Standardisation of rapid in‐gel digestion by mass spectrometry

Abstract In‐gel digestion has been standardised using a poly(propylene) disposable. We designed a four‐step rapid and simple in‐gel digestion protocol which is carried out in one self‐contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in‐gel digestion, we developed a rapid on‐column peptide acetylation protocol. Results show that trypsin in‐gel uptake is increased and in‐gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2‐D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.