storage was successfully monitored. The present protocol has promising potential for microbial control and pathogen detection in the food industry.

Determination of ω-end functionalities in tailored poly(2-alkyl-2-oxazoline)s by liquid chromatography and mass spectrometry.

The in-depth analytical characterization of polymers, in particular regarding intended biomedical applications, is becoming increasingly important to elucidate their structure-property relationships. Specifically, end group analysis of e.g. polymers featuring a 'stealth effect' towards the immune system is of particular importance because of their use in coupling reactions to bioactive compounds. Herein, we established a liquid chromatography (LC) protocol to analyse bicyclo[6.1.0]nonyne-functionalized poly(2-alkyl-2-oxazoline)s (POx)s as promising functional polymers that can be applied in strain-promoted click reactions. This work involved the synthesis of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) by living cationic ring-opening polymerization (CROP) with different molar masses ranging from 2 up to 17.5 kDa and, to our knowledge, the first liquid chromatographic analysis of PMeOx. The developed analytical protocol enables the quantitative determination of post-polymerization reaction sequences with respect to the conversion of the ω-end groups. All synthesized polymers were straightforwardly analysed on a C18-derivatized silica monolithic column under reversed-phase chromatographic conditions with a binary mobile phase gradient comprising a mixture of acetonitrile and water. Subsequent mass spectrometry of collected elution fractions enabled the confirmation of the desired ω-end group functionalities and the identification of synthetic by-products.

Evaluation of the Qvella FAST System and the FAST-PBC cartridge for rapid species identification and antimicrobial resistance testing directly from positive blood cultures.

Blood culture diagnostics require rapid and accurate identification (ID) of pathogens and antimicrobial susceptibility testing (AST). Standard procedures, involving conventional cultivation on agar plates, may take up to 48 hours or more until AST completion. Recent approaches aim to shorten the processing time of positive blood cultures (PBC). The FAST System is a new technology, capable of purifying and concentrating bacterial/fungal pathogens from positive blood culture media and producing a bacterial suspension called "liquid colony" (LC), which can be further used in downstream analyses (e.g., ID and AST). Here, we evaluated the performance of the FAST System LC generated from PBC in comparison to our routine workflow including ID by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using Sepsityper, AST by automatized MicroScan WalkAway plus and directly inoculated disk diffusion (DD), and MICRONAUT-AM for yeast/fungi. A total of 261 samples were analyzed, of which 86.6% (226/261) were eligible for the comparative ID and AST analyses. In comparison to the reference technique (culture-grown colonies), ID concordance of the FAST System LC and Sepsityper was 150/154 (97.4%) and 123/154 (79.9%), respectively, for Gram positive; 67/70 (95.7%) and 64/70 (91.4%), respectively, for Gram negative. For AST, categorical agreement (CA) of the FAST System LC in comparison to the routine workflow for Gram-positive bacteria was 96.1% and 98.7% for MicroScan and DD, respectively. Similar results were obtained for Gram-negative bacteria with 96.6% and 97.5% of CA for MicroScan and DD, respectively. Taken together, the FAST System LC allowed the laboratory to significantly reduce the time to obtain correct ID and AST (automated MicroScan) results 1 day earlier and represents a promising tool to expedite the processing of PBC.

On-Chip Discovery of Allergens from the Exudate of Large Yellow Croaker (Larimichthys Crocea) Muscle Food by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

It is extremely crucial to establish facile, accurate, and fast methods for testing allergenic proteins (allergens) in seafood. The current study focuses on the evaluation of fish muscle exudate proteins in an effort to discover potential allergens in fish exudate for allergy tests. Large yellow croaker (Larimichthys crocea) was studied as a seafood model. Magnetic beads (MBs) modified with an IgE antibody were utilized to isolate allergens existing in the exudate sample. Immunoglobulin E (IgE) in blood is a class of antibodies that is mainly associated with allergic reactions. Potential allergens in the muscle exudate were fished by IgE-biofunctional MBs in microfluidic channels. The protein-attached MBs were isolated under a magnetic field, eluted, and collected. The collected eluent was digested and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify allergens. Eight allergens from large yellow croaker exudate were identified, i.e., parvalbumin beta, parvalbumin, protein S100, histone H4, cytochrome c, fatty acid binding protein 3 (FABP3), microsomal glutamate S-transfer 3 (MGST3), and C-C motif chemokine 21 (CCL21). The presently proposed microfluidic-magnetic-based allergen extraction protocol enables a facile and rapid test of potentials of seafood allergies, providing a solution to circumvent food safety issues, especially for allergic populations.

