2 GLOBAL PEPTIDE SEQUENCING AND QUANTIFICATION OF PROTEINS IN PORCINE PARTHENOTES BY PROTEOMICS

Abstract

Mass spectrometry (MS) is a powerful emerging tool in proteomics for global identification and quantification of proteins and their differential analysis. The aim of this study was to utilize MS for global peptide sequencing and the relative quantification of peptide sequences in porcine embryos produced by parthenogenesis. Oocytes (n = 6000) recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and parthenogenetically activated by a single electric pulse of 1.36 kV cm^-1. After 24 h of in vitro culture in NCSU23 medium supplemented with 0.4% BSA, presumptive zygotes were sampled in Laemmli buffer and stored in liquid nitrogen until analysis. SDS-PAGE was used as the first step to separate intact proteins into 16 fractions. Each fraction was then in-gel tryptic-digested and analyzed by nanoLC-nano-ESI-MS/MS to sequence the peptide components in each fraction by LTQ ion-trap mass spectrometer (Finnigan, Fresno, CA, USA) after peptide purification by C18 ZipTips (Millipore, Billerica, MA, USA). Synthetic peptides from Myoglobin (LFTGHPETL*EK) and MAP3 Kinase (IPTGTV*HNQAK) protein having an isotope-labeled carbon (*) were used as internal standards for relative quantification. In total, 54 060 peptides were sequenced. Of these, 259 proteins were identified in the NCBI protein database for Sus scrofa using Spectrum Mill software (Agilent Technologies, Palo Alto, CA, USA). Of these, 199 proteins were identified with high confidence having at least 2 peptide sequences matching each protein. These included heat shock protein families, ribosomal proteins, cytoskeletal proteins, mitochondrial enzymes, disulfide isomerase proteins, glutathione S-transferase, DNA methyltransferase, STAT1, Ras-related protein Rab 1A, voltage-dependent ion channel proteins, zinc finger proteins and elongation factors, etc., which are involved in several important metabolic and cell signaling pathways. Several proteins including DNA methyltransferase 1, peroxidoxin 2, ubiquitin carboxyl-terminal hydrolase L1, serpin, heat shock proteins, cytoskeletal proteins, mitochondrial enzymes, elongation factor 1, bone morphogenetic protein, glucose-related proteins, etc., were among the highly abundant proteins. Our results thus showed that proteomic strategy can be successfully applied to analyze porcine embryo proteome. To our knowledge, this is the first time that several proteins have been sequenced and quantified in porcine parthenotes by proteomic means. In the long run, this study may help the understanding of mechanisms underlying embryonic development and identification of biomarkers of embryo quality.

This work was supported by grants from the Research Project on the Production of Bio-organs (No. 200503030202), Ministry of Agriculture and Forestry, and Korea Health 21 R&D Project (No. A060769), Ministry of Health and Welfare, Republic of Korea.