N-α acetylation (residual acetic acid)

Free α-amine + residual acetic acid (from acetonitrile hydrolysis, or carry-over from purification) → N-α-acetyl peptide. +42 Da. Common artifact in HPLC-purified peptides.

Why it happens (mechanism)

AcOH + amine + acid catalyst → amide (slow at RT, faster hot). Acetonitrile + water/acid hydrolyzes to AcOH; HPLC mobile phase containing acetonitrile → trace AcOH → acetylation during concentration steps.

When it strikes (triggers)

MeCN/TFA HPLC + slow concentration. Trace AcOH in DMF/NMP. N-terminal Gly is more reactive than bulkier Xxx. Stored in glass vials with rubber septa (acetic acid leaching).

How to spot it (MS signature)

+42.01 Da. Distinguish from +42 'allylation' or 'over-tBu' by location (only at N-terminus, only on free α-amine peptides).

How to prevent it

• Quench TFA with ether precipitation, then redissolve in cold water — minimize acetic acid exposure during workup.
• Use methanol-based HPLC (instead of MeCN) for Asp/Glu-rich or N-terminal-amine-free peptides — but caution for esterification side reaction.
• Lyophilize quickly; store as lyophilized powder, not solution.

If it already happened (salvage)

• Acetyl is hard to remove cleanly. Re-synthesize.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 7, §7.1.