γ-enolase (ENO2) is methylated at the Nτ position of His-190 among enolase isozymes.

Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.

MALDI-TOF Mass Spectrometry for interrogating ubiquitin enzymes.

The attachment of ubiquitin to a substrate (ubiquitination or ubiquitylation) impacts its lifetime and regulates its function within the cell. Several classes of enzymes oversee the attachment of ubiquitin to the substrate: an E1 activating enzyme that makes ubiquitin chemically susceptible prior to the following stages of conjugation and ligation, respectively mediated by E2 conjugating enzymes (E2s) and E3 ligases (E3s). Around 40 E2s and more than 600 E3s are encoded in the human genome, and their combinatorial and cooperative behaviour dictate the tight specificity necessary for the regulation of thousands of substrates. The removal of ubiquitin is orchestrated by a network of about 100 deubiquitylating enzymes (DUBs). Many cellular processes are tightly controlled by ubiquitylation, which is essential in maintaining cellular homeostasis. Because of the fundamental role(s) of ubiquitylation, there is an interest in better understanding the function and specificity of the ubiquitin machinery. Since 2014, an expanding array of Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Mass Spectrometry (MS) assays have been developed to systematically characterise the activity of a variety of ubiquitin enzymes in vitro. Here we recapitulate how MALDI-TOF MS aided the in vitro characterization of ubiquitin enzymes and the discovery of new and unexpected of E2s and DUBs functions. Given the versatility of the MALDI-TOF MS approach, we foreseen the use of this technology to further expand our understanding of ubiquitin and ubiquitin-like enzymes.

2D-DIGE Analysis of Liver Disease in Mice.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms. Thus, 2D-DIGE analysis provides highly complementary data to non-gel-based shotgun mass spectrometry (MS) methods (e.g., liquid chromatography (LC)-MS/MS)-allowing, for example, identification of novel protein biomarkers for HCC or increasing insights into the molecular mechanisms underlying hepatocarcinogenesis.

Development and validation of a decision tree classification model for the essential hypertension based on serum protein biomarkers.

Background: Essential hypertension (EH) is a key risk factor for cardiovascular disease. However, the etiology of EH is complex and unknown. So far, there is no good protein biomarker for screening EH. The purpose of this study was to discover potential biomarkers for EH by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and establish a decision-tree classification model. Methods: A total of 108 patients with clinically confirmed EH and 105 HC were enrolled in the present study from September 2020 to April 2021 and were randomly divided into the training group and the blind-test group. The serum protein expression profiles were performed using MALDI-TOF MS combined with magnetic beads with weak cation exchange (MB-WCX). The training group, which comprised 54 EH patients and 53 HC, was used to screen the statistically differential protein peaks by SPSS 19.0 and construct a decision-tree classification model by C5.0 algorithms of SPSS Modeler 18.0. All protein peak intensities of samples in the blind-test group, which comprised 54 EH patients and 52 HC, were used to verify the diagnostic capabilities of the model by classification model. Results: EH patients had higher age, systolic and diastolic blood pressures than HC group. The intensities of 60 protein peaks differed significantly between the EH patients and HC. An optimal decision-tree classification model of EH was successfully established with mass-to-charge ratios of 1,326.7, 1,785.3, 4,228.0, and 8,963.8 as differential protein peaks by the software analysis. The decision-tree classification model was able to distinguish between EH patients and HC and had a sensitivity of 94.44%, a specificity of 94.33%, an accuracy of 94.39%, and an area under the receiver operating characteristic (ROC) curve of 0.96. The blind-test results indicated a sensitivity of 87.04%, a specificity of 88.46%, an accuracy of 87.74%, and an area under the ROC curve of 0.928. Conclusions: MALDI-TOF MS combined with MB-WCX can be used to screen for serum differential protein expression profiles in EH patients. The decision-tree classification model based on mass-to-charge ratios of 1,326.7, 1,785.3, 4,228.0, and 8,963.8 could provide a new and reliable method for screening and identifying EH with high sensitivity and specificity.

