Automated Data Integration and Determination of Posttranslational Modifications with the Protein Inference Engine
This chapter describes using the Protein Inference Engine (PIE) to integrate various types of data – especially top down and bottom up mass spectrometer (MS) data – to describe a protein’s posttranslational modifications (PTMs). PTMs include cleavage events such as the n-terminal loss of methionine and residue modifications like phosphorylation. Modifications are key elements in many biological processes, but are difficult to study as no single, general method adequately characterizes a protein’s PTMs; manually integrating data from several MS experiments is usually required. The PIE is designed to automate this process using a guess and refine process similar to how an expert manually integrates data. The PIE repeatedly “imagines” a possible modification set, evaluates it using available data, and then tries to improve on it. After many rounds of refinement, the resulting modification set is proposed as a candidate answer. Multiple candidate answers are generated to obtain both best and near-best answers. Near-best answers are crucial in allowing for proteins with more than one supported modification pattern (isoforms) and obtaining robust results given incomplete and inconsistent data.
The goal of this chapter is to walk the reader through installing and using the downloadable version of PIE, both in general and by means of a specific, detailed example. The example integrates several types of experimental and background (prior) data. It is not a “perfect-world” scenario, but has been designed to illustrate several real-world difficulties that may be encountered when trying to analyze imperfect data.
Key wordsPTM MCMC Simulated annealing Proteomics Top-down Bottom-up Data integration PIE
- 19.Strader, M. B., Verberkmoes, N. C., Tabb, D. L., Connelly, H. M., Barton, J. W., Bruce, B. D., et al. (2004) Characterization of the 70S ribosome from Rhodopseudomonas palustris using an integrated “top-down” and “bottom-up” mass spectrometric approach. J. Proteome Res. 3, 965–978.PubMedCrossRefGoogle Scholar
- 21.Kertesz, V., Connelly, H. M., Erickson, B. K., and Hettich, R. L. (2009) PTMSearchPlus: software tool for automated protein identification and post-translational modification characterization by integrating accurate intact protein mass and bottom-up mass spectrometric data searches. Anal. Chem. 81, 8387–8395.PubMedCrossRefGoogle Scholar
- 26.Frottin, F., Martinez, A., Peynot, P., Mitra, S., Holz, R. C., Giglione, C., and Meinnel, T. (2006) The proteomics of N-terminal methionine cleavage. Mol. Cell. Proteomics 51, 2336–2349.Google Scholar