Peptide pI Calculator
Calculate the theoretical isoelectric point (pI) of a peptide directly from an amino acid sequence. FASTA format and terminal modifications are supported. For full molecular weight and charge analysis, use the complete peptide analysis tool.
pI and charge calculations use the IPC_peptide pKa set (Kozlowski, 2016). Molecular weight uses average isotopic masses (UWPR mass table). Results are theoretical approximations — always verify with experimental data.
ℹ️ Frequently Asked Questions
What is the isoelectric point (pI)?
The isoelectric point is the pH at which the peptide carries zero net charge. At pH values below the pI, the peptide carries a net positive charge; above the pI, it carries a net negative charge.
At pH = pI → net charge ≈ 0
The pI is critical for predicting peptide behavior in ion-exchange chromatography, isoelectric focusing (IEF), solubility optimization, and electrophoresis.
How is pI calculated?
The pI is determined using the Henderson-Hasselbalch equation and a binary search algorithm. For each pH value, the net charge is computed from all ionizable groups (D, E, C, Y, H, K, R side chains and the free N/C-terminal groups). The algorithm searches for the pH where net charge crosses zero (resolution: ±0.01 pH units).
Terminal modifications shift the pI: acetylation removes the positive N-terminal charge, and amidation removes the negative C-terminal charge.
Which pKa set does this calculator use?
This calculator uses the IPC_peptide pKa set from Kozlowski (2016), published in Biology Direct 11:55. This set was computationally optimized using a basin-hopping algorithm on 16,882 experimentally measured peptide isoelectric points.
The nine pKa values used:
NH₂ = 9.564 · COOH = 2.383
Asp = 3.887 · Glu = 4.317 · His = 6.018
Cys = 8.297 · Lys = 10.517 · Tyr = 10.071 · Arg = 12.503
Reference: doi:10.1186/s13062-016-0159-9
How do terminal modifications affect pI?
N-terminal Acetylation neutralizes the free amino group (pKa 9.564), removing a positive charge contributor. This generally lowers the pI.
C-terminal Amidation neutralizes the free carboxyl group (pKa 2.383), removing a negative charge contributor. This generally raises the pI.
When both modifications are applied simultaneously, the shift depends on the balance of ionizable side chains in the sequence.
Why does my calculated pI differ from other tools?
Different pI calculators use different pKa sets. For example, ExPASy ProtParam uses the Bjellqvist set, while Bachem uses Lehninger values. Differences of ±0.5 pH units are common between tools — this reflects inherent uncertainty in theoretical pI estimation, not errors.
For experimental peptide work, the IPC_peptide set used here has been benchmarked as the most accurate for short peptides (RMSD ~0.25 pH units).