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Proteomics. 2008 Dec;8(23-24):4898-906. doi: 10.1002/pmic.200800295.

A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing.

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


We experimentally demonstrate the use of an in-house developed pI calculator which takes into account peptide PTM such as phosphorylation and N-terminal acetylation. The pI calculator was utilized for a large set of peptides derived from a complex zebrafish lysate fractionated using peptide IEF, whereby a good correlation between the calculated (theoretical) pI and the experimental pI could be established. This pI calculator permits the implementation of optimal pK values depending on the experimental conditions and a reliable calculation of peptide pI which can be utilized as a filtering technique in validating peptide identifications. Our data reveal that the shift due to a phosphorylation or N-terminal acetylation is highly dependent on the presence of acidic or basic residues in the peptide. Furthermore, using this pI calculator, we revealed previously unknown position-specific pKs of asparagine and carbamidomethylated cysteine depending on their location in the peptide. Collectively, this peptide pI calculator is a welcome addition to the versatility and robustness of IEF for the separation and confident identification of (post-translationally modified) peptides.

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