Reveal New Genes Of Sulfolobus Solfataricus P2 Using Proteogenomics Approach

Sulfolobus solfataricus P2 is a kind of extremely thermophilic archaea.Its genome sequencing was accomplished in 2001,which is the first full sequenced archaea.Therefore,its gene annotation is more complete than other archaea.However,a large number of proteins are still annotated as prediction proteins,and the number of proteins in its proteome is less than the number of open reading frames in genome.In addition,because Archaea has some unique starting codons,which may cause errors in the translation from genome to proteome.Those errors need to be verified and corrected.Proteogenomics is a method of genome annotation based on proteome,genome and transcriptome data.It has been successfully used in many species.In order to further improve the gene annotation of Sulfolobus solfataricus P2,we will identify its proteome,and use the proteogenomics analysis to explore and verify new genes.The coverage of proteomic identification of thermophilic archaea is generally low.In this paper,we first optimized the identification of Sulfolobus solfataricus proteomics:1)adding 1%DDM solubilizer to increase the protein extraction;2)extracting membrane protein and soluble protein from bacteria in the early,middle and later stages of logarithmic growth;3)using various search software to improve the identification rate of protein.Finally,we identified 2123 proteins,including 1613 soluble proteins and 153 membrane proteins,accounting for 72.2%of the known proteome,which is the highest coverage of the Sulfolobus solfataricus P2 proteomic data.Then through the proteogenomics analysis,we found 155 new peptides,corresponding to 71 new genes.We choose five of the new genes to run RT-PCR,and all of them were confirmed.Blastp result showed that all the proteins encoded by the new genes are homologous with other sulfolobus.Some of them are homologous with the known functional proteins,including ligase,ATPase and antitoxin proteins.Some new proteins are homologous with predicted proteins,and for these protein we use CD-search to analyze their domains.We found that there are two new proteins with specific domain,GENE302 with PCC1 superfamily domain,GENE2953 with Sif superfamily domain.The PCC1 superfamily domain has the function of synthesizing t6a.SIF superfamily is one of the subunits of KEOPS,a protein complex that helps protein secretion.It is speculated that these two new proteins may have certain functions related to these domains.In this paper,the protein identification of Sulfolobus solfataricus P2 was increased.71 new genes were found by proteomics,and some of them were verified and function predicted.The results of this study expand the information of genome annotation of Sulfolobus solfataricus P2,and provide theoretical basis and data support for protein function research of Archaea.