Peptidylarginine deiminase (PAD): A promising target for chronic diseases treatment.

In 1958, the presence of citrulline in the structure of the proteins was discovered for the first time. Several years later they found that Arginine converted to citrulline during a post-translational modification process by PAD enzyme. Each PAD is expressed in a certain tissue developing a series of diseases such as inflammation and cancers. Among these, PAD2 and PAD4 play a role in the development of rheumatoid arthritis (RA) by producing citrullinated autoantigens and increasing the production of inflammatory cytokines. PAD4 is also associated with the formation of NET structures and thrombosis. In the crystallographic structure, PAD has several calcium binding sites, and the active site of the enzyme consists of different amino acids. Various PAD inhibitors have been developed divided into pan-PAD and selective PAD inhibitors. F-amidine, Cl-amidine, and BB-Cl-amidine are some of pan-PAD inhibitors. AFM-30a and JBI589 are selective for PAD2 and PAD4, respectively. There is a need to evaluate the effectiveness of existing inhibitors more accurately in the coming years, as well as design and production of novel inhibitors targeting highly specific isoforms.

Isolated auto-citrullinated regions of PADI4 associate to the intact protein without altering their disordered conformation.

PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.

Crystal structure of human peptidylarginine deiminase type VI (PAD6) provides insights into its inactivity.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Šresolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4.

Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.

Selective inhibition of peptidyl-arginine deiminase (PAD): can it control multiple inflammatory disorders as a promising therapeutic strategy?

Peptidyl arginine deiminases (PADs) are a family of post-translational modification enzymes that irreversibly citrullinate (deiminate) arginine residues of protein and convert them to a non-classical amino acid citrulline in the presence of calcium ions. It has five isotypes, such as PAD1, PAD2, PAD3, PAD4, and PAD6, found in mammalian species. It has been suggested that increased PAD expression in various tissues contributes to the development of multiple inflammatory diseases, including rheumatoid arthritis (RA), cancer, diabetes, and neurological disorders. Elevation of PAD enzyme expression depends on several factors like rising intracellular Ca2+ levels, oxidative stress, and proinflammatory cytokines. PAD inhibitors originating from natural or synthetic sources can be used as a novel therapeutic approach concerning inflammatory disorders. Here, we review the pathological role of PAD in several inflammatory disorders, factors that trigger PAD expression, epigenetic role and finally, decipher the therapeutic approach of PAD inhibitors in multiple inflammatory disorders.

Deimination, Intermediate Filaments and Associated Proteins.

Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.

Mapping Citrullinated Sites in Multiple Organs of Mice Using Hypercitrullinated Library.

Protein citrullination (or deimination), an irreversible post-translational modification, has been implicated in several physiological and pathological processes, including gene expression regulation, apoptosis, rheumatoid arthritis, and Alzheimer's disease. Several research studies have been carried out on citrullination under many conditions. However, until now, challenges in sample preparation and data analysis have made it difficult to confidently identify a citrullinated protein and assign the citrullinated site. To overcome these limitations, we generated a mouse hyper-citrullinated spectral library and set up coordinates to confidently identify and validate citrullinated sites. Using this workflow, we detect a four-fold increase in citrullinated proteome coverage across six mouse organs compared with the current state-of-the art techniques. Our data reveal that the subcellular distribution of citrullinated proteins is tissue-type-dependent and that citrullinated targets are involved in fundamental physiological processes, including the metabolic process. These data represent the first report of a hyper-citrullinated library for the mouse and serve as a central resource for exploring the role of citrullination in this organism.

Peptidyl arginine deiminases: detection and functional analysis of protein citrullination.

Citrullination is a post-translational modification of arginine that is catalyzed by the protein arginine deiminases (PADs). Abnormal citrullination is observed in many autoimmune diseases and cancers. Anti-citrullinated protein antibodies (ACPA) are hallmarks of RA and used as diagnostic markers for disease diagnosis. Even though citrullination is associated with many different pathologies, its role remains unclear due to the challenges associated with the detection of citrullinated proteins since the mass change is only 0.984 Da. Moreover, the functional effects of protein citrullination remain mostly unknown. Herein, we discuss a brief overview of PAD structure and function, recent advances in the detection of citrullinated proteins in complex biological systems and the functional consequences of protein citrullination.

Protein Arginine Deiminases (PADs): Biochemistry and Chemical Biology of Protein Citrullination.

Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS), and certain cancers. In this Account, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate-specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate. Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline, and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and lead-optimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.

Biochemical characterization of peptidylarginine deiminase-like orthologs from thermotolerant Emericella dentata and Aspergillus nidulans.

