Simultaneous detection of N-terminal fragment ions in a protein mixture using a ruthenium(II) complex.
Use of a bis(terpyridine)ruthenium(II) derivative as an N-terminal labeling reagent resulted in the simultaneous detection and individual determination of all the N-terminal fragments of the proteins in a mixture without requiring any separation. All of the N-termini of the guanidinated proteins were labeled selectively by the ruthenium complex (<Ru>-CO-labeling). After chymotryptic digestion, the fragments were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and post-source decay (PSD). The <Ru>-CO moiety exclusively enhanced N-terminal fragment ions in mass spectra and enabled easy N-terminal sequencing. In a mixture containing three different proteins (lysozyme, ubiquitin, and insulin), all of the N-terminal fragment ions labeled with the ruthenium complex were found to produce uniformly intense peaks without the detection of the other unlabeled fragments. The N-terminal sequences of these ions were determined individually by PSD analysis. Application to unknown proteins from Thermus thermophilus HB8 with two-dimensional electrophoretic separation resulted in the successful determination of the N-terminal sequence and easy identification of the target protein.
Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Akihiro
A
Ito
Taka-aki
TA
Okamura
Hitoshi
H
Yamamoto
Norikazu
N
Ueyama
Minoru
M
Yamaguchi
Hiroki
H
Kuyama
Eiji
E
Ando
Susumu
S
Tsunasawa
Kojiro
K
Ake
Ryoji
R
Masui
Seiki
S
Kuramitsu
Takashi
T
Nakazawa
Shigemi
S
Norioka
D045424:Complex Mixtures / Q000032:analysis* / Q000737:chemistry; D007477:Ions; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012428:Ruthenium / Q000737:chemistry*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D045424:Complex Mixtures; D007477:Ions; D011506:Proteins; D012428:Ruthenium