Peptide mass calculator

In-silico proteolytic digestion with peptide mass calculation for mass spectrometry experiment planning.

Job name

Input

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What is a peptide mass calculator?

A peptide mass calculator performs in-silico proteolytic digestion: it takes a protein sequence, cleaves it at sites recognized by a chosen protease, and computes the molecular mass of each resulting peptide fragment. The output is a theoretical peptide mass fingerprint (PMF) — the set of masses expected from digesting a known protein with a specific enzyme.

Peptide mass fingerprinting is a core technique in mass spectrometry-based proteomics. Before running an experiment, researchers use in-silico digestion to predict which peptides a protein will produce, estimate whether those peptides fall within the instrument's detection range, and plan enzyme selection. After an experiment, comparing observed masses against theoretical digests helps confirm protein identity.

How to use the peptide mass calculator online

ProteinIQ's peptide mass calculator runs entirely in the browser — paste a sequence, pick an enzyme, and get results instantly. Multiple proteins can be digested in a single batch.

Input

Input	Description
`Protein sequence`	One or more protein sequences in FASTA format, raw single-letter code, or fetched from RCSB by PDB ID.

Enzyme selection

Each protease recognizes specific residues and cleaves the peptide backbone at predictable positions. The calculator includes 12 enzymes:

Enzyme	Cleavage rule
`Trypsin`	After K or R, unless followed by P (default)
`Trypsin (no P exception)`	After K or R, regardless of the next residue
`Lys-C`	After K
`Arg-C`	After R
`Asp-N`	Before D (N-terminal cleavage)
`Glu-C (phosphate)`	After E
`Glu-C (bicarbonate)`	After D or E
`Chymotrypsin`	After F, Y, W, M, or L, unless followed by P
`Pepsin (pH 1.3)`	After F or L
`Proteinase K`	After A, E, F, I, L, T, V, W, or Y
`CNBr`	After M (cyanogen bromide chemical cleavage)
`Formic acid`	After D

Trypsin is the most common choice in proteomics because it produces peptides in the 500–3000 Da range that ionize well in electrospray and MALDI sources.

Settings

Setting	Description
`Missed cleavages`	Number of internal cleavage sites the enzyme fails to cut (0–5, default 0). Real digests are rarely complete — setting 1 or 2 missed cleavages better reflects experimental conditions.
`Mass type`	`Monoisotopic` (default) uses the mass of the most abundant isotope of each element. `Average` uses the weighted mean across natural isotopic distributions. Monoisotopic is standard for high-resolution instruments (Orbitrap, FT-ICR); average suits lower-resolution data (older MALDI-TOF).
`Ion type`	Charge state for the reported mass. `[M+H]⁺` (singly protonated, default) is typical for MALDI. `[M+2H]²⁺` and `[M+3H]³⁺` reflect multiply charged species common in ESI. `[M]` reports neutral mass; `[M-H]⁻` is for negative-ion mode.
`Cysteine modification`	Chemical modification applied to all cysteine residues. `None (reduced)` leaves cysteines unmodified. Common alkylation reagents: `Iodoacetamide (CAM)` (+57.02 Da), `Iodoacetic acid (CM)` (+58.01 Da), `4-Vinylpyridine` (+105.06 Da), `Acrylamide` (+71.04 Da).
`Methionine oxidation`	When enabled, adds +16.00 Da to every methionine to account for oxidation, a common artifact in sample preparation.
`Min mass` / `Max mass`	Filter peptides outside a mass window (in Da). Useful for focusing on the detection range of a specific instrument, e.g., 500–4000 Da for MALDI-TOF.
`Sort by`	`Position in protein` (default) lists peptides from N-terminus to C-terminus. `Peptide mass` sorts ascending by mass.

Output columns

Column	Description
`Protein ID`	Identifier parsed from the FASTA header.
`Position`	Start and end residue numbers in the original protein (1-based).
`Peptide sequence`	Amino acid sequence of the cleaved fragment.
`Length`	Number of residues.
`Mass (Da)`	Calculated mass in Daltons, incorporating selected modifications and ion type.
`Missed cleavages`	Number of internal cleavage sites within this peptide (0 for fully cleaved fragments).

Results can be exported as CSV, JSON, or Excel.

How it works

The calculator applies three steps to each input protein:

Cleavage site identification: The sequence is scanned for residues matching the selected enzyme's specificity rules. Each rule is a combination of a residue match (e.g., K or R for trypsin) and an optional exception (e.g., not followed by P). Cleavage occurs either C-terminal or N-terminal to the matched residue, depending on the enzyme.
Peptide generation: The sequence is split at all identified cleavage sites to produce fully cleaved peptides (0 missed cleavages). When missed cleavages are allowed, adjacent peptide fragments are merged: 1 missed cleavage joins each pair of consecutive fragments, 2 missed cleavages joins triplets, and so on.
Mass calculation: Each peptide's mass is the sum of its residue masses plus one water molecule ( $\text{H}_2\text{O}$ = 18.011 Da monoisotopic) representing the free N- and C-termini. Modification delta masses are added per modified residue. The final mass is adjusted for the selected ion type by adding or removing proton masses and dividing by charge state where applicable.

All residue masses follow NIST standard values. Monoisotopic masses use the most abundant isotope of each element (e.g., $^{12}\text{C}$ , $^{1}\text{H}$ , $^{14}\text{N}$ , $^{16}\text{O}$ , $^{32}\text{S}$ ); average masses use the natural isotopic distribution weighted mean.

Limitations

Post-translational modifications beyond cysteine alkylation and methionine oxidation are not modeled. Glycosylation, phosphorylation, and other PTMs require manual mass adjustment.
Cleavage rules are deterministic. In practice, protease efficiency varies by local sequence context, tertiary structure accessibility, and digestion conditions. The calculator does not model incomplete digestion probabilities.
Non-specific cleavage (e.g., pepsin at higher pH values) is approximated by a simplified rule set rather than probabilistic models.