Journal
JOURNAL OF PROTEOME RESEARCH
Volume 7, Issue 10, Pages 4336-4345Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr8002672
Keywords
isoelectric focusing; shotgun proteomics; tandem mass spectrometry; isoelectric point; fluorescent p/markers
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The use of isoelectric focusing as first dimension of separation is a new trend in shotgun proteomics. In all applications using this approach, peptides are separated into equitable fractions, whereas theoretical distribution of peptides according to p1 is heterogeneous. We present the development of a new tool and strategy that generates a fractionation scheme resulting in almost even distribution of peptides per fraction, based on theoretical and experimental data. The plCarver software tool also increases the throughput of the approach by reducing the number of fractions and merging the peptide-poor regions. A set of isoelectric point fluorescent peptide markers was also developed in combination with the pICarver program to calibrate the pH gradient of commercially available strips. These markers enhanced the precision of pICarver predications. The overall strategy allowed detecting false positive identification and post-translational modifications. The software tool is freely available on www. expasy.org/tools/pICarver.
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