Deamidation: Asparagine and Glutamine Mechanism, Rates and Detection

Short answer: Deamidation is a spontaneous, non-enzymatic reaction in which the side-chain –NH2 of an asparagine (Asn) or glutamine (Gln) residue is replaced by –OH, turning it into an acidic residue [1]. In peptides at neutral pH, asparagine usually reacts through a five-membered succinimide ring that then opens to give a mixture of isoaspartate and aspartate, in a ratio of roughly 3:1 [1][3]. Each deamidation adds +0.984 Da to the molecule's mass and introduces a negative charge [1].

This article is educational chemistry for laboratory readers. It does not describe effects in people or animals and is not guidance for human or animal use.

What deamidation is

Deamidation of asparagine is one of the most frequent modifications seen in proteins and peptides [1]. It happens on its own, without an enzyme, and it is a major chemical pathway by which peptides and proteins degrade [1][4].

Two residues are involved:

Asparagine (Asn, N): the faster and far more studied case [1][6].
Glutamine (Gln, Q): a similar series of reactions occurs, but it is much slower than for asparagine [1]. Because the rate is so much lower, Gln deamidation has been studied far less [6].

Aspartic acid (Asp) residues can follow a closely related route. A similar attack on the Asp side chain leads to dehydration and the same succinimide intermediate [1]. Asn residues form succinimides about 10 times faster than comparable Asp residues [1]. In one model hexapeptide, swapping the Asn for Asp cut the rate of succinimide formation 34-fold [2]. (Our amino acid codes guide lists the one- and three-letter symbols used here.)

The deamidation mechanism, step by step

At physiological pH and temperature, asparagine deamidation runs through a ring intermediate [1]:

Ring closure: the nitrogen atom of the next peptide bond (on the C-terminal side of Asn) attacks the carbonyl group of the Asn side chain, forming a five-membered succinimide ring [1]. (See our peptide bond guide for how that backbone link is built.)
Ring opening: the succinimide is then hydrolyzed fairly quickly at either of its two carbonyl groups [1]. This gives L-isoaspartate (isoAsp) and normal L-aspartate in a ratio of approximately 3:1 [1].
Racemization: the succinimide can also racemize at its alpha-carbon, producing D-succinimidyl, D-aspartyl and D-isoaspartyl forms [1].

An isoaspartyl site is a beta-linked Asp–Xaa peptide bond, an atypical link in the backbone [12]. The net result is a mixture of L- and D-forms of normal and iso-aspartyl peptides [2]. (For more on D-forms, see our D-amino acids guide.)

A classic model study measured how fast each step goes. The hexapeptide Val-Tyr-Pro-Asn-Gly-Ala deamidated with a half-life of only 1.4 days at 37 °C and pH 7.4, giving an aspartyl succinimide [2]. Under the same conditions the succinimide was hydrolyzed with a half-time of 2.3 hours and racemized with a half-time of 19.5 hours [2].

Why pH changes the route

The pathway depends on pH:

Neutral to alkaline pH: in the model hexapeptide Val-Tyr-Pro-Asn-Gly-Ala, deamidation between pH 5 and 12 ran entirely through the cyclic imide intermediate, giving both Asp and isoAsp peptides [4].
Acidic pH: the route switched to direct hydrolysis of the Asn side-chain amide, which gave only the Asp peptide [4]. A separate pentapeptide series showed the same pattern, with the Asp peptide as the only product in acid [3].

The same hexapeptide study found marked dependence on pH, temperature and buffer composition, with buffer catalysis between pH 7 and 11 [4].

What controls the rate

The next residue in the sequence

The residue on the C-terminal side of Asn matters most [3]. In pentapeptides tested at neutral and alkaline pH, bigger and more branched side chains in that position slowed deamidation by as much as 70-fold compared with glycine [3]. The Asn-Gly (N-G) sequence had the greatest rate [3]. In the hexapeptide model, replacing the glycine after Asn with a bulky leucine or proline cut the degradation rate 33- to 50-fold [2].

The residue on the N-terminal side of Asn had little or no effect on the rate in neutral and alkaline solutions [3]. In acidic solution, the sequence did not affect the rate at all [3].

Peptide bond cleavage at Asn

Deamidation is not the only reaction at asparagine. Every pentapeptide in which Ser followed Asn also underwent a peptide cleavage reaction in neutral and alkaline solutions, splitting into a tripeptide and a dipeptide [3]. That cleavage ran at about 10% of the deamidation rate [3]. Cleavage products were also seen for the Asn-Leu and Asn-Pro hexapeptides [2]. (See our dipeptide and tripeptide guides for those terms.)

