Abstract
The shotgun proteomic strategy based on digesting proteins into peptides and sequencing them using tandem mass spectrometry and automated database searching has become the method of choice for identifying proteins in most large scale studies. However, the peptide-centric nature of shotgun proteomics complicates the analysis and biological interpretation of the data especially in the case of higher eukaryote organisms. The same peptide sequence can be present in multiple different proteins or protein isoforms. Such shared peptides therefore can lead to ambiguities in determining the identities of sample proteins. In this article we illustrate the difficulties of interpreting shotgun proteomic data and discuss the need for common nomenclature and transparent informatic approaches. We also discuss related issues such as the state of protein sequence databases and their role in shotgun proteomic analysis, interpretation of relative peptide quantification data in the presence of multiple protein isoforms, the integration of proteomic and transcriptional data, and the development of a computational infrastructure for the integration of multiple diverse datasets.
Highlights
The shotgun proteomic strategy based on digesting proteins into peptides and sequencing them using tandem mass spectrometry and automated database searching has become the method of choice for identifying proteins in most large scale studies
4 Peptide and protein identification part of the analysis presented in Ref. 27 was repeated in this work using the Human International Protein Index (IPI) version 2.35 protein sequence database
Shotgun proteomic technology has matured to a point where it can be used for routine identification and, when coupled with stable isotope labeling, accurate relative quantification of thousands of peptides in a single experiment
Summary
The shotgun proteomic strategy based on digesting proteins into peptides and sequencing them using tandem mass spectrometry and automated database searching has become the method of choice for identifying proteins in most large scale studies. If the database contains several peptides with a similar molecular weight and having a high degree of sequence homology, determination of the correct peptide sequence among the alternatives becomes difficult or even impossible (in the case of Ile/Leu substitutions) This can result in the assignment of incorrect ( homologous) peptide sequences to MS/MS spectra, which in turn can result in incorrect protein identifications. More than 30% of all proteins that are detected in a typical shotgun proteomic experiment, including many low molecular weight or low abundance proteins, are identified by a single peptide
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