High sequence-coverage detection of proteolytic peptides using a bis(terpyridine)ruthenium(II) complex.
The use of a bis(terpyridine)ruthenium(ii) complex for peptide labeling (Ru-CO labeling) supplied high intensity peaks in mass spectrometry (MS) analysis that overcame the contribution of protonation or sodiated adduction to peptides. Ru-CO-labeled insulin A- and B-chains were detected simultaneously in comparable peak abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The mass spectra of chymotryptic peptide fragments of Ru-CO-labeled insulin also simultaneously indicated both N-terminal fragment ions, and amino acid sequences were determined easily by matrix-assisted laser desorption/ionization post-source-decay (MALDI-PSD). The sensitivity of detecting Ru-CO-labeled peptide fragment ions was not dependent on the length or the sequences of the peptides. The Ru-CO labeling method was applied to tryptic myoglobin fragments. The method indicated that each fragment ion is detected nearly equal in abundance and enabled the desired fragment ions to be distinguished from matrix clusters or their in-source fragments in lower mass regions. The desired fragment ions can be found in the mass region higher than 670.70 (= Ru-CO). This method provided a high sequence coverage (96%) by peptide mass fingerprinting (PMF). Application of this method to a protein mixture (myoglobin, lysozyme and ubiquitin) successfully achieved high sequence-coverage characterization (>90%) of these proteins simultaneously.
Ito
Akihiro
A
Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Okamura
Taka-Aki
TA
Masui
Ken
K
Kaneko
Maki
M
Masui
Ryoji
R
Ake
Kojiro
K
Kuramitsu
Seiki
S
Yamaguchi
Minoru
M
Kuyama
Hiroki
H
Ando
Eiji
E
Norioka
Shigemi
S
Nakazawa
Takashi
T
Tsunasawa
Susumu
S
Yamamoto
Hitoshi
H
Ueyama
Norikazu
N
D000818:Animals; D007328:Insulin / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
C524337:(4'-(4-(N-succinimidyloxycarbonyl)phenyl)terpyridine)(terpyridine)ruthernium(II); D007328:Insulin; D009211:Myoglobin; D009942:Organometallic Compounds; D010446:Peptide Fragments