title: Selective detection of peptide-oligonucleotide heteroconjugates utilizing capillary HPLC-ICPMS.
abstract: A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as (31)P(+), the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.
authors.affiliation: University of Cincinnati/Agilent Technologies Metallomics Center of the Americas, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
authors.forename: Brittany
authors.initials: B
authors.lastname: Catron
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Caruso
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D010759:Phosphorus Isotopes / Q000032:analysis / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D010446:Peptide Fragments; D010759:Phosphorus Isotopes; D012710:Serum Albumin, Bovine