Key points
- A post-translational modification (PTM) is a chemical change made to a peptide or protein after the ribosome has built it. Hundreds are known; a few dozen account for most of what is seen in practice.
- Each modification has a characteristic mass shift, which is how modifications are detected. Acetylation (+42.011) and trimethylation (+42.047) differ by only 0.036 Da and need high resolution to separate.
- Many bioactive peptides are only active after modification. C-terminal amidation, needed by roughly half of all peptide hormones, is the clearest example.
- Modifications change charge, mass and hydrophobicity, so they shift the values every calculator on this site reports for the unmodified sequence.
Why modifications matter
The genetic code specifies 20 amino acids. That is a limited chemical palette: no phosphate groups, no lipids, no sugars, no way to switch a residue's charge on demand. Post-translational modification extends it enormously, and it does so reversibly, which is what makes signalling possible.
For peptides specifically, modification is often the difference between an inert chain and an active hormone. A precursor protein is cleaved into fragments, the fragments are modified, and only then does a functional peptide exist. Understanding this sequence of events explains why a peptide synthesised from the gene-encoded sequence alone may be completely inactive.
Mass shifts of common modifications
Because a modification adds or removes atoms, it changes the peptide's mass by a fixed amount. This table gives the monoisotopic shifts used when searching mass spectra. The wider context of how these are detected is in the guide to peptide mass spectrometry.
| Modification | Residues | Δ mass (Da) | Effect on charge |
|---|---|---|---|
| C-terminal amidation | C-terminus | −0.984 | Removes one negative charge |
| Deamidation | Asn, Gln | +0.984 | Adds one negative charge |
| Citrullination | Arg | +0.984 | Removes one positive charge |
| Methylation | Lys, Arg | +14.016 per methyl | Keeps the positive charge |
| Hydroxylation | Pro, Lys | +15.995 | None |
| Acetylation | Lys, N-terminus | +42.011 | Removes one positive charge |
| Trimethylation | Lys | +42.047 | Fixes a permanent positive charge |
| γ-Carboxylation | Glu | +43.990 | Adds one negative charge |
| Sulfation | Tyr | +79.957 | Adds one negative charge |
| Phosphorylation | Ser, Thr, Tyr | +79.966 | Adds up to two negative charges |
| Ubiquitination remnant after trypsin | Lys | +114.043 | Removes one positive charge |
| O-GlcNAc / HexNAc | Ser, Thr, Asn | +203.079 | None |
| Farnesylation | Cys | +204.188 | None; adds strong hydrophobicity |
| Myristoylation | N-terminal Gly | +210.198 | Removes the N-terminal charge |
| Palmitoylation | Cys | +238.230 | None; adds strong hydrophobicity |
Three pairs in this table are traps. Acetylation and trimethylation differ by 0.036 Da and are routinely confused on low-resolution instruments. Sulfation and phosphorylation differ by 0.009 Da, which is beyond most instruments entirely; the two are usually told apart by behaviour instead, since sulfate is lost very easily during fragmentation while phosphate is more stable. And deamidation and citrullination both add 0.984 Da, but they happen on different residues and have opposite effects on charge.
C-terminal amidation
Roughly half of all known peptide hormones end in an amide rather than a free acid, among them oxytocin, vasopressin, substance P, calcitonin, gastrin and many insect and amphibian peptides. The modification is almost always essential: the amidated form can be orders of magnitude more potent than the free acid.
Biosynthesis is indirect. The precursor carries an extra glycine at the C-terminus, and a bifunctional enzyme, peptidylglycine α-amidating monooxygenase (PAM), converts that glycine into the amide. The copper- and ascorbate-dependent first step hydroxylates the glycine α-carbon; the second cleaves it, releasing glyoxylate and leaving the amide behind. A peptide whose gene sequence ends in Gly-Lys-Arg is therefore a strong candidate for an amidated hormone.
For anyone working with such peptides, the practical consequences are concrete. The amide is 0.984 Da lighter than the acid, so the calculated molecular weight must be corrected. It also removes an acidic group, which can shift the isoelectric point by several units: linear oxytocin has a calculated pI near 5.4, while the native amidated and disulfide-closed hormone is near 9. In synthesis, the amide comes from the choice of resin, as described in the SPPS guide.
Phosphorylation
Phosphorylation is the most studied modification in biology, and the central mechanism of cellular signalling. A kinase transfers the γ-phosphate of ATP onto a hydroxyl group, and a phosphatase removes it. The human genome encodes over 500 kinases and around 200 phosphatases.
In animals, phosphorylation occurs mainly on serine, threonine and tyrosine, in roughly a 90:10:0.05 ratio. Tyrosine phosphorylation is rare but disproportionately important, since it controls growth factor receptor signalling and is the target of many cancer drugs.
The phosphate group is doubly ionisable, with a first pKa near 1–2 and a second near 5.5–6.5, so at physiological pH it carries close to two negative charges. That is a substantial change to a peptide's electrostatics, and it is why phosphorylation can switch a binding site on or off.
Analytically, phosphopeptides present specific difficulties. They ionise less efficiently than their unmodified counterparts and are often suppressed in a complex mixture, so they are usually enriched first, typically with titanium dioxide or immobilised metal affinity chromatography. During CID fragmentation, phosphoserine and phosphothreonine readily lose phosphoric acid (−97.977 Da), which produces a characteristic neutral loss peak but can also leave too little information to pinpoint the modified residue. Electron-based fragmentation, ETD or EThcD, is generally preferred for localisation.
