173 SIMULTANEOUS QUANTIFICATION OF MULTIPLE PROTEINS IN PORCINE PARTHENOTES BY MULTIPLE SELECTED REACTION MONITORING

Abstract

Simultaneous quantification of multiple proteins could be a very useful tool in cell signaling studies. Parthenogentic embryos do not develop to term, although their in vitro developmental ability to the blastocyst stage and to form pluripotent stem cells upon culture is comparable to those of sperm- or somatic nucleus-introduced embryos. This study was designed to quantify the expression of eight selected proteins by multiple selected reaction monitoring (mSRM) of trypsinized preparations from porcine zygotes (n = 1500) produced by parthenogenesis. The trypsinized samples were prepared, spiked with isotopically labeled synthetic MAP3K (IPTGTV*HNQAK) peptides as internal standards for quantification (asterisk denotes isotopic amino acid residue), and analyzed by reverse phase LC-MS/MS. Upon the appearance of the target peptide, SRM data were acquired within fragmention mass ±1.5 m/z and each SRM transition, with its respective retention time, was validated as indicative of each specific peptide. Data were processed by integrating the appropriate peaks for the native and internal standard peptide, followed by calculation of the ratio of peak areas to estimate the relative abundance of the peptide. Analysis of data showed that the relative abundance of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), STAT2, calpastatin, complement cytolysis inhibitor, plakoglobin, serotransferrin, and epsilon-globin were 203.72, 138.52, 60.50, 18.63, 31.23, 0.85, 10.33, and 0.12, respectively. These data were consistent with those observed by reverse phase LC-MS/MS combined with 1-dimensional (1-D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of cellular lysates from porcine embryos (n = 6000). The data presented here, therefore, provide mSRM of embryos for the first time in any species and suggest that mSRM in combination with reverse-phase LC-MS/MS combined with 1-D SDS-PAGE could be a powerful tool for proteome analysis of porcine embryos to obtain vital new information for understanding the basic biology of embryonic development.

This study was supported by grants from Biogreen 21, RDA, and the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.