title: Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.
abstract: The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.
authors.affiliation: Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, CNRS UMR 5254, Helioparc, 2 av. Pr. Angot, F-64053 Pau, France.
authors.forename: Christina
authors.initials: C
authors.lastname: Rappel
authors.forename: Dirk
authors.initials: D
authors.lastname: Schaumlöffel
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007328:Insulin / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D008187:Lutetium / Q000737:chemistry*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods; D004369:Pentetic Acid / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D011868:Radioisotopes / Q000737:chemistry*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D007328:Insulin; D010455:Peptides; D011868:Radioisotopes; D009240:N-Formylmethionine Leucyl-Phenylalanine; C042205:methionyl-leucyl-phenylalanine; D008187:Lutetium; D004369:Pentetic Acid