Mass Spectrometric Detection of Carboxyethylpyrrole in Proteins

Abstract

Purpose : Carboxyethylpyrrole (CEP) oxidative modifications stimulate angiogenesis in vivo and are elevated in age-related macular degeneration (AMD) ocular tissues and plasma. Mice immunized with CEP develop a dry AMD-like phenotype. To date, in vivo CEP protein adducts have only been detected by immunoreactivity. The purpose of this research is to develop corroborating mass spectrometric methods to detect and measure CEP modified proteins in plasma.

Methods : AMD and control plasma proteins were hydrolyzed with pronase E and leucine aminopeptidase to release CEP-Lys. Authentic standards of CEP-Lys and CEP-¹⁵N₂¹³C₆-Lys were prepared by organic synthesis. Protein was quantified by amino acid analysis. CEP-Lys in enzymatic hydrolysates was enriched by immunoaffinity chromatography using anti-CEP mAb coupled to protein G agarose. CEP-Lys was quantified by LC MS/MS-multiple reaction monitoring (MRM) using an AB Sciex API 3000 triple quadrupole mass spectrometer. The 14 most abundant proteins in plasma were enriched using Seppro IgY14, digested with trypsin and tryptic peptides fractionated by anti-CEP affinity chromatography. Peptides were analyzed by LC MS/MS on MALDI TOF/TOF and Orbitrap Elite mass spectrometers. Proteins were identified using the UniProt human database and the Mascot search engine.

Results : CEP-Lys (m/z 269.3) was detected by LC MS/MS-MRM using two transitions (m/z 206.2 and m/z 148.2) in both AMD and control plasma proteins following complete enzymatic hydrolysis and CEP immunoaffinity enrichment. Analyses of plasma suggest about 40 fmol CEP-Lys per mg plasma protein. Analyses of CEP-Lys standards suggest (i) about 20 fmol as the limit of detection in our LC MS/MS-MRM system and (ii) 25-30% average recovery from anti-CEP fractionation. LC MS/MS analysis of the Seppro IgY14-bound plasma fraction following tryptic digestion and anti-CEP affinity chromatography resulted in the identification of over 40 proteins. Identification of plasma protein CEP modification sites will require larger sample amounts than used in the current analyses.

Conclusions : For the first time, endogenous CEP protein adducts in plasma have been demonstrated by mass spectrometry. This technology could lead to improved methods for quantifying this very low abundancy protein modification in biological specimens.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.