title: Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics.
abstract: In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy. For amino labeling, cysteinyl peptides were immobilized on Sepharose-6B resin and labeled with DOTA-NHS ester followed by metallation with lanthanides. Thiol labeling was carried out using lanthanide-containing metal-coded affinity tags (MeCAT) after elution of peptides from the resin. Complete dual labeling of the standard peptides was demonstrated by liquid chromatography electrospray ionization mass spectrometry, whereas more than 80% of the detected peptides of BSA and HSA were completely dual-labeled. Parallel detection by LC coupled to inductively coupled plasma mass spectrometry (ICP-MS) delivered reliable quantitative information. Thus, the versatile lanthanide choice in both labeling steps allowed estimating primary amino and thiol stoichiometries for the studied samples using different lanthanides. On the other hand, enhancement of ICP-MS signal was achieved as expected when all positions were labeled with the same lanthanide. Finally, linear calibrations of the signal for most of the labeled peptides by standard additions of digested BSA showed a suitable behaviour for quantitative applications and demonstrated the pre-concentration capability of the employed resin.
authors.affiliation: Department of Chemistry, Humboldt-Universitaet zu Berlin, Berlin, Germany.
authors.forename: Ahmed H
authors.initials: AH
authors.lastname: El-Khatib
authors.forename: Diego
authors.initials: D
authors.lastname: Esteban-Fernández
authors.forename: Michael W
authors.initials: MW
authors.lastname: Linscheid
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012709:Serum Albumin / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D013388:Succinimides / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D000588:Amines; C502492:DOTA-NHS; D028581:Lanthanoid Series Elements; D010455:Peptides; D012709:Serum Albumin; D013388:Succinimides; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D003545:Cysteine