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Proteomics. 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.

Author information

1
Department of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD, USA.

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

PMID:
23828821
PMCID:
PMC4864592
DOI:
10.1002/pmic.201300064
[Indexed for MEDLINE]
Free PMC Article

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