Buffers, salts and solvents

Buffers: a high concentration of organic and inorganic buffers strongly increased the rate in the model peptide Boc-Asn-Gly-Gly-NH2 [7]. General base catalysis was observed for small bases such as ammonia [3].
Ionic strength: the deamidation rates of the pentapeptides were not affected by ionic strength [3].
Organic solvents: adding organic solvents to an aqueous solution of the peptide generally decreased the reaction rate [7]. Aprotic salts and polyethylene glycol had only a minor influence [7].

Structure

One study measured deamidation rates for 306 asparaginyl sequences in model peptides at pH 7.4 and 37 °C, and set up a library of 913 amide-containing peptides for other researchers [5]. The same authors also compared a whole protein (rabbit muscle aldolase) with matching model peptides to measure how protein structure changes the rate [5]. Across peptides and proteins, deamidation half-times under physiological conditions range from about 1 to 1,000 days [1].

Deamidation in solid and lyophilized samples

Water content still matters in dried samples. Two patterns have been reported in freeze-dried amorphous proteins and peptides [8]:

a rate constant that rises steadily as water content rises, and
a "hockey stick" pattern, where the rate barely depends on water at low water content and then speeds up above a threshold [8].

In one solid-state study, model peptides were incorporated into lyophilized poly(vinyl pyrrolidone) solids [9]. Their observed deamidation rate constants dropped by four orders of magnitude in glassy solids, which pointed to matrix mobility as a key factor [9]. In the same work, cyclic beta-turn peptides degraded 1.2 to 8 times more slowly than their linear analogs [9]. (Our lyophilized peptides guide covers freeze-drying in more detail.)

How deamidation is detected

Mass spectrometry: a deamidated peptide is 0.984 Da heavier than the original, the mass difference between –OH and –NH2 groups [1]. Because the modification is stable in the gas phase, tandem MS spectra can locate the deamidated site [1]. (See our mass spectrometry peptide testing guide.)
Asp vs isoAsp: isoaspartyl and aspartyl forms have no mass difference, so telling them apart by mass alone is challenging [10]. Special methods, such as enzyme-mediated 18O labeling, have been developed to flag the isoAsp form [10].
Charge-based separation: deamidation adds a negative charge, and charge isoforms of a protein can be resolved by cation-exchange HPLC [1]. (Our isoelectric point guide explains why an extra negative charge shifts a peptide's behavior.)
Peptide mapping: in a common workflow, the protein is digested with trypsin and the peptides are separated by reverse-phase HPLC before MS analysis [1]. (See our what is HPLC peptide testing guide.)

One analytical caution: deamidation was found to increase after reduction, alkylation and tryptic digestion, the standard proteomics sample-preparation steps [1]. Standard sample preparation can therefore add deamidation of its own, a warning for straightforward attempts to measure it [1].

Deamidation as a peptide impurity

A review of related impurities in peptide medicines lists succinimide formation among the typical peptide degradation mechanisms, alongside beta-elimination, diketopiperazine and pyroglutamate formation [11]. The review notes that these degradation-related impurity types can also be found in finished peptide drug products [11]. (For how impurities show up in a purity figure, see our peptide purity guide.)

FAQ

What is deamidation in simple terms?
It is a spontaneous, non-enzymatic change in which the side-chain –NH2 of an asparagine or glutamine residue is replaced by –OH, giving an acidic, negatively charged residue [1].

What does asparagine become after deamidation?
A mixture dominated by L-isoaspartate, with normal L-aspartate in a ratio of roughly 3:1 at neutral pH, plus smaller amounts of D-forms [1][3].

Which sequence deamidates fastest?
In the pentapeptide series studied, Asn followed by glycine (N-G) had the greatest rate [3].

How much does deamidation change a peptide's mass?
+0.984 Da per deamidated residue [1].

Does glutamine deamidate too?
Yes, but much more slowly than asparagine [1][6]. Its products are alpha- and gamma-glutamic acid [6].

Bottom line

Deamidation turns asparagine (and, much more slowly, glutamine) into an acidic residue [1]. At neutral pH it runs through a succinimide ring that opens to roughly 3 parts isoaspartate to 1 part aspartate [1][3], while in acid it goes by direct hydrolysis to aspartate only [4]. The residue after Asn sets most of the rate, with N-G the fastest in the series studied [3]. Water content and matrix mobility shape the rate in solid samples [8][9]. Analysts detect it by a +0.984 Da mass shift and a new negative charge, though isoAsp and Asp share the same mass [1][10].

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