Pressure-assisted tryptic digestion using a syringe.
A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix-assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37 degrees C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure-assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure-assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA).
Yang
Hyo-Jik
HJ
Department of Chemistry, Chungnam National University, Daejeon, South Korea.
Hong
Jangmi
J
Lee
Sunyoung
S
Shin
Seongjae
S
Kim
Jinhee
J
Kim
Jeongkwon
J
D000097:Acetonitriles / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods; D011312:Pressure; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods; D013594:Syringes; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D000097:Acetonitriles; D009211:Myoglobin; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D014357:Trypsin; C032159:acetonitrile