Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.
We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.
Kosaka
Toshiyuki
T
Biomedical Research Laboratories, Sankyo Co, Ltd, 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, Japan. kosaka@shina.sankyo.co.jp
Yoneyama-Takazawa
Tomoko
T
Kubota
Kazuishi
K
Matsuoka
Tatsuji
T
Sato
Ikuya
I
Sasaki
Takamitsu
T
Tanaka
Yorihisa
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017357:Cyclotrons; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014020:Tissue Extracts / Q000737:chemistry
D011506:Proteins; D014020:Tissue Extracts; D012710:Serum Albumin, Bovine