What amino acid codes can I type into the calculator?

You can use the 20 standard one-letter codes: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y . The tool also accepts ambiguous codes B (Asx), Z (Glx), X (unknown), and U (selenocysteine), but these have uncertain masses and may reduce accuracy.

How do I enter more than one sequence at a time?

Put each sequence on its own line. If you want to name them, start a line with > followed by a label, then put the sequence on the next line. This is standard FASTA format. The calculator will process all sequences together and show results for each one.

What does the asterisk (*) do after S, T, or Y?

Typing * right after an S, T, or Y marks that residue as phosphorylated. The tool adds +79.966 Da (monoisotopic) to that position automatically. This is a quick shortcut so you do not need to add phosphorylation through the modification library.

What is the difference between monoisotopic mass and average mass?

Monoisotopic mass uses the lightest common isotope of each element (like ¹²C and ¹⁴N). It gives the sharpest, most exact value and is best for high-resolution instruments. Average mass blends all natural isotopes by their abundance. It works better for low-resolution instruments or very large proteins where individual isotope peaks merge together.

Why does the calculator add water to the mass?

When amino acids join together, each peptide bond loses one water molecule (H₂O). But the full peptide chain still has a free H at the N-terminus and a free OH at the C-terminus. So you add one water molecule (18.011 Da monoisotopic) to the sum of all residue masses to get the correct total mass.

What does missed cleavages mean?

Missed cleavages are cut sites that the enzyme skipped. In a real experiment, enzymes do not always cut at every possible spot. Setting missed cleavages to 1 or 2 shows you the longer peptide fragments that form when the enzyme fails to cut at one or two sites. A value of 0 means every site was cut perfectly.

How do I add a custom modification that is not in the list?

Select Custom… from the modification dropdown. Type a name for your modification, enter the mass change in Daltons, choose whether it applies to monoisotopic mass, average mass, or both, and list the target amino acids (or type "Any"). Then press Add Modification .

What is the difference between global and local modification location?

Global applies the modification to every matching residue in the sequence. Local applies it only to the specific positions you list. For example, if you pick phosphorylation with local positions 3 and 7, only the residues at positions 3 and 7 get the +79.966 Da shift.

Can I undo or remove a modification after adding it?

Yes. Every modification you add appears in the tracking table below the library section. Each row has a pencil icon to edit it and a trash icon to delete it. Clicking the pencil loads the modification back into the form so you can change it and re-add it.

What does the ion mode setting do?

Ion mode sets the charge state used to calculate the m/z value. [M+H]⁺ adds one proton for a singly charged ion. [M+2H]²⁺ adds two protons and divides by two. [M−H]⁻ removes one proton for negative mode. [M] gives the neutral mass with no charge adjustment.

How is m/z calculated from the neutral mass?

For [M+H]⁺ , the formula is m/z = M + 1.00728. For multiply charged ions like [M+2H]²⁺ , it is m/z = (M + 2 × 1.00728) ÷ 2. For [M−H]⁻ , it is m/z = M − 1.00728. The number 1.00728 is the mass of a proton.

What is the isotope distribution chart?

The isotope distribution chart shows the pattern of peaks you would see in a mass spectrometer. The tallest bar is the base peak (100%). Smaller bars at higher m/z values represent heavier isotope combinations (M+1, M+2, etc.) caused by natural amounts of ¹³C, ¹⁵N, and other heavy isotopes.

Why are some modifications marked with a warning triangle?

Modifications marked with ⚠ in the tracking table are flagged as incomplete or experimental. Their mass values come from limited data and may be less accurate. You must check the Show incomplete / experimental modifications box before you can select them from the dropdown.

What does the cysteine treatment option do?

It adds a mass shift to every cysteine residue to account for chemical treatment. For example, Iodoacetamide adds +57.021 Da per cysteine (carbamidomethylation). Choose None / Reduced if your cysteines are untreated and have free thiol groups.

How do I export my results?

After calculating, scroll to the Export section. Click Copy Results to copy a text summary to your clipboard. Click Export CSV to download the fragment table as a spreadsheet file. Click Chart SVG or Chart PNG to save the isotope distribution chart as an image.

What does the minimum and maximum mass filter do?

These filters hide digest fragments that fall outside your chosen mass range. Only peptides with a mass between the minimum and maximum values appear in the fragment table. This is useful when your instrument can only detect ions within a certain mass window.

What enzymes are available besides trypsin?

The calculator includes over 20 enzymes and chemical cleavage agents. Some examples are Lys C (cuts after K), Asp N (cuts before D), Chymotrypsin (cuts after F, Y, W), CNBr (cuts after M), Pepsin , Proteinase K , and Thermolysin . Choose None to skip digestion entirely.

What does the color-coded sequence visualization show?

The visualization highlights each modified residue with a colored background and marks every enzyme cleavage site with a vertical bar | . A legend below the sequence explains what each color means. This makes it easy to see at a glance where modifications and cuts fall along your peptide chain.

Does the calculator work on full proteins or only short peptides?

It works on both. You can enter sequences of any length, from a few amino acids up to full-length proteins with thousands of residues. For large proteins, you will usually want to pick an enzyme to digest the sequence into smaller fragments that a mass spectrometer can measure.

What does the instrument resolution setting do?

The instrument resolution setting is found under Advanced Instrument Settings . It lets you record the resolving power of your mass spectrometer, such as 60,000 for an Orbitrap. You can pick a preset or type a custom value. This helps you document which instrument conditions match your calculated results.

Why does my sequence show orange highlighted characters in the preview?

Orange highlighted characters are invalid . They are not recognized amino acid codes. Common mistakes include typing the number 1 instead of the letter I, or including spaces or punctuation. Remove or replace these characters so the calculator can process your sequence correctly.

Can I apply the same modification to only one chain of a multi-chain protein?

Yes. When you add a modification, type the chain name in the Subunit / Chain field. This labels the modification so you know which chain it belongs to. For single-chain inputs, leave this field blank.

Chemistry calculators

Peptide Mass Calculator

Updated Jul 25, 2026 By Jehan Wadia

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