A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing.
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.
Gauci
Sharon
S
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
van Breukelen
Bas
B
Lemeer
Simone M
SM
Krijgsveld
Jeroen
J
Heck
Albert J R
AJ
D000107:Acetylation; D000818:Animals; D002457:Cell Extracts; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D011934:Reagent Strips; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism
D002457:Cell Extracts; D010748:Phosphopeptides; D011934:Reagent Strips