Comparison of the Vitek MS and Bruker Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Systems for Identification of Chryseobacterium Isolates from Clinical Specimens and Report of Uncommon Chryseobacterium Infections in Humans.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry is becoming more popular and is replacing traditional identification methods in the clinical microbiology laboratory. We aimed to compare the Vitek mass spectrometry (MS) and Bruker Biotyper systems for the identification of Chryseobacterium isolated from clinical specimens and to report uncommon Chryseobacterium infections in humans. The microbial database from a hospital was searched for records between 2005 and 2016 to identify cultures that yielded Chryseobacterium Species identification by the Vitek MS and Bruker Biotyper systems was compared to identification by 16S rRNA gene sequencing. Over the study period, 140 Chryseobacterium isolates were included. Based on 16S rRNA gene sequencing, 78 isolates were C. indologenes, 39 were C. gleum, 12 were uncommon Chryseobacterium species (C. arthrosphaerae, C. culicis, C. cucumeris, C. bernardetii, C. artocarpi, and C. daecheongense), and the remaining 11 isolates were only identified at the genus level. The Vitek MS and Bruker Biotyper systems correctly identified 98.7% and 100% of C. indologenes isolates, respectively. While the Bruker Biotyper accurately identified 100% of C. gleum isolates, the Vitek MS system correctly identified only 2.6% of isolates from this species. None of the uncommon Chryseobacterium species were successfully identified by either of these two systems. The overall accuracies of Chryseobacterium identification at the species level by the Vitek MS and Bruker Biotyper systems were 60.5% and 90.7%, respectively. An upgrade and correction of the Vitek MS and Bruker Biotyper databases is recommended to correctly identify Chryseobacterium species.

Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of blood isolates of Vibrio species.

Among 56 blood isolates of Vibrio species identified by sequencing analysis of 16S rRNA and rpoB genes, the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system correctly identified all isolates of Vibrio vulnificus (n = 20), V. parahaemolyticus (n = 2), and V. fluvialis (n = 1) but none of the isolates of serogroup non-O1/O139 (non-serogroup O1, non-O139) V. cholerae (n = 33) to the species level. All of these serogroup non-O1/O139 V. cholerae isolates were correctly identified using the newly created MALDI-TOF MS database.

Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of blood isolates of Acinetobacter species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper) was able to accurately identify 98.6% (142/144) of Acinetobacter baumannii isolates, 72.4% (63/87) of A. nosocomialis isolates, and 97.6% (41/42) of A. pittii isolates. All Acinetobacter junii, A. ursingii, A. johnsonii, and A. radioresistens isolates (n = 28) could also be identified correctly by Bruker Biotyper.

Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Nocardia, Rhodococcus, Kocuria, Gordonia, Tsukamurella, and Listeria species.

We evaluated whether the Bruker Biotyper matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system provides accurate species-level identifications of 147 isolates of aerobically growing Gram-positive rods (GPRs). The bacterial isolates included Nocardia (n = 74), Listeria (n = 39), Kocuria (n = 15), Rhodococcus (n = 10), Gordonia (n = 7), and Tsukamurella (n = 2) species, which had all been identified by conventional methods, molecular methods, or both. In total, 89.7% of Listeria monocytogenes, 80% of Rhodococcus species, 26.7% of Kocuria species, and 14.9% of Nocardia species (n = 11, all N. nova and N. otitidiscaviarum) were correctly identified to the species level (score values, ≥ 2.0). A clustering analysis of spectra generated by the Bruker Biotyper identified six clusters of Nocardia species, i.e., cluster 1 (N. cyriacigeorgica), cluster 2 (N. brasiliensis), cluster 3 (N. farcinica), cluster 4 (N. puris), cluster 5 (N. asiatica), and cluster 6 (N. beijingensis), based on the six peaks generated by ClinProTools with the genetic algorithm, i.e., m/z 2,774.477 (cluster 1), m/z 5,389.792 (cluster 2), m/z 6,505.720 (cluster 3), m/z 5,428.795 (cluster 4), m/z 6,525.326 (cluster 5), and m/z 16,085.216 (cluster 6). Two clusters of L. monocytogenes spectra were also found according to the five peaks, i.e., m/z 5,594.85, m/z 6,184.39, and m/z 11,187.31, for cluster 1 (serotype 1/2a) and m/z 5,601.21 and m/z 11,199.33 for cluster 2 (serotypes 1/2b and 4b). The Bruker Biotyper system was unable to accurately identify Nocardia (except for N. nova and N. otitidiscaviarum), Tsukamurella, or Gordonia species. Continuous expansion of the MALDI-TOF MS databases to include more GPRs is necessary.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate Aeromonas dhakensis from A. hydrophila, A. caviae, and A. veronii.

