title: Absolute quantification of intact proteins via 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide-europium labeling and HPLC coupled with species-unspecific isotope dilution ICPMS.
abstract: Quantitative proteomics requires novel analytical methodology to fill the gap related to absolute protein abundance in different physiological conditions. In this paper, we demonstrate a proof-of-concept study for absolute protein quantification. 1,4,7,10-Tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide (MMA-DOTA) loaded with Eu was used to label lysozyme, insulin, and ribonuclease A, and they were subsequently quantified using HPLC coupled with (153)Eu species-unspecific isotope dilution inductively coupled plasma mass spectrometry (ICPMS). Labeling procedures were optimized using electrospray ionization mass spectrometry (ESI-MS) based on the labeling efficiency and specificity of the three intact proteins, which suggested that 10-fold or higher MMA-DOTA to cysteine sulphydryl rates at pH from 6.8 to 7.6 and 47 degrees C for 40 min were optimal conditions for the conjugation of the reduced-form proteins and that a 5-fold excess of Eu with respect to the DOTA present in the MMA-DOTA-conjugated proteins and pH 5.8 are optimal for Eu labeling. Subsequently, these three MMA-DOTA-Eu-labeled proteins were digested with trypsin, and the tryptic peptides were quantified via HPLC coupled with (153)Eu species-unspecific isotope dilution ICPMS. The results for the protein studied indicated that not only could 100% digestion efficiency not be achieved but also the resulting peptides needed a chromatographic separation at higher resolution. On the other hand, the labeled intact proteins were quantified without tryptic digestion. The average recovery was found to be 97.9% in six independent experiments, and the precision was evaluated to be 5.8% at the 10 pmol L(-1) level. The detection limits (3sigma) were determined to be 0.819, 1.638, and 0.819 fmol for lysozyme, the A chain of insulin, and ribonuclease A, respectively, using ICPMS with a normal concentric pneumatic nebulizer. These results demonstrated that high-quality absolute protein quantification could be achieved through labeling the intact proteins but not the tryptic peptides, implying that intact proteins may be more feasible and practical targets than tryptic peptides for ICPMS-based absolute protein quantification.
authors.affiliation: Department of Chemistry and the Key Laboratory of Analytical Sciences, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Yan
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Limin
authors.initials: L
authors.lastname: Yang
authors.forename: Qiuquan
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: C549509:1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide-europium; D002614:Chelating Agents; D003432:Cross-Linking Reagents; D007328:Insulin; D008301:Maleimides; D009942:Organometallic Compounds; D010446:Peptide Fragments; D011506:Proteins; C043592:maleimide; D005063:Europium; D012259:Ribonuclease, Pancreatic; D009113:Muramidase; D014357:Trypsin