Asn / Gln deamidation
Asn (and to a lesser extent Gln) loses NH₃ to become Asp via the same succinimide intermediate as aspartimide formation. +1 Da, but accompanied by α/iso-Asp isomerization — very common in long peptides on storage.
Why it happens (mechanism)
Asn-Xxx backbone amide attacks the side-chain amide carbonyl, expelling NH₃ and forming a succinimide. Hydrolysis of the imide (route A vs. B) gives α-Asp (~25%) or iso-Asp (~75%). Net change: -NH₂ → -OH, so +0.984 Da. Gln does it via a 6-membered glutarimide, much slower than Asn.
When it strikes (triggers)
Asn followed by Gly is the textbook hot spot (also Ser/Thr/His). Basic pH > 7 accelerates 100-fold over neutral. Heat. Long aqueous storage. Note: Asn(Trt) is *not* immune — Trt removal during cleavage exposes the side chain, and even some Trt-protected forms cyclize directly under base.
How to spot it (MS signature)
+0.984 Da (or +0.98 in average mass). Hard to distinguish from natural ¹³C₁ isotope envelope at low resolution. HRMS / good MS1 resolution required. Iso-Asp is often invisible to trypsin (missed cleavage at iso-Asp-Xxx) — diagnostic in proteomics.
How to prevent it
- Avoid -NG- and -NS- sequences in design when feasible (replace with -QG- or -DG-).
- Keep cleavage cool (4 °C) and short (≤90 min) for Asn-rich peptides.
- Lyophilize promptly; store dry (NOT in solution) at -20 °C or -80 °C.
- Avoid pH > 7 in any handling buffer for Asn-rich peptides.
- Use Asn(Trt) and Gln(Trt) — keeps the side-chain amide masked through synthesis.
If it already happened (salvage)
- Not really salvageable — the +1 Da product *is* a different molecule (Asp instead of Asn). Re-synthesize with the precautions above, or accept and characterize the deamidation-prone hot-spots upfront.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 6, §6.2.