Among 217 Aeromonas isolates identified by sequencing analysis of their rpoB genes, the accuracy rates of identification of A. dhakensis, A. hydrophila, A. veronii, and A. caviae were 96.7%, 90.0%, 96.7%, and 100.0%, respectively, by the cluster analysis of spectra generated by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate between Mycobacterium masilliense (M. abscessus subspecies bolletti) and M. abscessus (sensu stricto).

Among 36 Mycobacterium masilliense and 22 M. abscessus isolates identified by erm(41) PCR and sequencing analysis of rpoB and 23S rRNA genes, the rate of accurate differentiation between these two subspecies was 100% by cluster analysis of spectra generated by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Comparison of the accuracy of matrix-assisted laser desorption ionization-time of flight mass spectrometry with that of other commercial identification systems for identifying Staphylococcus saprophyticus in urine.

Among 30 urinary isolates of Staphylococcus saprophyticus identified by sequencing methods, the rate of accurate identification was 100% for Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 86.7% for the Phoenix PID and Vitek 2 GP systems, 93.3% for the MicroScan GP33 system, and 46.7% for the BBL CHROMagar Orientation system.

Characterization of the phenotypes of methicillin- and vancomycin-susceptible Staphylococcus argenteus after vancomycin passages.

OBJECTIVES: Staphylococcus argenteus is generally more susceptible to antibiotic treatments than Staphylococcus aureus; however, the study showed that the daptomycin/vancomycin-resistant S. argenteus was isolated from a patient with repeated antibiotic treatments. In this study, the methicillin- and vancomycin-susceptible S. argenteus isolates were used to characterize the phenotypes of S. argenteus after vancomycin passages in vitro. METHODS: Eleven S. argenteus isolates were used for passaging under different concentrations of vancomycin. The minimal inhibitory concentration (MIC) of vancomycin was determined by the agar dilution assay, and the biofilm mass of the passaged variants was quantified by the crystal violet staining assay and observed under the confocal microscope. RESULTS: The MIC of vancomycin for eight of 11 S. argenteus isolates was increased from ≤2 µg/mL to ≤4-8 µg/mL after vancomycin passages. Two variants with the high-level vancomycin-intermediate (vancomycin MIC ≤8 µg/mL) phenotype were identified, and the parental strains of these variants did not have the heterogeneous vancomycin-intermediate population determined by the population profile analysis. Further, three S. argenteus isolates showed an increase in biofilm production and icaA transcription after the low-dose (2 µg/mL) vancomycin passages. CONCLUSIONS: S. argenteus is capable of acquiring a vancomycin-tolerant phenotype and/or converting to a strong biofilm producer after vancomycin passages, which could contribute to the decrease of their antibiotic susceptibility.

Current status of MALDI-TOF mass spectrometry in clinical microbiology.

Mass spectrometry (MS) is a type of analysis used to determine what molecules make up a sample, based on the mass spectrum that are created by the ions. Mass spectrometers are able to perform traditional target analyte identification and quantitation; however, they may also be used within a clinical setting for the rapid identification of bacteria. The causative agent in sepsis is changed over time, and clinical decisions affecting the management of infections are often based on the outcomes of bacterial identification. Therefore, it is essential that such identifications are performed quickly and interpreted correctly. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer is one of the most popular MS instruments used in biology, due to its rapid and precise identification of genus and species of an extensive range of Gram-negative and -positive bacteria. Microorganism identification by Mass spectrometry is based on identifying a characteristic spectrum of each species and then matched with a large database within the instrument. The present review gives a contemporary perspective on the challenges and opportunities for bacterial identification as well as a written report of how technological innovation has advanced MS. Future clinical applications will also be addressed, particularly the use of MALDI-TOF MS in the field of microbiology for the identification and the analysis of antibiotic resistance.

Accurate differentiation of novel Staphylococcus argenteus from Staphylococcus aureus using MALDI-TOF MS.

