Identification of human liver diacetyl reductases by nano-liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry.
Several forms of diacetyl-reducing enzyme were found to exist in the human liver cytosol. Three (DAR-2, DAR-5, and DAR-7) of them were purified as a single band on SDS-PAGE by a combination of a few kinds of column chromatographies. The in-gel tryptic digests of the purified enzymes were analyzed by nano-liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), which provided peptide masses at a ppm-level accuracy. The enzymes, DAR-2, DAR-5, and DAR-7, were identified as alcohol dehydrogenase beta subunit (ADH2), carbonyl reductase (CBR1), and aldehyde reductase (AKR1A1), respectively, by peptide mass fingerprinting. In addition, an alternating-scan acquisition of nano-LC/FT ICR mass spectra, i.e., switching of normal acquisition conditions and in-source fragmentation conditions scan by scan, provided sets of parent and fragment ion masses of many of the tryptic peptides in a single LC/MS run. The peptide sequence-tag information at the ppm-level accuracy was used to further confirm the protein identities. It was demonstrated that nano-LC/FT ICR MS can be used for rigorous protein identification at a subpicomole level as an alternative technique to nano-LC/MS/MS.
Tanaka
Y
Y
Biopharmaceutics, Tohoku Pharmaceutical University, 4-1, Komatsushima 4-chome, Aoba-ku, Sendai, 981-8558, Japan. ytanaka@tohoku-pharm.ac.jp
Sato
I
I
Iwai
C
C
Kosaka
T
T
Ikeda
T
T
Nakamura
T
T
D000094:Acetoin Dehydrogenase / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019992:Databases as Topic; D005583:Fourier Analysis; D006801:Humans; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D021241:Spectrometry, Mass, Electrospray Ionization
D000094:Acetoin Dehydrogenase