Modifications of lysine
The lysine side chain, with its single reactive primary amine, carries more different modifications than any other residue. Several are central to the regulation of chromatin:
- Acetylation neutralises the positive charge. On histones this loosens their grip on DNA and is generally associated with active transcription.
- Methylation can be mono-, di- or trimethyl and does not neutralise the charge. Its meaning depends entirely on position: trimethylation of lysine 4 of histone H3 marks active promoters, while trimethylation of lysine 9 marks silenced chromatin.
- Ubiquitination attaches the small protein ubiquitin through an isopeptide bond to the lysine ε-amine. Chains of ubiquitin mark proteins for degradation by the proteasome. After a tryptic digest, a two-residue remnant stays behind, adding 114.043 Da, which is how ubiquitination sites are mapped.
- Hydroxylation of lysine, and of proline, is essential in collagen. Both require vitamin C as a cofactor, and the failure of collagen cross-linking without it is the biochemical basis of scurvy.
Lipid modifications
Attaching a fatty acid or isoprenoid converts a soluble peptide into one that partitions into membranes. Myristoylation adds a 14-carbon acyl group to an N-terminal glycine and is usually permanent; palmitoylation adds a 16-carbon group to a cysteine through a thioester and is reversible, which allows proteins to cycle on and off membranes.
Lipidation has become a major strategy in peptide drug design for a different reason. Attaching a fatty acid lets a peptide bind reversibly to serum albumin, which acts as a circulating reservoir and dramatically extends the plasma half-life. This is the principle behind several long-acting peptide therapeutics.
What modifications do to calculated values
Every tool on this site computes values for the unmodified linear sequence. Modifications change those values in predictable ways:
| Modification | Mass | pI and net charge | Hydrophobicity |
|---|---|---|---|
| C-terminal amide | −0.984 Da | Removes an acidic group; pI rises | Slightly more hydrophobic |
| N-terminal acetyl | +42.011 Da | Removes a basic group; pI falls | Slightly more hydrophobic |
| Phosphorylation | +79.966 Da | Adds up to two negative charges; pI falls sharply | More hydrophilic |
| Lysine acetylation | +42.011 Da | Removes a positive charge; pI falls | More hydrophobic |
| Disulfide bond | −2.016 Da per bond | Removes two thiols; pI usually rises | Not reflected in a linear profile |
| Lipidation | +204 to +238 Da | Little direct effect | Much more hydrophobic; aggregation risk |
The hydrophobicity column is worth a caveat. The Kyte–Doolittle scale assigns values to the 20 standard residues only, so a modified residue keeps its unmodified score. For phosphorylated or lipidated peptides, a hydropathy profile understates the change considerably.
Modifications that are artefacts
Not every mass shift found in a sample is biology. Several of the most frequently observed ones are introduced during handling or analysis:
- Methionine oxidation (+15.995) occurs readily in air and during sample preparation. Its presence in a sample says more about storage than about the cell it came from.
- Deamidation (+0.984) accelerates during long digestions at pH 8, a standard proteomics condition.
- Carbamidomethylation (+57.021) is introduced deliberately to block cysteines, but iodoacetamide can also react with other residues if used in excess or for too long.
- Carbamylation (+43.006) comes from cyanate in old or warmed urea solutions and modifies lysines and the N-terminus. It is very close in mass to γ-carboxylation and to trimethylation plus a proton, so it is a genuine source of misassignment.
- Sodium and potassium adducts (+21.982 and +37.956) are not modifications at all, only ions carried along with the peptide.
Distinguishing real modifications from artefacts is mostly a matter of sample history: which buffers, how long, at what pH and temperature, described further in the stability and storage guide.
Frequently asked questions
How many post-translational modifications exist?
The Unimod database, the standard reference for mass spectrometry, lists well over a thousand entries, though many are chemical derivatisations and artefacts rather than biological modifications. A few dozen account for the overwhelming majority of biologically important cases.
Are modifications reversible?
Some are, and that is their purpose: phosphorylation, acetylation, methylation, ubiquitination and palmitoylation all have dedicated enzymes that remove them. Others, such as amidation, γ-carboxylation and myristoylation, are effectively permanent.
Can modified peptides be synthesised chemically?
Yes. Phosphorylated, acetylated, methylated and lipidated residues are available as protected building blocks for Fmoc synthesis, and amidation follows from the choice of resin. Glycosylation is far harder and usually requires specialist chemistry.
Why does my synthetic peptide not match the natural one?
The most common reasons are a missing C-terminal amide, missing disulfide bonds, or a modification such as pyroglutamate at the N-terminus. Compare the measured mass with the calculated one and see whether the difference matches a shift in the table above.
References
- Walsh CT, Garneau-Tsodikova S, Gatto GJ (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angewandte Chemie International Edition 44:7342–7372.
- Eipper BA, Stoffers DA, Mains RE (1992) The biosynthesis of neuropeptides: peptide α-amidation. Annual Review of Neuroscience 15:57–85.
- Olsen JV, Blagoev B, Gnad F, et al. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648.
- Creasy DM, Cottrell JS (2004) Unimod: protein modifications for mass spectrometry. Proteomics 4:1534–1536.