AIM: We evaluated a Staphylococcus argenteus-specific diagnostic profile of matrix-assisted laser desorption/ionization time-of-flight mass spectrometer for accurate identification of this novel bacterium. MATERIALS & METHODS: Staphylococcus argenteus was identified based on negative crtM gene and presence of specific sequence types. A classification model was generated by cluster analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometer results and ClinProTools software for 25 S. argenteus and 25 methicillin-susceptible S. aureus (MSSA). The performance of the classification model was validated against 72 S. argenteus and 72 MSSA isolates. RESULTS: With cluster analysis and classification model, differentiation of 72 S. argenteus from 72 MSSA had 100.0% accuracy by chemical extraction method and 87.5% sensitivity and 100.0% specificity by direct smear method. CONCLUSION: The classification model could accurately differentiate S. argenteus from MSSA.

Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Clinical and Environmental Isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei is not represented in the current version of Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system. A total of 66 isolates of B. pseudomallei, including 30 clinical isolates collected from National Taiwan University Hospital (NTUH, n = 27) and Peking Union Medical College Hospital (PUMCH, n = 3), and 36 isolates of genetically confirmed strains, including 13 from clinical samples and 23 from environmental samples, collected from southern Taiwan were included in this study. All these isolates were identified by partial 16S rDNA gene sequencing analysis and the Bruker Biotyper MALDI-TOF MS system. Among the 30 isolates initially identified as B. pseudomallei by conventional identification methods, one was identified as B. cepacia complex (NTUH) and three were identified as B. putida (PUMCH) by partial 16S rDNA gene sequencing analysis and Bruker Biotyper MALDI-TOF MS system. The Bruker Biotyper MALDI-TOF MS system misidentified 62 genetically confirmed B. pseudomallei isolates as B. thailandensis or Burkholderia species (score values, 1.803-2.063) when the currently available database (DB 5627) was used. However, using a newly created MALDI-TOF MS database (including B. pseudomallei NTUH-3 strain), all isolates were correctly identified as B. pseudomallei (score values >2.000, 100%). An additional 60 isolates of genetically confirmed B. cepacia complex and B. putida were also evaluated by the Bruker Biotyper MALDI-TOF MS system using the newly created database and none of these isolates were identified as B. pseudomallei. MALDI-TOF MS is a versatile and robust tool for the rapid identification of B. pseudomallei using the enhanced database.

Applicability of an in-House Saponin-Based Extraction Method in Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Bacterial and Fungal Species in Positively Flagged Blood Cultures.

We used an in-house saponin-based extraction method to evaluate the performance of the Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) system for the identification of bacteria and fungi in 405 positively flagged blood culture bottles. Results obtained from MALDI-TOF/MS were compared with those obtained using conventional phenotypic identification methods. Of the 405 positively flagged blood culture bottles, 365 showed monomicrobal growth and were correctly identified to the species (72.1%) or genus (89.6%) level using the Bruker Biotyper system. The remaining 40 positively flagged blood culture bottles showed polymicrobial growth. Of them, 82.5% (n = 33) of the isolates were correctly identified to the species level and 92.5% (n = 37) to the genus level using the Bruker Biotyper system. The overall accuracy of identification to the genus level in flagged blood cultures was 89.5% for Gram-positive organisms, 93.5% for Gram-negative pathogens and 71.9% for fungi. Confidence scores were ≥1.500 for 307 (75.8%) bottles, ≥1.700 for 249 (61.5%) bottles and ≥2.000 for 142 (35.1%) bottles. None of the yeast cultures yielded scores ≥1.700. Using an identification-score cutoff of ≥1.500, the MALDI Biotyper correctly identified 99.2% of Gram-positive bacteria, 97.6% of Gram-negative bacteria and 100% of yeast isolates to the genus level and 77.6% of Gram-positive bacteria, 87.1% of Gram-negative bacteria and 100.0% of yeast isolates to the species level. The overall rate of identification using our protocol was 89.9% (364/405) for genus level identification and 73.1% (296/405) for species level identification. Yeast isolates yielded the lowest confidence scores, which compromised the accuracy of identification. Further optimization of the protein extraction procedure in positive blood cultures is needed to improve the rate of identification.

Peroxisome Proliferator-Activated Receptor γ Level Contributes to Structural Integrity and Component Production of Elastic Fibers in the Aorta.

