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General reference for researchers and laboratory staff; not a handling protocol or safety data sheet. Follow your institution’s chemical hygiene plan and the supplier’s safety data sheet.
Short answer
Iodoacetamide is an alkylating reagent used in proteomics to cap free cysteine thiols after disulfide bonds are reduced. The cap it leaves, carbamidomethyl, adds 57.021464 Da to cysteine and is the standard fixed modification in database searches. It is also a toxic, sensitising chemical, and a 2017 systematic comparison found it alkylates methionine and other residues off-target.
Definition and scope
Iodoacetamide (2-iodoacetamide) has the formula C2H4INO and a molecular weight of 184.96, with CAS number 144-48-9 (PubChem compound 3727, read 2026-11-07). In mass-spectrometry sample preparation it is one of several cysteine-alkylating reagents. This page covers what the reagent does, how that appears in search settings, how it compares with other alkylating agents, and its hazard classification. It deliberately gives no concentrations, incubation times or bench steps; those belong in a validated laboratory protocol.
How it is used in a proteomics workflow
Cysteine residues can form disulfide bonds. In a bottom-up workflow the disulfides are first reduced (typically with DTT, TCEP or a thiol such as b-mercaptoethanol), then the freed thiols are alkylated so they cannot re-form bonds. Muller and Winter describe reduction and alkylation of cysteine as part of virtually any proteomics workflow (Mol Cell Proteomics 2017). For where this step sits relative to digestion and cleanup, see the sample-preparation section of choosing DDA or DIA, sample prep and run QC.
What carbamidomethyl means in the search
Unimod entry 4, Carbamidomethyl, is described as an iodoacetamide derivative with composition H(3)C(2)NO and a monoisotopic mass of 57.021464 Da. Its classified site is cysteine; Unimod also lists artefact sites including lysine, the peptide N-terminus, histidine, aspartate, glutamate, serine, threonine and tyrosine (read 2026-11-07). This is why a search engine is told to expect carbamidomethyl-cysteine as a fixed modification, and why unexpected +57 Da shifts on other residues point to over-alkylation. The search-configuration side is covered in protein identification by database search.
Iodoacetamide compared with other alkylating reagents
The most complete head-to-head evidence is Muller and Winter (Mol Cell Proteomics 2017;16(7):1173-1187; PMID 28539326). They compared four alkylating reagents on HeLa cell digests across 13 reduction and alkylation conditions. The table restates only what that study reports.
| Reagent | Iodine-containing | What the study reported |
|---|---|---|
| Iodoacetamide | Yes | Alkylates methionine (carbamidomethylated side chain), producing a prominent neutral loss and fewer identified methionine-containing peptides |
| Iodoacetic acid | Yes | Same methionine problem, via carboxymethylated side chains |
| Chloroacetamide | No | Included in the comparison; grouped with the non-iodine reagents in the iodine versus non-iodine contrast |
| Acrylamide | No | Gave the best results as alkylation reagent |
The authors observed off-site alkylation at seven amino acids and the peptide N-terminus, and more than 9-fold differences in identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and non-iodine-containing reagents. These results come from one cell line and specific conditions; treat them as a reason to check methionine behaviour in your own data, not as a universal ranking.
Hazard classification and lab management
PubChem’s aggregated GHS classification (read 2026-11-07) carries the signal word Danger. Percentages are the share of ECHA notifications reporting each statement:
- H301, toxic if swallowed (99%)
- H317, may cause an allergic skin reaction (93.8%)
- H334, may cause allergy or asthma symptoms or breathing difficulties if inhaled (89.6%)
- H413, may cause long-lasting harmful effects to aquatic life (61.5%)
For chemical inventory, labelling and waste decisions, the classification above means this is a sensitiser and an acute oral toxicant, so it belongs in the laboratory’s hazardous-chemical register with the supplier’s current safety data sheet attached.
Common misunderstandings
- “Alkylation only touches cysteine.” Unimod lists artefact sites beyond cysteine and Muller and Winter measured off-target modification at seven amino acids plus the N-terminus.
- “The conventional reagent is the best-performing one.” Iodoacetamide is conventional, but in that study a non-iodine reagent (acrylamide) gave the best results.
- “A +57 Da shift is always a cysteine.” It is the fixed modification for cysteine, but the same mass on other residues is an off-target product.
FAQ
What does iodoacetamide do in proteomics?
It alkylates reduced cysteine thiols, forming carbamidomethyl-cysteine (+57.021464 Da) so disulfide bonds cannot re-form before digestion and analysis.
What is carbamidomethyl?
The modification iodoacetamide leaves on cysteine, Unimod entry 4, composition H(3)C(2)NO. It is normally set as a fixed modification in the search.
Is iodoacetamide hazardous?
Yes. Aggregated GHS notifications on PubChem include toxic if swallowed, skin sensitisation, respiratory sensitisation and long-lasting aquatic harm, with the signal word Danger.
Are there alternatives?
Iodoacetic acid, chloroacetamide and acrylamide were compared with it by Muller and Winter (2017); acrylamide gave the best results in that study.
Sources
- PubChem compound 3727, Iodoacetamide (NCBI), read 2026-11-07.
- Unimod entry 4, Carbamidomethyl, read 2026-11-07.
- Muller T, Winter D. Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents. Mol Cell Proteomics 2017;16(7):1173-1187. PMID 28539326, read 2026-11-07.
