Selective Extraction and Enrichment of Multiphosphorylated Peptides Using Polyarginine-Coated Diamond Nanoparticles

Abstract

Despite recent advances in phosphopeptide research, detection and characterization of multiply phosphorylated peptides have been a challenge. This work presents a new strategy that not only can effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Polyarginine-coated diamond nanoparticles are the solid-phase extraction supports used for this purpose. The supports show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 microL) of tryptic digests of proteins such as beta-casein, alpha-casein, and nonfat milk at a concentration as low as 1 x 10 (-9) M. The concentration is markedly lower than that can be achieved by using other currently available technologies. We quantified the enhanced selectivity and detection sensitivity of the method using mixtures composed of mono- and tetraphosphorylated peptide standards. This new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in complex and dilute analytes.

Analytical strategies for phosphoproteomics

“…Recently, a new and interesting method employing polyarginine-coated beads was introduced for purification of multiple phosphorylated peptides prior to MS analysis [91]. This method, however, has yet to be tested for its efficiency in larger phosphoproteomics studies and in those where only low amounts of starting material (micrograms) are available.…”

Time-Resolved Luminescence Nanothermometry with Nitrogen-Vacancy Centers in Nanodiamonds

“…18 They were covalently conjugated with poly-L-arginine using water-soluble carbodiimide cross-linkers. 23 GNRs surface-coated with cetyltrimethylammonium bromide (CTAB) were obtained from Nanopartz and used without further modification and purification.…”

Application of Nanodiamonds in Biomolecular Mass Spectrometry

“…PA-coated NDs can be prepared via an EDC-mediated coupling reaction following the similar procedures used for covalent linking of PL [33]. The amino acid arginine residues are typical recognition sites in protein–protein interactions involving phosphate or sulfate moieties, and their high affinity for phosphate groups are able to generate stable complexes having “covalent-like” stability.…”

“…The amino acid arginine residues are typical recognition sites in protein–protein interactions involving phosphate or sulfate moieties, and their high affinity for phosphate groups are able to generate stable complexes having “covalent-like” stability. Accordingly, Wu et al [33] have proved that PA-coated NDs can be a very useful tool for isolating multiphosphorylated peptides, leading to improved detection sensitivity in the complex solutions. Figure 7 shows an example of extracting and enriching phosphopeptides from the tryptic digests of nonfat milk, which contains abundant phosphoproteins.…”

“…(b, c) MALDI mass spectra of the same sample obtained using PA-coated NDs to extract and enrich phosphopeptides from the 100 μL tryptic digest product after (b) 100-fold and (c) 1000-fold dilution. The phosphopeptides without oxidation are denoted by Arabic numerals, while the phosphopeptides containing oxidized methionine residues are marked with # in spectra b and c. Reproduced from reference [33] with permission from the American Chemical Society.…”