Peptide Mass Calculator

The default of 111.1 Da is the average residue mass (amino acid minus water). In contrast, the protein MW calculator uses ~128.16 Da for the full amino acid mass. The difference is because this calculator adds back one terminal water separately and subtracts (n-1) water molecules for peptide bonds, while the protein calculator subtracts water from the full amino acid masses.

In ESI-MS, peptides acquire multiple protons to form [M+zH]^z+ ions. Higher charge states produce lower m/z values. For example, a 2000 Da peptide appears at m/z 1001 for z=2, m/z 668 for z=3, and m/z 501 for z=4. Most mass spectrometers have an optimal m/z range of 200-2000, so multiply charged ions are essential for detecting larger peptides.

PMF is a protein identification technique where a protein is digested with a specific protease (usually trypsin), the peptide masses are measured by mass spectrometry, and the experimental mass list is compared against theoretical digests of all proteins in a database. Accurate mass measurement (within 10-50 ppm) is crucial for reliable identification.

Disulfide bonds can be determined by: (1) comparing reduced vs. non-reduced mass — each broken disulfide adds 2.016 Da, (2) alkylation assays with iodoacetamide that cap free cysteines (+57.02 Da each), (3) Ellman's assay for free thiols, or (4) structural prediction from sequence analysis and homology modeling.

Common mass shifts include: methylation (+14.016 Da), dimethylation (+28.031), oxidation of Met (+15.995), deamidation of Asn/Gln (+0.984), ubiquitination via GlyGly tag (+114.043), SUMOylation, glycosylation (variable, +162+ Da per hexose), and palmitoylation (+238.23). Each PTM creates a characteristic mass shift detectable by MS.

The proton mass is 1.00728 Da (atomic mass units), slightly more than 1.00000 Da. This difference arises from nuclear binding energy. While the difference seems negligible, it becomes significant for multiply charged ions: for z=10, the correction is 0.0073 Da per m/z unit, which matters for high-resolution mass spectrometry with sub-ppm accuracy.

Cyclic peptides form an additional bond (removing one more water molecule) compared to linear peptides of the same sequence. Subtract 18.015 Da from the calculated mass for head-to-tail cyclization. For other cyclization types (disulfide, thioether, lactone), the mass loss varies. Many antimicrobial peptides and natural products are cyclic.

For confident peptide identification: PMF requires 10-50 ppm accuracy, LC-MS/MS with fragmentation requires 5-20 ppm for precursor and 0.02-0.5 Da for fragments, and top-down proteomics requires sub-ppm accuracy. Modern Orbitrap and FT-ICR mass spectrometers routinely achieve 1-5 ppm accuracy.

Stable isotope labeling shifts peptide masses predictably: SILAC uses ¹³C₆-Lys (+6.020 Da) and ¹³C₆¹⁵N₄-Arg (+10.008 Da), TMT tags add ~229 Da per peptide, and ¹⁸O labeling during digestion adds +2.004 or +4.008 Da. These mass shifts enable quantitative proteomics by comparing labeled and unlabeled peptide pairs.