Mining the Human Tissue Proteome for Protein Citrullination

Abstract

Citrullination is a posttranslational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations, and while the modification has been linked to several diseases, including rheumatoid arthritis (RA) and cancer, its physiological or pathophysiological roles remain largely unclear. In part, this is owing to limitations in available methodology to robustly enrich, detect, and localize the modification. As a result, only a few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass-spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ∼70 million tandem mass spectra yielded ∼13,000 candidate spectra, which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized ∼2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new, and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in the brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins, arguing that the development of specific enrichment methods would be required in order to study the full extent of cellular protein citrullination.

Keywords: Data evaluation; Omics; Post-translational modifications*; Tandem Mass Spectrometry; Tissues*; citrullination; human proteome; peptidylarginine deiminase; synthetic peptides.

Fig. 1.

Workflow for the identification and validation of citrullinated peptides in human tissue proteomes.

Fig. 2.

Example HCD tandem mass spectra illustrating criteria for the validation of peptide citrullination. (A) A valid Cit peptide is characterized by the detection of fragment ions covering the presumed Cit modification site as well as the detection of more than one neutral loss ion from Cit fragment ions. NL, neutral loss of isocyanic acid. (B) Ambiguous assignments may be represented by a single diagnostic neutral loss ion and the absence of site-determining fragment ions. Here, the y11 ion determining the Cit site is missing, but a neutral loss from the precursor ion as well as a b2/b3 pair can be detected that localizes the Cit site. (C) An ambiguous assignment is also made when no neutral loss ion is detected but the peptide sequence does not contain any residues that may be deamidated (N or Q). (D) Finally, an ambiguous assignment is also made when the sequence not covered by fragment ions contains N/Q residues, raising the probability that the peptide may be deamidated rather than citrullinated.

Fig. 3.

Validation of peptide citrullination by synthetic peptides. (A) HCD spectrum of an endogenously doubly citrullinated peptide from gamma-adducin from human brain. (B) Reference spectrum of the corresponding synthetic peptide. The two spectra are virtually identical, thus validating the Cit modification. (C and D) Spectra of synthetic peptides in which citrullinated Arg residues are replaced by deamidated Asn residues. These spectra are very different from the endogenous peptide shown in Panel A, thus illustrating how such permutations can aid in the validation of Cit peptides. R(+1), citrullination; N(+1), deamination.

Fig. 4.

Diagnostic fragment ions in HCD spectra of citrullinated peptides. (A) Comparison of the number of unique Cit peptides identified by database searching (all tissues) to those whose HCD spectra contain citrulline immonium ion (130.0975 m/z). It is apparent that only a minority of all Cit peptide spectra contain the immonium ion. (B) In case a Cit immonium ion can be detected, there is a higher chance that the peptide is a genuine Cit peptide. MI, manual inspection. (C) Comparison of the prevalence of diagnostic ions in HCD spectra of synthetic peptides. It is apparent, that the neutral loss (NL) ion is of more diagnostic value than the detection of the immonium ion.

Fig. 5.

Decision tree summarizing the experience obtained in this study and aiding the identification and validation of citrullinated peptides from HCD spectra. NL, neutral loss of isocyanic acid.

Fig. 6.

Analysis of protein citrullination across 30 human tissues. (A) Distribution of Cit peptides and proteins as validated by manual inspection (MI), comparison to synthetic reference peptide spectra (spectrum comparison, SC), or both. (B) Comparison of PAD isoform expression and distribution of Cit PSMs and proteins across 30 human tissues. PSMs, peptide spectrum matches; iBAQ, intensity-based absolute quantification.

Fig. 7.

Discovery of citrullinated proteins across human tissues. (A) Ranked list of the ten most highly citrullinated proteins in this study. Numbers on top of columns indicate the number of tissues in which the protein was found to be citrullinated. (B) Ranked list of how many Cit proteins were detected in how many tissues. (C) Abundance distributions of all proteins identified in each of 26 human tissues in which citrullination was detected. Blue circles indicate citrullinated proteins, and blue horizontal lines indicate the median abundance of the (likely partially) modified proteins.