June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
High-Energy Collisional Dissociation Fragmentation of CEP and CML Peptides
Author Affiliations & Notes
  • Geeng-Fu Jang
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Jack S Crabb
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Lei Zhang
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Joe G Hollyfield
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Depts of Ophthalmology and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
  • Robert G Salomon
    Department of Chemistry, Case Western Reserve University, Cleveland, Ohio, United States
  • John W Crabb
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Depts of Ophthalmology and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Geeng-Fu Jang, None; Jack Crabb, None; Lei Zhang, None; Joe Hollyfield, None; Robert Salomon, None; John Crabb, None
  • Footnotes
    Support  NIH grants EY025388 and EY016813, FFB Center Grant to the Cole Eye Institute, NIH-NEI P30 Core Grant (IP30EY025585-01A1) and Unrestricted Grant from The Research to Prevent Blindness, Inc., awarded to the Cole Eye Institute.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5258. doi:
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    • Get Citation

      Geeng-Fu Jang, Jack S Crabb, Lei Zhang, Joe G Hollyfield, Robert G Salomon, John W Crabb; High-Energy Collisional Dissociation Fragmentation of CEP and CML Peptides. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5258.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Carboxyethylpyrrole (CEP) oxidative modifications stimulate angiogenesis in vivo and are elevated in ocular tissues and blood plasma from donors with age-related macular degeneration (AMD). To date, in vivo CEP protein adducts have only been detected by and described in full length reports using immunoreactivity. Carboxymethyllysine (CML), an advanced glycation end product that also stimulates angiogenesis, is also elevated in AMD plasma. To demonstrate CEP and CML modified proteins in AMD plasma using mass spectrometry, we performed LC MS/MS on tryptic peptides of plasma proteins using high-energy collisional dissociation (HCD) fragmentation on a Thermo Orbitrap Elite mass spectrometer.

Methods : The 14 most abundant proteins in plasma were enriched using Seppro IgY14, digested with trypsin and CEP peptides were enriched by anti-CEP chromatography. Alternatively, AMD plasma proteins were denatured, reduced, alkylated, and the most abundant protein, human serum albumin (HSA), enriched by reverse-phase chromatography (RP-HPLC). Albumin-enriched plasma and authentic synthetic CEP-BSA were trypsinized and peptides were fractionated by RP-HPLC then analyzed by LC MS/MS. CEP peptides were identified using the UniProt human or human_EST database and the Mascot search engine, search parameters including CEP-K, oxidized CEP-K and CML (adducts of 122.0368, 138.0318 and 58.0055 at ε-Lys, respectively). Spectra with signature m/z’s were extracted, and the resulting spectra were manually sequenced.

Results :
HCD fragmentation provides high mass accuracy on the Orbitrap and informative immonium (IM) and fragment ions of CEP-K and CML in < m/z 300. Initially, tryptic CEP-BSA peptides were analyzed and three signature CEP-K m/z’s were identified, 251.1396 (CEP-K+-H2O), 223.1447 (IM) or 206.1181 (IM-NH3). Surprisingly, oxidized CEP-K peptides in CEP-BSA were also discovered with corresponding signature m/z’s, 267.1345, 239.1396 or 222.1130. Five CML peptides from HSA and one from IgG were identified with signature CML m/z’s, 187.1083 (CML+-H2O), 159.1134 (IM) or 142.0868 (IM-NH3).

Conclusions : For the first time, oxidized CEP-K peptides in CEP-BSA were observed. This finding raises the possibility that these oxidized forms of CEP may exist in nature and be associated with distinct biological activities yet to be defined. Our CML peptide results demonstrate modified forms of HSA and IgG.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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