Loss of integrity and massive disruption of elastic fibers are key features of abdominal aortic aneurysm (AAA). Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to attenuate AAA through inhibition of inflammation and proteolytic degradation. However, its involvement in elastogenesis during AAA remains unclear. PPARγ was highly expressed in human AAA within all vascular cells, including inflammatory cells and fibroblasts. In the aortas of transgenic mice expressing PPARγ at 25% normal levels (Pparg(C) (/-) mice), we observed the fragmentation of elastic fibers and reduced expression of vital elastic fiber components of elastin and fibulin-5. These were not observed in mice with 50% normal PPARγ expression (Pparg(+/-) mice). Infusion of a moderate dose of angiotensin II (500 ng/kg per minute) did not induce AAA but Pparg(+/-) aorta developed flattened elastic lamellae, whereas Pparg(C/-) aorta showed severe destruction of elastic fibers. After infusion of angiotensin II at 1000 ng/kg per minute, 73% of Pparg(C/-) mice developed atypical suprarenal aortic aneurysms: superior mesenteric arteries were dilated with extensive collagen deposition in adventitia and infiltrations of inflammatory cells. Although matrix metalloproteinase inhibition by doxycycline somewhat attenuated the dilation of aneurysm, it did not reduce the incidence nor elastic lamella deterioration in angiotensin II-infused Pparg(C/-) mice. Furthermore, PPARγ antagonism downregulated elastin and fibulin-5 in fibroblasts, but not in vascular smooth muscle cells. Chromatin immunoprecipitation assay demonstrated PPARγ binding in the genomic sequence of fibulin-5 in fibroblasts. Our results underscore the importance of PPARγ in AAA development though orchestrating proper elastogenesis and preserving elastic fiber integrity.

Fishing for biomarkers: analyzing mass spectrometry data with the new ClinProTools software.

Recently, applications of mass spectrometry in the field of clinical proteomics have gained tremendous visibility in the scientific and clinical community. One major objective is the search for potential biomarkers in complex body fluids like serum, plasma, urine, saliva, or cerebral spinal fluid. For this purpose, efficient visualization of large data sets derived from patient cohorts is crucial to provide clinical experts an interactive impression of the data quality. Additionally, it is necessary to apply statistical analysis and pattern matching algorithms to attain validated signal patterns that may allow for later applications in sample classification. We introduce the new ClinProTools bioinformatics software, which performs all major steps of profiling, screening, and monitoring applications in clinical proteomics. ClinProTools is the data interpretation software of the mass spectrometry-based ClinProt solutions for biomarker analysis. ClinProTools performs data pretreatment, visualization, statistics, pattern determination, pattern evaluation, and classification of spectra. This article will focus on ClinProTool's powerful and intuitive visualization options for clinical proteomics applications.

Identification of Weissella species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Although some Weissella species play beneficial roles in food fermentation and in probiotic products, others such as Weissella confusa are emerging Gram-positive pathogens in immunocompromised hosts. Weissella species are difficult to identify by conventional biochemical methods and commercial automated systems and are easily misidentified as Lactobacillus and Leuconostoc species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly being used for bacterial identification. Little, however, is known about the effectiveness of MALDI-TOF MS in identifying clinical isolates of Weissella to the species level. In this study, we evaluated whether the MALDI-TOF MS Bruker Biotyper system could accurately identify a total of 20 W. confusa and 2 W. cibaria blood isolates that had been confirmed by 16s rRNA sequencing analysis. The MALDI-TOF Biotyper system yielded no reliable identification results based on the current reference spectra for the two species (all score values <1.7). New W. confusa spectra were created by randomly selecting 3 W. confusa isolates and external validation was performed by testing the remaining 17 W. confusa isolates using the new spectra. The new main spectra projection (MSP) yielded reliable score values of >2 for all isolates with the exception of one (score value, 1.963). Our results showed that the MSPs in the current database are not sufficient for correctly identifying W. confusa or W. cibaria. Further studies including more Weissella isolates are warranted to further validate the performance of MALDI-TOF in identifying Weissella species.

Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii.

