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. 2013 Sep;13(17):2682-91.
doi: 10.1002/pmic.201300064. Epub 2013 Aug 7.

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation

Zhicheng Jin  1 , Zongming Fu, Jun Yang, Juan Troncosco, Allen D Everett, Jennifer E Van Eyk
Affiliations

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation

Zhicheng Jin et al. Proteomics. 2013 Sep.

Abstract

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high-resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys-C, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID-triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.

Keywords: Biomedicine; Citrullination; GFAP; MBP; Mass spectrometry; NRGN.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Figure 1
Figure 1
Conversion of peptidylarginine to peptidylcitrulline by PAD enzymes in the presence of calcium
Figure 2
Figure 2
Comparison of CID and HCD spectra of a citrullinated peptide of human GFAP in Glu-C digests. A: CID spectrum of the citrullinated peptide, GHLKR*NIVVKTVE of human GFAP at m/z 747.44, z: +2; B: HCD spectrum of this citrullinated peptide at m/z 747.44.
Figure 3
Figure 3
Extracted ion chromatogram of the in vivo citrullinated peptide and intact peptide of NRGN in Lys-C digests of the AD3 brain sample. A: HPLC peak of the unmodified peptide at m/z 909.9275 with mass tolerance of 10 ppm. Right: isotopic clusters of the +2 ion. B: HPLC peak of the citrullinated peptide at m/z 910.4195 with mass tolerance of 10 ppm. Right: isotopic clusters of the +2 ion. C: Overlap of HPLC peaks in A and B. From integrated peak area, occupancy rate of citrullinated peptide at R68 residue was 8.6% for the AD3 sample. D: Citrullination occupancy rate for NRGN at R68, MBP at R92 and R124. Occupancy rate was calculated based on integrated peak area of citrullinated peptide vs. the sum of citrullinated and intact peptides.
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References

    1. Arita K, Hashimoto H, Shimizu T, Nakashima K, Yamada M, et al. Structural basis for Ca2+-induced activation of human PAD4. Nat Struct Mol Biol. 2004;11:777–783. - PubMed
    1. Harauz G, Musse AA. A tale of two citrullines - Structural and functional aspects of myelin basic protein deimination in health and disease. Neurochem Res. 2007;32:137–158. - PubMed
    1. Wegner N, Lundberg K, Kinloch A, Fisher B, Malmstrom V, et al. Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunol Rev. 2010;233:34–54. - PubMed
    1. Nicholas AP, Sambandam T, Echols JD, Tourtellotte WW. Increased citrullinated glial fibrillary acidic protein in secondary progressive multiple sclerosis. J Comp Neurol. 2004;473:128–136. - PubMed
    1. Ishigami A, Maruyama N. Importance of research on peptidylarginine deiminase and citrullinated proteins in age-related disease. Geriatr Gerontol Int. 2010;10:S53–S58. - PubMed

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