Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate

Gun Wook Park, Heeyoun Hwang, Kwang Hoe Kim, Ju Yeon Lee, Hyun Kyoung Lee, Ji Yeong Park, Eun Sun Ji, Sung Kyu Robin Park, John R. Yates, Kyung Hoon Kwon, Young Mok Park, Hyoung Joo Lee, Young Ki Paik, Jin Young Kim, Jong Shin Yoo

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Abstract

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

Original languageEnglish
Pages (from-to)4082-4090
Number of pages9
JournalJournal of Proteome Research
Volume15
Issue number11
DOIs
Publication statusPublished - 2016 Nov 4

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)

Cite this

Park, G. W., Hwang, H., Kim, K. H., Lee, J. Y., Lee, H. K., Park, J. Y., Ji, E. S., Park, S. K. R., Yates, J. R., Kwon, K. H., Park, Y. M., Lee, H. J., Paik, Y. K., Kim, J. Y., & Yoo, J. S. (2016). Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate. Journal of Proteome Research, 15(11), 4082-4090. https://doi.org/10.1021/acs.jproteome.6b00376