Nanoscale Liquid Chromatography-tandem Mass Spectrometry Research Articles

69 DIFFERENTIAL AND QUANTITATIVE ANALYSIS OF DOG OVIDUCTAL FLUID

The major reproductive peculiarity of the bitch is that ovulation releases prophase I (germinal vesicle, GV, immature) oocytes. Resumption of meiotic maturation, as well as fertilisation and embryonic development to the morula stage occur in the oviduct. Because the dog is a biomedical model for human diseases and also a model for endangered canid species, the development of assisted reproduction techniques would be of great interest. To date, in vitro-produced canine embryos remain exceptional and no puppy has been born. The main limiting factors of in vitro embryo production are the low oocyte maturation rates, the poor oocyte quality and the high polyspermy. A better knowledge of the composition of oviductal fluid during the periovulatory period may help to mimic the in vivo conditions for in vitro oocyte culture and, thereafter, their fertilisation and embryonic development. The objective of this study was to analyse the oviductal fluid by a label-free quantitative proteomic workflow based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein separation, nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis and quantitative method using spectral counting. Ovarian cycles were followed by vaginal smears, ultrasonography and progesterone blood assays. Oviductal fluids were collected from 3 beagle bitches, after ovariectomies performed 3.5 days after ovulation. After dissection, the ampulla and isthmus were separated and flushed with 50 μL of PBS. Oviductal fluids were submitted to 1D SDS-PAGE and all bands were digested with trypsin. Peptide extracts were analysed on an Ettan multidimensional LC (MDLC) system coupled to a linear ion trap quadrupole (LTQ) mass spectrometer. After protein identification using Mascot server and with Swiss-Prot and National Center for Biotechnology Information (NCBI) databases, bioinformatic processing of data and statistic analysis (t-test with P < 0.05) were performed using the spectral counting quantitative module of the Scaffold software. Using this strategy, 427 proteins were qualitatively identified in canine oviductal fluid. Three proteins were specific of the ampulla, 10 specific of the isthmus and 414 were found in both oviductal parts. Among these common proteins, some were differentially expressed, from 1.25 to 9 times higher (HV303_Human, RLA2_Horse, SPRL1_Human, SODC_CANFA, PROF1_Human, ARF4_Bovin and TRXR1_Bovin). The gene ontology analysis displayed biological pathways specific to the biology of reproduction (6 proteins; RUVB1_Human, OVGP1_Pig, STAT3_Human, PLAK_Human, GPX3_Rat and DYL1_Human). These candidate proteins and especially oviduct-specific glycoprotein and glutathione peroxidase, will now be validated by immunodetection methods.

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments.

BackgroundIsotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high.ResultsWe designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L) pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein identification process and, consequently, on the amount of potentially critical information in clinical studies. The EIPeptiDi tool is available at with a demo data set.ConclusionEIPeptiDi significantly increases the number of peptides identified and quantified in analyzed samples, thus reducing the number of unassigned H/L pairs and allowing a better comparative analysis of sample data sets.

Identification of a Novel Two-Partner Secretion Locus in Moraxella catarrhalis

Although Moraxella catarrhalis continues to be a significant cause of disease in both children and adults, the steps involved in pathogenesis remain poorly understood. We have identified three open reading frames in the M. catarrhalis genome that encode homologues of the two-partner secretion system (TPS). The sequenced M. catarrhalis hemagglutinin-like locus of strain 7169 has a unique gene organization composed in the order of mchA1, mchB, and mchA2, where mchA1 is divergent. MchA1 and MchA2 are 74% identical at the amino acid level and diverge only in the C-terminal regions. The TPS motif identified in the common N-terminal regions of MchA1 and MchA2 was found to be homologous to the filamentous hemagglutinin of Bordetella pertussis, and MchB has homology to other TpsB transporters. The presence of MchA1 and MchA2 in outer membrane protein preparations and concentrated culture supernatants (CCSs) of strain 7169 was confirmed by immunoblotting using specific antisera. Nanoscale liquid chromatography-tandem mass spectrometry peptide sequencing of the antibody-reactive bands from the CCSs was performed and demonstrated that 13 different peptides mapped to identical regions of MchA1 and MchA2. Quantitative adherence assays revealed a decrease of binding to primary normal human bronchial epithelial cells by the mch mutants 7169mchB and 7169mchA1A2B compared to that by the wild-type strain. These studies show that MchA1, MchA2, and MchB are components of a novel TPS identified in M. catarrhalis and suggest that these proteins may be involved in colonization.

Heavy Water Labeling of Keratin as a Non-Invasive Biomarker of Skin Turnover In Vivo in Rodents and Humans

Measurement of skin turnover has been problematic in humans. Heavy water (2H2O) labeling has recently been developed as a safe, simple method to study in vivo kinetics of many biosynthetic processes, including DNA and protein synthesis. Here, we apply this approach to the measurement of 2H incorporation into skin keratin and show close agreement between keratin and keratinocyte turnover data in the epidermis of rodents. Elevated turnover rates of both keratin and keratinocytes were observed in the epidermis of the flaky skin mouse, although topical treatments effective in human psoriasis had no effect on either turnover rate in these mice. In humans, keratin turnover was monitored non-invasively by serial tape stripping during and after 2H2O labeling. Kinetic data were consistent with previous estimates of epidermal turnover, with a lag time of 18 days before label appeared at the skin surface and a transit time of 4-5 weeks. Variability in skin keratin turnover rates was present among healthy individuals. In summary, 2H2O labeling of skin keratin represents a non-invasive approach for assessing skin turnover dynamics in pre-clinical models and in human subjects.

Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in Brassicaceae

An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish ( Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (μmol mg −1 min −1) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 μmol mg −1 min −1. The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (p I) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish , respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.