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Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):407-12. doi: 10.1073/pnas.1108399108. Epub 2011 Dec 22.

Database independent proteomics analysis of the ostrich and human proteome.

Author information

1
Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Abstract

Mass spectrometry (MS)-based proteome analysis relies heavily on the presence of complete protein databases. Such a strategy is extremely powerful, albeit not adequate in the analysis of unpredicted postgenome events, such as posttranslational modifications, which exponentially increase the search space. Therefore, it is of interest to explore "database-free" approaches. Here, we sampled the ostrich and human proteomes with a method facilitating de novo sequencing, utilizing the protease Lys-N in combination with electron transfer dissociation. By implementing several validation steps, including the combined use of collision-induced dissociation/electron transfer dissociation data and a cross-validation with conventional database search strategies, we identified approximately 2,500 unique de novo peptide sequences from the ostrich sample with over 900 peptides generating full backbone sequence coverage. This dataset allowed the appropriate positioning of ostrich in the evolutionary tree. The described database-free sequencing approach is generically applicable and has great potential in important proteomics applications such as in the analysis of variable parts of endogenous antibodies or proteins modified by a plethora of complex posttranslational modifications.

PMID:
22198768
PMCID:
PMC3258627
DOI:
10.1073/pnas.1108399108
[Indexed for MEDLINE]
Free PMC Article

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