Standardisation of rapid in-gel digestion by mass spectrometry.
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.
Granvogl
Bernhard
B
Department für Biologie I, Ludwig-Maximilians-Universität, München, Germany.
Gruber
Patrick
P
Eichacker
Lutz Andreas
LA
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000737:chemistry*; D045332:Photosystem II Protein Complex / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000592:standards*; D011817:Rabbits
D009124:Muscle Proteins; D045332:Photosystem II Protein Complex