Microchip coupled with MALDI-TOF MS for the investigation of bacterial contamination of fish muscle products.

Bacterial contamination is a significant concern in food safety. Traditional methods, though being a gold standard for bacterial detection, are time-consuming. In this work, we managed to establish a simple and versatile magnetic-assisted microfluidic method for rapid bacterial detection of fish muscle products, by manipulating anti-human IgG functionalized magnetic beads in a zig-zag shaped microfluidic channel, increasing the probability for bacteria capture. The captured bacteria were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method is capable of isolating Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae from 5 µL of sablefish sarcoplasmic protein sample, and detecting Escherichia coli in the range of 6.0 to 6.0×104 CFU/mL with a detection limit of 6 CFU/mL. Bacterial growth on salmon sashimi during its period of storage was successfully monitored. The current protocol holds great potential for pathogen detection and microbial control in the food industry.

Capture and identification of bacteria from fish muscle based on immunomagnetic beads and MALDI-TOF MS.

In the present study, E. coli was taken as a model bacterium, anti-E. coli functionalized magnetic beads were constructed and used to capture E. coli from aqueous extracts of fish sarcoplasmic protein (FSP) and fish muscle protein of sablefish. The excellency of the reproducibility of the present protocol was demonstrated by capturing E. coli from sablefish FSP extracts. The presence of 10 CFU/mL E. coli is still detectable. A microbial safety test on the surface of fish muscle was successfully performed. The bacterial identification accuracy from samples with different matrices was found to be excellent with RSD = 3%. High specific detection of target bacteria in complex biological samples was testified by spiking Staphylococcus aureus and Klebsiella pneumoniae in samples as interference. Ten biomarker ions were discovered for E. coli's recognition. It is promising to apply the present protocol in bacterial analysis in muscle food samples to ensure their safety.

Identification of sacrococcygeal and pelvic abscesses infected with invasive Mycoplasma hominis by MALDI-TOF MS.

BACKGROUND: Mycoplasma hominis is the smallest prokaryotic microorganism with no cell wall, high pleomorphism, and slower reproduction than bacteria. It is difficult for clinical technicians to find M. hominis through the negative Gram staining of specimens. Therefore, it is likely to miss detection in routine clinical smear etiological examination. M. hominis is generally considered to be a common colonizing bacterium in urogenital tract with low pathogenicity, and it is usually difficult to invade submucosal tissue and enter the bloodstream. METHODS: The abscesses of the patient were examined histopathologically, and the pus in the abscesses was extracted for etiological examination. MALDI-TOF MS was used to identify and confirmed the pathogens in the specimens. The commercial Mycoplasma isolation, culture, and drug sensitivity kit was used to determine antibiotic susceptibility. RESULTS: No pathogens were found after pathological and smear microscopic examination of the puncture fluid from the sacrococcygeal and pelvic abscesses. Until 48 h later, small, translucent, and gray-white colonies were observed in the blood plate culture results. The laboratory physician ultimately determined that the pathogen was M. hominis by MALDI-TOF MS. CONCLUSION: We report a case of extra-urogenital cystic abscesses infected by M. hominis, in order to improve clinicians' comprehensive understanding of the pathogenicity of Mycoplasma. In addition, the clinical laboratory technician should pay attention to the role of Wright-Giemsa staining of puncture fluid smear in the preliminary detection and the application of MALDI-TOF MS in identification of uncommon pathogenic microorganisms.

Direct microorganism species identification and antimicrobial susceptibility tests from positive blood culture bottles using rapid Sepsityper Kit.

