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 (-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).

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).

Distinction of Leu and Ile Using a Ruthenium(II) complex by MALDI-LIFT-TOF/TOF-MS analysis.

The novel N-terminal labeling method using a ruthenium(II) complex derivative characteristically indicated a(n) and d(n) (N-terminal) fragment ions in high sensitivity by MS/MS analysis (MALDI-LIFT or ESI-CID). Although these fragment ions depended on a fragmentation process by MS/MS analytical methods to some degree, each case indicated similar side-chain cleavage patterns. The labeling method allows accurate distinction of amino acid residues by MS/MS analysis even if the residues are structural isomers such as leucine and isoleucine.

Rapid and sensitive amino-acid sequencing of cloning Thermus thermophilus HB8 ferredoxin by proteomics.

Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin.