Absolute Quantification of Peptides by Isotope Dilution Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry and Gas Chromatography/Mass Spectrometry

作者：Liu, Rui; Hou, Xiandeng; Lv, Yi; McCooeye, Margaret; Yang, Lu^*; Mester, Zoltan

来源：Analytical Chemistry, 2013, 85(8): 4087-4093.

DOI：10.1021/ac400158u

• PHOTOCHEMICAL VAPOR GENERATION
• ICP-MS
• QUANTITATIVE PROTEOMICS
• PROTEIN QUANTIFICATION
• COLLISION CELL
• NANOHPLC-ICPMS
• IMMUNOASSAY
• HYDROLYSIS
• SULFUR
• SELENOMETHIONINE

Absolute quantitation of peptides/proteins in dilute calibration solutions used in various diagnostic settings is a major challenge. Here we report the absolute quantitation of peptides by non-species-specific isotope dilution liquid chromatography-inductively coupled plasma mass spectrometry (ID LC-ICPMS) based on stoichiometric Eu tagging. The method was validated by species-specific isotope dilution gas chromatography/mass spectrometry (GC/MS) analysis of constituent amino acids of the target peptide. Quantitative labeling of bradykinin peptide was accomplished with a commercially available 2',2 ''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-NHS-ester) and subsequently tagged with Eu. A Eu-151-enriched spike was used for the non-species-specific ID LC-ICPMS determination of bradykinin. The non-species-specific ID LC-ICPMS method was cross-validated by a species-specific ID GC/MS approach, which is based on the determination of phenylalanine in bradykinin to derive the concentration of the peptide in the sample. The hydrolysis of the peptide into amino acids was achieved by microwave digestion with 4 M methanesulfonic acid, and derivatization of phenylalanine with methyl chloroformate (MCF) was performed prior to its detection by GC/MS based on a C-13-enriched phenylalanine spike. The accuracy of the method was confirmed at various concentration levels with a typical precision of better than 5% relative standard deviation (RSD) at 20 pmol for non-species-specific ID LC-ICPMS and 500 pmol for species-specific ID GC/MS. A detection limit (3 SD) of 7.2 fmol estimated for ID LC-ICPMS with a 10 mu L injection volume from three procedure blanks was obtained for bradykinin, confirming the suitability of the method for the direct determination of peptides at trace levels. To the best of our knowledge, the proposed method is the first ICPMS peptide quantification strategy which employs an independent validation strategy using species-specific ID GC/MS for amino acid quantitation.

• 出版日期2013-4-16
• 单位成都理工大学; 四川大学