Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys
Abstract Background Shotgun proteomics utilizes a database search strategy to compare detected mass spectra to a library of theoretical spectra derived from reference genome information. As such, the robustness of proteomics results is contingent upon the completeness and accuracy of the gene annota...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-11-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-017-4279-0 |
| _version_ | 1819015896018976768 |
|---|---|
| author | J. Michael Proffitt Jeremy Glenn Anthony J. Cesnik Avinash Jadhav Michael R. Shortreed Lloyd M. Smith Kylie Kavanagh Laura A. Cox Michael Olivier |
| author_facet | J. Michael Proffitt Jeremy Glenn Anthony J. Cesnik Avinash Jadhav Michael R. Shortreed Lloyd M. Smith Kylie Kavanagh Laura A. Cox Michael Olivier |
| author_sort | J. Michael Proffitt |
| collection | DOAJ |
| description | Abstract Background Shotgun proteomics utilizes a database search strategy to compare detected mass spectra to a library of theoretical spectra derived from reference genome information. As such, the robustness of proteomics results is contingent upon the completeness and accuracy of the gene annotation in the reference genome. For animal models of disease where genomic annotation is incomplete, such as non-human primates, proteogenomic methods can improve the detection of proteins by incorporating transcriptional data from RNA-Seq to improve proteomics search databases used for peptide spectral matching. Customized search databases derived from RNA-Seq data are capable of identifying unannotated genetic and splice variants while simultaneously reducing the number of comparisons to only those transcripts actively expressed in the tissue. Results We collected RNA-Seq and proteomic data from 10 vervet monkey liver samples and used the RNA-Seq data to curate sample-specific search databases which were analyzed in the program Morpheus. We compared these results against those from a search database generated from the reference vervet genome. A total of 284 previously unannotated splice junctions were predicted by the RNA-Seq data, 92 of which were confirmed by peptide spectral matches. More than half (53/92) of these unannotated splice variants had orthologs in other non-human primates, suggesting that failure to match these peptides in the reference analyses likely arose from incomplete gene model information. The sample-specific databases also identified 101 unique peptides containing single amino acid substitutions which were missed by the reference database. Because the sample-specific searches were restricted to actively expressed transcripts, the search databases were smaller, more computationally efficient, and identified more peptides at the empirically derived 1 % false discovery rate. Conclusion Proteogenomic approaches are ideally suited to facilitate the discovery and annotation of proteins in less widely studies animal models such as non-human primates. We expect that these approaches will help to improve existing genome annotations of non-human primate species such as vervet. |
| first_indexed | 2024-12-21T02:39:01Z |
| format | Article |
| id | doaj.art-17845563f9824873ac245b29ea628bd8 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-21T02:39:01Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-17845563f9824873ac245b29ea628bd82022-12-21T19:18:43ZengBMCBMC Genomics1471-21642017-11-0118111010.1186/s12864-017-4279-0Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeysJ. Michael Proffitt0Jeremy Glenn1Anthony J. Cesnik2Avinash Jadhav3Michael R. Shortreed4Lloyd M. Smith5Kylie Kavanagh6Laura A. Cox7Michael Olivier8Department of Genetics, Texas Biomedical Research InstituteDepartment of Genetics, Texas Biomedical Research InstituteDepartment of Chemistry, University of WisconsinDepartment of Genetics, Texas Biomedical Research InstituteDepartment of Chemistry, University of WisconsinDepartment of Chemistry, University of WisconsinDepartment of Pathology and Comparative Medicine, Wake Forest School of MedicineDepartment of Genetics, Texas Biomedical Research InstituteDepartment of Genetics, Texas Biomedical Research InstituteAbstract Background Shotgun proteomics utilizes a database search strategy to compare detected mass spectra to a library of theoretical spectra derived from reference genome information. As such, the robustness of proteomics results is contingent upon the completeness and accuracy of the gene annotation in the reference genome. For animal models of disease where genomic annotation is incomplete, such as non-human primates, proteogenomic methods can improve the detection of proteins by incorporating transcriptional data from RNA-Seq to improve proteomics search databases used for peptide spectral matching. Customized search databases derived from RNA-Seq data are capable of identifying unannotated genetic and splice variants while simultaneously reducing the number of comparisons to only those transcripts actively expressed in the tissue. Results We collected RNA-Seq and proteomic data from 10 vervet monkey liver samples and used the RNA-Seq data to curate sample-specific search databases which were analyzed in the program Morpheus. We compared these results against those from a search database generated from the reference vervet genome. A total of 284 previously unannotated splice junctions were predicted by the RNA-Seq data, 92 of which were confirmed by peptide spectral matches. More than half (53/92) of these unannotated splice variants had orthologs in other non-human primates, suggesting that failure to match these peptides in the reference analyses likely arose from incomplete gene model information. The sample-specific databases also identified 101 unique peptides containing single amino acid substitutions which were missed by the reference database. Because the sample-specific searches were restricted to actively expressed transcripts, the search databases were smaller, more computationally efficient, and identified more peptides at the empirically derived 1 % false discovery rate. Conclusion Proteogenomic approaches are ideally suited to facilitate the discovery and annotation of proteins in less widely studies animal models such as non-human primates. We expect that these approaches will help to improve existing genome annotations of non-human primate species such as vervet.http://link.springer.com/article/10.1186/s12864-017-4279-0ProteogenomicsProteomicsLiverVervetRNA-SeqMorpheus |
| spellingShingle | J. Michael Proffitt Jeremy Glenn Anthony J. Cesnik Avinash Jadhav Michael R. Shortreed Lloyd M. Smith Kylie Kavanagh Laura A. Cox Michael Olivier Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys BMC Genomics Proteogenomics Proteomics Liver Vervet RNA-Seq Morpheus |
| title | Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys |
| title_full | Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys |
| title_fullStr | Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys |
| title_full_unstemmed | Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys |
| title_short | Proteomics in non-human primates: utilizing RNA-Seq data to improve protein identification by mass spectrometry in vervet monkeys |
| title_sort | proteomics in non human primates utilizing rna seq data to improve protein identification by mass spectrometry in vervet monkeys |
| topic | Proteogenomics Proteomics Liver Vervet RNA-Seq Morpheus |
| url | http://link.springer.com/article/10.1186/s12864-017-4279-0 |
| work_keys_str_mv | AT jmichaelproffitt proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT jeremyglenn proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT anthonyjcesnik proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT avinashjadhav proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT michaelrshortreed proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT lloydmsmith proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT kyliekavanagh proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT lauraacox proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys AT michaelolivier proteomicsinnonhumanprimatesutilizingrnaseqdatatoimproveproteinidentificationbymassspectrometryinvervetmonkeys |