High sequence-coverage detection of proteolytic peptides using a bis(terpyridine)ruthenium(ii) complex

Abstract

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.

Profiling the Protein Targets of Unmodified Bio‐Active Molecules with Drug Affinity Responsive Target Stability and Liquid Chromatography/Tandem Mass Spectrometry

“…Sequence coverage was calculated by dividing the number of amino acids observed by the number of amino acids in the entire sequence. Naturally, high protein sequence coverage thus has a higher confidence than a low sequence coverage . This forms the interpretation of diverse transition patterns of sequence coverage with pronase.…”

Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells

“…Interestingly, Keyes et al developed ruthenium(II) polypyridyl luminophores anchored to peptide sequences as a new class of stimulated emission depletion (STED) microscopy probes for the imaging of key cell organelles . Ueyama et al also described a peptide‐labeling approach by using Ru II terpyridine complexes to implement the mass spectrometry detection of proteolytic peptides …”

“…[26] Ueyama et al also described a peptide-labeling approach by using Ru II terpyridine complexes to implement the mass spectrometry detection of proteolytic peptides. [27] As far as it concerns RGD-type peptides, only a few examples are described. Thus, Sadler et al reported the synthesis of a Ru II arene complex attached to the linear RGD tripeptide [28] that dissociated from the peptide by irradiation with visible light to form an aqua complex that generated monofunctional adducts with the guanine bases of DNA.…”

Size‐based protein separations by microchip electrophoresis using an acid‐labile surfactant as a replacement for SDS

“…The widely applied method of ''bottom-up'' proteomics by peptide analysis via MS of a tryptic digest does not provide 100% coverage of the protein [14][15][16]. Although this method is well established and has been an indispensable tool in the proteomics field, it is difficult to determine modifications made to the mature protein when compared with the sequence that is predicted from the open-reading frame [17].…”