Minimizing Resolution of Isotopically Coded Peptides in Comparative Proteomics

Abstract

Stable isotopes are now widely used to quantify concentration changes in proteomics. This paper focuses on the resolution of isotopically coded peptides and how isotope effects occurring during chromatographic separations can be minimized. Heavy isotope derivatizing agents used in this work were the commercially available ²H₈-ICAT reagent and ¹³C₄-succinic anhydride. The ICAT reagent derivatizes cysteine-containing peptides, whereas the succinic anhydride reacts with primary amine groups in peptides. It was observed during reversed-phase chromatography of peptides from a BSA tryptic digest differentially labeled with the ²H₀- and ²H₈-ICAT reagents that resolution of the isoforms exceeded 0.5 with 20% of the peptides in the digest. Three-fourths of the peptides in this group contained two cysteine residues and were doubly labeled. Only 23% of the peptides labeled with a single ICAT residue had a resolution greater than 0.4. The resolution of peptides differentially labeled with ¹³C- and ¹²C-succinate never exceeded ±0.01, even in the case of peptides from the BSA digest labeled with 2 mol of succinate. Because this value is within the limits of the method used to determine resolution, it was concluded the ¹³C- and ¹²C-coded isoforms of labeled peptides did not resolve. The isotope ratio in the case of ¹³C/¹²C coding could be determined from a single mass spectrum taken at any point in the elution profile. This enabled isotope ratio analysis to be completed early in the elution of a peptide from chromatography columns. Keywords: comparative proteomics • quantification • GIST • ICAT • isotope effects • isotope ratio • labeling • signature peptides • mass spectrometry