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Monograph 0003 
Acetylation of Proteins and Peptides

Acetylation of proteins and peptides in the context of peptide MS/MS sequencing is performed for a number of reasons.  It allows one to count the number of lysine residues in a protein.  After acetylation the amino terminus of the protein should be modified giving the peptide a mass shift of 42 u, assuming of course that the N-terminal  residue is not blocked.  All internal lysine residues will also be acetylated.  If the mass shift of the peptide is 126 u following an acetylation then one may assume that there are three amino groups available for acetylation.  There are at least three common solutions for a mass shift of 126 u after an acetylation.  The peptide could contain two internal lysines and an amino terminus, or three internal lysines and a blocked amino terminus, or a lysine at the amino terminus with one internal lysine residue.  This is useful information in a de novo peptide MS/MS sequencing experiment.  In a method made famous by Donald F. Hunt, PhD at the University of Virginia acetylated and non-acetylated peptides are compared in MS CID fragmentation experiments and the acetylated peak shifts are noted.  

Another useful aspect of acetylation is that the amino acids Glutamine and Lysine have nearly isobaric mass and an acetylation experiment will differentiate them by 42 u.  One might assume that following a trypsin digest of a protein that all lysine residues would reside at the C-terminus of these tryptic peptides avoiding any ambiguity.  However there are instances where trypsin will not cleave after lysine and incomplete enzymatic cleavages are a common occurrence.  Therefore, one cannot assume that a mass difference of 128 u (residue mass for Q/K) in the middle of a tryptic peptide is a glutamine residue without first doing an acetylation.

Acetylation Protocol

  1. Make Acetylation Reagent.  20 無 Acetic Anhydride + 60 無 Methanol

  2. Reconstitute 1 nmol of peptide (or less) in 20無 of 50 mM ammonium bicarbonate.

  3. Add 50無 of acetylation reagent (from step 1) to 20無 of peptide solution (from step 2). Let stand at room temperature for one hour.

  4. Lyophilize to dryness.

  5. Analyze the product of the acetylation reaction by mass spectrometry.


Method Notes:  

1) Please note this protocol may also acetylate cysteine residues if they are not S-carboxymethylated first.

2) Acetylation using acetic anhydride and methanol can also lead to the formation of methyl esters. (This note provided by acetylation and mass spectrometry expert Mark S. Bolgar. Thanks Mark!)

Imortant : Acetic Anhydride is a very caustic reagent.  Consult your material safety data sheets (MSDS) and get the permission of your safety officer at you company or institution before performing this reaction.  Read our disclaimer, follow the link at the bottom of the page.  Always wear the appropriate safety apparel.


  1. The protocol above was adapted from am MS/MS peptide sequencing course taught by Donald F. Hunt, PhD at the University of Virginia.

  2. Hisada M, Konno K, Itagaki Y, Naoki H, Nakajima T.  Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom.  Rapid Commun Mass Spectrom. 2000;14(19):1828-34.  PMID: 11006592; UI: 20462980

  3. Suckau D, Mak M, Przybylski M.  Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping.  Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5630-4.  PMID: 1608973; UI: 92302295.  Download the article from PNAS

  4. Riordan, J.F. & Vallee, B. L. (1972) Methods Enzymol. 25, 494-499


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Last updated:  Saturday, February 17, 2007 10:20:33 PM