We evaluated the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the MALDI Bruker Biotyper system (microflex LT; Bruker Daltonik GmbH, Bremen, Germany), on the identification of 50 isolates of clinically encountered molds, including Penicillium marneffei (n = 28), Paecilomyces species (n = 12), Fusarium solani (n = 6), Rhizopus species (n = 3), and Pseudallescheria boydii (n = 1). The isolates were identified to species levels by sequence analysis of the internal transcribed spacer (ITS) regions using primers ITS1 and ITS4. None of the 28 genetically well characterized isolates of P. marneffei were identified as P. marneffei by MALDI-TOF MS, because P. marneffei was not present in either Bruker general library (DB 5627) or Bruker filamentous fungi library V1.0. However, the rate of accurate identification as P. marneffei (score value ≥ 2.000) was 85.7% based on newly created database from one P. marneffei strain (NTUH-3370) by MALDI Biotyper system. Sequencing analysis of these 22 non-P. marneffei isolates of molds revealed seven Paecilomyces variotii, six F. solani, four Paecilomyces lilacinus, and one each of Paecilomyces sinensis, Rhizopus arrhizus, R. oryzae, R. microspores, and P. boydii. Although all the seven P. variotii isolates, four of the six F. solani, two of the four P. lilacinus, and two of the three isolates of Rhizopus species, and the P. boydii isolate had concordant identification results between MALDI-TOF MS and sequencing analysis, the score values of these isolates were all of <1.700. This study indicated that the MALDI Bruker Biotyper is ineffective for identifying P. marneffei and other unusual molds because of the current database limitations. Therefore, it is necessary to continuously update the MALDI-TOF MS databases.

Comparison of the accuracy of two conventional phenotypic methods and two MALDI-TOF MS systems with that of DNA sequencing analysis for correctly identifying clinically encountered yeasts.

We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex, and C. rugosa complex.

Rapid identification of M. abscessus and M. massiliense by MALDI-TOF mass spectrometry with a comparison to sequencing methods and antimicrobial susceptibility patterns.

AIM: Development of the matrix-assisted laser desorption ionization-TOF (MALDI-TOF) mass spectrometry method to rapidly identify Mycobacteria abscessus and Mycobacteria massiliense from M. abscessus complex in clinical microbiology laboratories. MATERIALS & METHODS: Of 128 M. abscessus complex clinical isolates, sequence analysis identified 64 as M. massiliense and 64 as M. abscessus. MALDI-TOF mass spectrometry with clustering analysis created a model to differentiate these two species. Multilocus sequence typing was used to confirm our model. RESULTS: Using a model containing five signals, 100% species recognition was achieved for 50 strains of each species. The sequence type (ST) cluster of M. abscessus was distinct from the cluster of M. massiliense. ST1 was prevalent (59%) among M. abscessus isolates, and ST117 was common (41%) among M. massiliense isolates. CONCLUSION: Molecular methods are more costly and time-consuming and may not be available in the clinical microbial laboratory. Subsequently, MALDI-TOF analysis could be a rapid identification method in the future.

Differential expression of porcine testis proteins during postnatal development.

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.

Hydrostatic pressure pre-treatment affects the protein profile of boar sperm before and after freezing-thawing.

A sublethal environmental stress, high-hydrostatic pressure (HHP) was reported to significantly improve the motility, viability and fertility parameters of frozen bull and boar semen. However, the mechanism of how HHP treatment improves survival rates at sperm cryopreservation remains unclear. The purpose of this study was to evaluate the effect of HHP treatment of fresh boar semen on the protein profile of boar sperm before and after freezing. Fresh, extended semen of eight boars was split, one part was treated with 200, 300 or 400bar for 90min using a custom made pressuring device before the start of the semen freezing procedure, and the other part was prepared without HHP treatment. After thawing, samples were checked for motility. The effect of HHP treatment on the post-thaw motility of frozen semen was significant (P=0.02). Post-thaw motility of each treatment groups increased compared to control (46% vs. 52%, 56% and 56%; control vs. 200bar, 300bar and 400bar treatments). Samples for protein analysis were collected from the 300bar treatment group before HHP treatment at room temperature (25+/-3 degrees C), at 5 degrees C of the cooling process and after thawing with or without HHP treatment. The sperm were lysed using a urea-pyranoside-dithiothreitol buffer to extract their proteins for protein analysis. Approximately 800microg total proteins were assayed by two-dimensional gel electrophoresis and stained with colloidal Coomassie blue. The levels of 125 protein spots were quantified. The results revealed that the levels of 7 protein spots differed significantly among treatments. The identities of various protein constituents were identified by mass spectrometry and database searching. Ubiquinol-cytochrome c reductase complex core protein 1, perilipin, and carbohydrate-binding protein AWN precursor were identified as HHP response proteins being significantly higher in HHP-treated samples. Testis-specific glyceraldehyde 3-phosphate dehydrogenase, outer dense fiber of sperm tails 2 isoform 10, cytosolic 5'-nucleotidase 1B, and quinone oxidoreductase represented the cooling and freezing related proteins. The differing levels of these identified proteins could be valuable for further exploring the protective mechanism of the HHP treatment in frozen-thawed porcine sperm.