INTRODUCTION: We evaluated the performance of Rapid Sepsityper Kit in species identification (ID) and antimicrobial susceptibility testing (AST). METHODS: Positive blood culture bottles (n = 227) containing single microorganisms were enrolled. We compared the direct method using Rapid Sepsityper Kit for ID and AST with the conventional method. The analyses of ID and AST were performed using MALDI Biotyper and BD Phoenix platform, respectively. RESULTS: The direct ID method correctly identified 89.4% (203/227) of samples, and Gram-negative bacilli (95.2%) had a higher ID rate than Gram-positive cocci (84.4%). Five cases were misidentified, and non-acceptable identification was high among Streptococcus species. Direct AST results were obtained from 168 isolates. Non-acceptable ID occurred among 24 isolates; 4 Streptococcus species, and 31 isolates, which did not grow in the direct AST method, were excluded. A total of 1714 antibiotic susceptibility tests (625 from 69 Gram-positive cocci and 1089 from 99 Gram-negative bacilli) were performed. The direct AST methods showed 98.3% (1685/1714) of categorical agreement (CA), 0.7% (12/1714) of very major errors, 0.2% (4/1714) of major errors, and 0.8% (13/1714) of minor errors. Complete CA was obtained for methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamase-producing Escherichia coli. CONCLUSIONS: The direct ID method using Rapid Sepsityper Kit and the direct AST method in combination with the BD Phoenix platform, which was associated with a reduction of turnaround time, may be a reliable approach for blood culture bottles. However, additional validation and further improvements, especially for Gram-positive cocci, would have an impact on microbiological diagnoses.

The investigation of storage situation of fish muscle via the analysis of its exudate by MALDI-TOF MS.

The potential of MALDI-TOF MS was investigated in terms of its capability to determine the change of exudates of fish muscle with different freeze-thaw cycles (0, 1 and 2), and frozen storage periods. The exudates were collected from dead chilled marine fish species, large yellow croacker (Larimichthys crocea, LC) and freshly slaughtered freshwater fish species, Japanese seabass (Lateolabrax japonicus) to be studied as models. 109 proteins, in which, 32 are extracellular proteins, and 15 are intracellular proteins, were identified by analyzing exudate of LC using MALDI-TOF MS and HPLC-MS/MS. The results show that the present method may be able to determine the change of fish muscle foods in a more sensitive mode than K value indicated quality control. The feasibility of verifying the storage situation of fish sample was performed by analyzing fish samples obtained from the local market. It is promising to estimate the storage situation of fishery products or other animal muscle foods by analyzing their muscle exudates based on the presently developed strategy.

MALDI-TOF MS and Magnetic Beads for Rapid Seafood Allergen Tests.

We developed a strategy using immunomagnetic separation (IMS) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to test seafood allergens. The protocol employed commercial magnetic beads (MBs) functionalized with anti-human IgE antibodies to carry out the IMS of IgEs in blood samples, followed by capture of allergens from seafood protein extracts for allergy analysis. After elution, the captured allergens were identified by MALDI-TOF MS and HPLC-MS/MS. The non-specific adsorption of MBs to biomolecules, the reproducibility and sensitivity of the protocol were investigated. The method shows consistent results with enzyme-linked immunosorbent assay tests. The false positive rate of the present method for the allergy test is 0%. The protocol was applied to detect the allergens in greasy-back shrimp for checking the allergenicity of patients' serum. Cooking fish as soup may effectively decrease the allergenicity. The method can be potentially used to identify unknown allergens of seafood to ensure the safety of allergic patients.

Comparative Evaluation of Bruker Biotyper and ASTA MicroIDSys for Species Identification in a Clinical Microbiology Laboratory.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for microbial identification, because of its speed and accuracy, since its introduction to clinical microbiology laboratories. In this study, we evaluated the performance of ASTA MicroIDSys, a newly developed MALDI-TOF, and compared it with the widely used Bruker Biotyper. Microbial identification with the Bruker Biotyper system was performed by using a direct smear method and the Bruker Biotyper database (reference library version 6.0.0.0). The isolates were also tested in parallel, using the ASTA MicroIDSys system with a direct smear method and the MicroIDSys database, CoreDB v1.26. A total of 914 clinical isolates were recovered from the clinical specimens. Identical results with confidence scores (≥2.0, for the Bruker Biotyper) and acceptable scores (≥140 for the ASTA MicroIDSys) were obtained for 840 (91.9%) isolates. The minor errors were defined as misidentification at the species level, and the rate was 1.1% (9/792) for Bruker Biotyper and 1.6% (13/792) for ASTA MicroIDSys. Major errors were defined as misidentification at the genus level, and the rate was 0.3% (2/792) for both Bruker Biotyper and ASTA MicroIDSys. ASTA MicroIDSys showed reliable performance for microbial identification, which was comparable to that of the Bruker Biotyper. Therefore, ASTA MicroIDSys can be applied for the identification of microorganisms in clinical microbiology laboratories.