Influence of the metals and ligands in dinuclear complexes on phosphopeptide sequencing by electron-transfer dissociation tandem mass spectrometry

Abstract

Phosphorylation is one of the most important protein modification, and electron transfer dissociation tandem mass spectrometry (ETD-MS/MS) is a potentially useful method for sequencing of phosphopeptide, including determination of phosphorylation site. Notably, ETD-MS/MS typically provide useful information when precursor contains more than three positive charges and has not yet closed as the method for large-scale phosphopeptide analysis, due to the difficulty of the production for acidic phopeptides having more than three positive charges. To increase the charge state of phosphopeptides, we have used the dinuclear metal complexes, which selectively bound to phosphate group in phosphopeptide with addition of positive charge(s). The dinuclear copper, zinc and gallium complexes have been tested and the type of metal present in the complex strongly affected to the affinity of phosphorylated compound and their ETD fragmentation. The dinulcear copper complex interact weakly with phosphate group and ETD induced peptide fragmentation is largely suppressed by the presence of Cu 2+ , which worked as an electron trap. The dinulcear gallium complex strongly bound to phosphate group. However, the ligand binding to gallium acted as an electron trap and the presence of dinulcear gallium complex in precursor for ETD-MS/MS hampers the sequencing of phosphopeptide, as in the case of dinulcear copper complexes. In contrast, dinulcear zinc complexes efficiently bind to phosphopeptides with increase in the charge state, facilitating phosphopeptide sequencing by ETD-MS/MS. The fragmentation of ligand and peptide backbone in dinulcear zincphosphopeptide complex are competitively induced by ETD. These processes are influenced by ligand structure and the detailed ETD fragmentation pathway were investigated using density functional theory calculations.

In Situ Pinpoint Photopolymerization of Phos-Tag Polyacrylamide Gel in Poly(dimethylsiloxane)/Glass Microchip for Specific Entrapment, Derivatization, and Separation of Phosphorylated Compounds

“…The fluorescence intensity decreased in the presence of Phos-tag acrylamide coordinated to metal ions upon introducing the elution buffer. The ability of Phos-tag to capture phosphorylated compounds differs depending on the phosphorylation site and molecular weight [ 33 ]. When Cu 2+ was used for coordination, the fluorescence intensity was found to be unstable.…”

Structural analysis of peptides modified with organo-iridium complexes, opportunities from multi-mode fragmentation

“…24 Europium-bound biomolecules and some other heavy metal adducts are also believed to be so-called "electron sinks" and quench electrons. 25 26,27 Despite the partial quenching of ECD electrons by iridium, low intensity fragments were observed in all ECD MS/MS spectra of Ir-containing reaction products (Figure 3+4, SI Figures SF5, SF7, SF15, and SF17. These fragments are summarised in the corresponding fragmentation maps (Figure 3, SF6, SF7, SF13, SF15, and SF17) and SI Tables ST2, ST3, ST4, ST8, ST10, and ST12).…”

Zinc N,N-bis(2-picolyl)amine Chelates Show Substitution-Dependent Cleavage of Phosphodiesters in Models as Well as of PNAzyme-RNA Bulges

“…However, although unsymmetrical 2,6-disubstituted pyridines are widespread building blocks in many fields of chemistry, there is a lack of efficient synthetic strategies when one of these substituents is an amino group. This has been a limitation in the preparative scale production of the intermediates for making metal ion chelating ligands that contain the 2-aminopicolyl structure, despite the favourable impact that these have on binding and reactivity [12][13][14][15][16][17][18][19][20]. The most common previous strategies have focused on the functionalisation of a methyl group in 2-methyl-6-amino-pyridine, after the amino group has been protected.…”

“…One group of metal chelating ligands which has been described in the literature for a variety of purposes is the N,N-bis(2-picolyl)amine head group. Metal ion complexes of these tridentate ligands have found applications in molecular imaging, phosphate binding and phosphodiester cleavage [12][13][14][15][16][17]. Of particular relevance to developing artificial nucleases, it has been shown that by modifying the second coordination sphere of the metal ion with 6-amino substituents on the pyridyl ring provides considerably enhanced activity towards phosphate diester cleavage over the parent pyridyl group [18][19][20].…”