Before you start

Reagents and materials

Totals are for 6 samples (+10% extra). Per-sample (≤ 100 µg) amounts are in the middle column.

Reagent	Per sample (≤ 100 µg)	Total
Protein in 8 M urea / 50 mM ABC (≤ 100 µg)	50 µL	50 µL
DTT, 100 mM (5–10 mM final)	3 µL	3 µL
Iodoacetamide, 200 mM, fresh and dark (15–20 mM final)	5 µL	5 µL
50 mM ABC (dilute urea below 1–2 M)	300 µL	300 µL
Trypsin, MS-grade (1:50 enzyme : protein)for 100 µg protein	2 µg	2 µg
Formic acid or TFA (acidify to pH < 3)	4 µL	4 µL

NH₄HCO₃	0.395 g
LC-MS-grade water	to 100 mL

Make fresh (pH ≈ 7.8–8.0; do not adjust).

Make fresh: aged urea forms cyanate, which carbamylates proteins. Do not heat above 37 °C.

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

Denature
Bring protein to 8 M urea in 50 mM ABC or Tris pH 8.
Reduce
Add DTT to 5–10 mM and incubate 30–60 min at 37 °C (not higher in urea).
Alkylate
Add iodoacetamide to 15–20 mM and incubate 30 min at room temperature in the dark.
Quench (optional)
Add DTT to quench excess iodoacetamide.
Dilute
Dilute with 50 mM ABC so urea is below 1–2 M.
Digest
Add trypsin at 1:50–1:100 (w/w) and incubate overnight (12–18 h) at 37 °C. A Lys-C pre-digest at 4 M urea improves efficiency.
Stop
Acidify with formic acid or TFA to pH < 3 (about 0.5–1%).
Desalt
Desalt peptides on C18 (StageTips or cartridges) before LC-MS/MS.

Avoid detergents (SDS, NP-40) and PEG in samples for LC-MS; if lysis needs SDS, use S-Trap, SP3 or FASP.
Wear gloves and clean surfaces to avoid keratin contamination.

Problem	What to check or change
Many missed cleavages	Lower urea before trypsin, check trypsin activity, extend digestion.
Carbamylation artefacts	Use fresh urea, avoid heating, keep incubation at ≤ 37 °C.
Polymer peaks	Remove detergents/PEG; use clean plasticware.

Iodoacetamide is toxic; TFA and formic acid are corrosive.