Carboxyvinylpyrrole Protein Adducts: Potential AMD Biomarkers

Abstract

Purpose : The AMD biomarker potential of carboxyethylpyrrole (CEP) adducts has been documented. Based on ELISA, CEP protein adducts are about 60% more abundant in AMD than normal donor plasma however mass spectrometry has yet to demonstrate any CEP-containing peptides. LC MS/MS analyses have revealed possible CEP-related carboxyvinylpyrrole (CVP) and oxidized CVP adducts in plasma that contain carboxyvinyl instead of carboxyethyl attached to a pyrrole ring. CVP contains two less hydrogens than CEP. We hypothesize that CEP adducts may be metabolized in vivo to other moieties still recognized by anti-CEP antibodies. Toward improved AMD biomarker technology, we are further characterizing CVP plasma protein adducts.

Methods : AMD and control plasma proteins were denatured in SDS, reduced, alkylated, precipitated with acetone and digested with trypsin. Following proteolysis, trypsin was heat-inactivated and the digest fractionated by anti-CEP chromatography. Peptides recovered from immuno-chromatography were further fractionated by C18 chromatography and analyzed by LC MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Putative CVP- and CVP lactam-containing peptides (120.0211 and 136.0160 Da at ε-Lys, respectively) were identified using the Mascot search engine and the UniProt human database. Peptides were semi-quantified by spectral counting. Organic synthesis of an authentic standard of CVP-Lys is on going.

Results : Sixteen proteins containing putative CVP modification were identified, with most of the modified peptides (n=38) found in both control and AMD plasma. More of these peptides (n=18) were identified in human serum albumin (HSA) than any other protein, with several detected repeatedly. Nine of the modified peptides were detected with corresponding putative CVP lactam modifications. Six HSA putative CVP peptides were more abundant in AMD plasma than in control plasma.

Conclusions : Current LC MS/MS identifications of multiple proteins with apparent CVP modifications demonstrate this modification is more readily detected than CEP and suggests it may provide more effective blood-borne AMD biomarkers. For example, targeted quantitative proteomic analysis of select peptides from an abundant plasma protein such as albumin could have biomarker utility. Accurate quantitation and confirmation of the identity of the modification awaits the availability of an authentic CVP standard.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.