Peptide Fragment Ion m/z Calculator (b/y Ions, MS/MS Spectrum Annotation)

How Fragment Ions Are Generated

In liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments, peptides are activated in the collision cell (collision-induced dissociation CID, higher-energy collisional dissociation HCD), causing peptide bond cleavage and generating a series of fragment ions. Fragmentation occurs preferentially at amide bonds (CO—NH); upon cleavage:

• The N-terminal portion retains a positive charge → b ion
• The C-terminal portion, with addition of one water molecule, carries a positive charge → y ion

For a peptide with n residues, the theoretical fragments are b₁ through b_{n-1} and y₁ through y_{n-1}, giving 2(n−1) singly charged ions in total. b₁ and y₁ are typically very weak or absent in real spectra; b2, y2, and higher-numbered ions are the primary targets for analysis.

Core Formulas (Monoisotopic Mass)

Physical constants are sourced from the NIST Chemistry WebBook and the Unimod database:

• Proton mass: 1.007276 Da
• Water mass: 18.010565 Da
• Residue mass of each amino acid: neutral amino acid molecular weight minus one water molecule (i.e., the actual mass contribution of that residue in the peptide chain)

b ion (z = +1): sum of residue masses from position 1 to i, plus one proton:

b_i = residue_mass[1] + residue_mass[2] + ... + residue_mass[i] + 1.007276

y ion (z = +1): sum of residue masses for the last i C-terminal residues, plus water and one proton:

y_i = residue_mass[n-i+1] + ... + residue_mass[n] + 18.010565 + 1.007276

Multiply charged fragment ions (z ≥ 2), m/z as measured by the mass spectrometer:

b_i(z) = [ residue_sum(1..i)      + z × 1.007276 ] / z
y_i(z) = [ residue_sum(n-i+1..n)  + 18.010565 + z × 1.007276 ] / z

Complementary relationship: the b_i(+1) and y_{n-i}(+1) ions from the same cleavage site always sum to the neutral peptide mass plus two protons:

b_i + y_{n-i} = M_peptide + 2 × 1.007276
where M_peptide = Σ(all n residue masses) + 18.010565

This relationship can be used to quickly verify that calculations are correct.

Mass Shifts for Common Modifications

Carbamidomethylation of Cys (CAM): +57.021464 Da

Produced by alkylation of free thiols with iodoacetamide (IAA). Nearly all standard shotgun proteomics workflows set this as a fixed modification; the Cys residue mass increases from 103.009185 Da to 160.030649 Da. This tool selects this option by default. If your sample preparation did not include alkylation (e.g., protocols without a reduction step), change the setting to “None”.

Oxidation of Met: +15.994915 Da

The most common variable modification in sample preparation; the methionine side-chain sulfur atom is oxidized. Oxidized and unoxidized forms of a Met-containing peptide often appear simultaneously; database searches typically treat this as a variable modification. This tool supports specifying exact positions (1-based numbering) so you can verify a particular peptide form against a spectrum.

Applicability and Limitations

1. High-resolution instruments only (Orbitrap, Q-TOF, etc.), using monoisotopic masses. Ion trap and other low-resolution instruments require average masses; this tool does not perform that conversion.
2. b and y ions only. CID/HCD also produces a ions (b−CO, −27.995 Da), water-loss ions (−18.011 Da), ammonia-loss ions (−17.027 Da), and others; these neutral losses must be considered during spectrum annotation.
3. Standard 20 amino acids only; selenocysteine (U), pyrrolysine (O), and non-standard amino acids are not supported.
4. Linear peptides only; cyclic peptides or non-peptide bond linkages are not applicable.
5. L and I have identical masses (113.084064 Da); CID/HCD cannot distinguish them — this is an intrinsic limitation of the technique, not of this tool.

Validation Example

Using the classic test peptide PEPTIDE (no modifications, 7 residues), residue masses (Da):

P = 97.052764   E = 129.042593   P = 97.052764
T = 101.047679   I = 113.084064   D = 115.026943   E = 129.042593

Calculating b2 and y2 (charge +1):

b2 = 97.052764 + 129.042593 + 1.007276 = 227.102633 Da
y2 = 115.026943 + 129.042593 + 18.010565 + 1.007276 = 263.087377 Da

Complementary relationship check (b6 and y1):

b6 = 97.052764+129.042593+97.052764+101.047679+113.084064+115.026943 + 1.007276
   = 653.314083 Da

y1 = 129.042593 + 18.010565 + 1.007276 = 148.060434 Da

b6 + y1 = 801.374517 Da

M_peptide = 781.349400 + 18.010565 = 799.359965 Da
M + 2H    = 799.359965 + 2 × 1.007276 = 801.374517 Da  ✓

The two values agree exactly, confirming the formulas are correct.