Peptidylarginine deiminases (PADs) are a group of hydrolases, mediating the deimination of peptidylarginine residues into peptidyl-citrulline. Equivocal protein citrullination by PADs of fungal pathogens has a strong relation to the progression of multiple human diseases, however, the biochemical properties of fungal PADs remain ambiguous. Thus, this is the first report exploring the molecular properties of PAD from thermotolerant fungi, to imitate the human temperature. The teleomorph Emericella dentata and anamorph Aspergillus nidulans have been morphologically and molecularly identified, with observed robust growth at 37-40 °C, and strong PAD productivity. The physiological profiles of E. dentata and A. nidulans for PADs production in response to carbon, nitrogen sources, initial medium pH and incubation temperature were relatively identical, emphasizing the taxonomical proximity of these fungal isolates. PADs were purified from E. dentata and A. nidulans with apparent molecular masses 41 and 48 kDa, respectively. The peptide fingerprints of PADs from E. dentata and A. nidulans have been analyzed by MALDI-TOF/MS, displaying a higher sequence similarity to human PAD4 by 18% and 31%, respectively. The conserved peptide sequences of E. dentata and A. nidulans PADs displayed a higher similarity to human PAD than A. fumigatus PADs clade. PADs from both fungal isolates have an optimum pH and pH stability at 7.0-8.0, with putative pI 5.0-5.5, higher structural denaturation at pH 4.0-5.5 and 9.5-12 as revealed from absorbance at λ280nm. E. dentata PAD had a higher conformationally thermal stability than A. nidulans PAD as revealed from its lower Kr value. From the proteolytic mapping, the orientation of trypsinolytic recognition sites on the PADs surface from both fungal isolates was very similar. PADs from both isolates are calcium dependent, with participation of serine and cysteine residues on their catalytic sites. PADs displayed a higher affinity to deiminate the peptidylarginine residues with a feeble affinity to work as ADI. So, PADs from E. dentata and A. nidulans had a relatively similar conformational and kinetic properties. Further molecular modeling analysis are ongoing to explore the role of PADs in citrullination of human proteins in Aspergillosis, that will open a new avenue for unraveling the vague of protein-protein interaction of human A. nidulans pathogen.

Structure, function, and inhibition of a genomic/clinical variant of Porphyromonas gingivalis peptidylarginine deiminase.

Citrullination is an essential post-translational modification in which the guanidinium group of protein and peptide arginines is deiminated by peptidylarginine deiminases (PADs). When deregulated, excessive citrullination leads to inflammation as in severe periodontal disease (PD) and rheumatoid arthritis (RA). Porphyromonas gingivalis is the major periodontopathogenic causative agent of PD and also an etiological agent of RA. It secretes a PAD, termed Porphyromonas PAD (PPAD), which is a virulence factor that causes aberrant citrullination. Analysis of P. gingivalis genomes of laboratory strains and clinical isolates unveiled a PPAD variant (PPAD-T2), which showed three amino-acid substitutions directly preceding catalytic Residue H236 (G231 N/E232 T/N235 D) when compared with PPAD from the reference strain (PPAD-T1). Mutation of these positions in the reference strain resulted in twofold higher cell-associated citrullinating activity. Similar to PPAD-T1, recombinant PPAD-T2 citrullinated arginines at the C-termini of general peptidic substrates but not within peptides. Catalytically, PPAD-T2 showed weaker substrate binding but higher turnover rates than PPAD-T1. In contrast, no differences were found in thermal stability. The 1.6 Å-resolution X-ray crystal structure of PPAD-T2 in complex with the general human PAD inhibitor, Cl-amidine, revealed that the inhibitor moiety is tightly bound and that mutations localize to a loop engaged in substrate/inhibitor binding. In particular, mutation G231 N caused a slight structural rearrangement, which probably originated the higher substrate turnover observed. The present data compare two natural PPAD variants and will set the pace for the design of specific inhibitors against P. gingivalis-caused PD.

Development of Activity-Based Proteomic Probes for Protein Citrullination.

Protein arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine to form peptidyl citrulline. This modification is increased in multiple inflammatory diseases and in certain cancers. PADs regulate a variety of signaling pathways including apoptosis, terminal differentiation, and transcriptional regulation. Activity-based protein profiling (ABPP) probes have been developed to understand the role of the PADs in vivo and to investigate the effect of protein citrullination in various pathological conditions. Furthermore, these ABPPs have been utilized as a platform for high-throughput inhibitor discovery. This review will showcase the development of ABPPs targeting the PADs. In addition, it provides a brief overview of PAD structure and function along with recent advances in PAD inhibitor development.

Highly Efficient Cell-Penetrating Probes of Protein Arginine Deiminases for Functional Proteomics.

Protein arginine deiminases (PADs) have recently emerged at the forefront of drug-discovery programs for several human disorders. Despite this, a precise understanding of their functional roles in human biology remains to be fully elucidated. This report highlights a recent development of next-generation activity-based PAD probes that are highly efficient, cell active, and metabolically stable. This discovery should rapidly expedite functional assignments of PAD biology in both normal and diseased cells, thereby leading to the development of PAD-targeted therapeutics in the near future.

The Pathophysiological Role of Neutrophil Extracellular Traps in Inflammatory Diseases.

Neutrophil pathogen-killing mechanism termed neutrophil extracellular traps (NETs) has been recently identified. NETs consist of chromatin and histones along with serine proteases and myeloperoxidase and are induced by a great variety of infectious and non-infectious stimuli. NETosis is a kind of programmed neutrophil death characterized by chromatin decondensation and release of nuclear granular contents, mainly driven by peptidylarginine deiminase 4 citrullination of histones. Although classically related to the protection against infectious pathogens, nowadays NETs have been described as a player of several pathophysiological processes. Neutrophil dysregulation has been demonstrated in the pathogenesis of most representative vascular diseases, such as acute coronary syndrome, stroke and venous thrombosis. Indeed, NETs have been identified within atherosclerotic lesions and arterial thrombi in both human beings and animal models. Moreover, an imbalance in this mechanism has been proposed as a critical source of modified and/or externalized autoantigens in autoimmune and inflammatory diseases. Finally, an update on the role of NETs in the pathogenesis of cancer has been included. In the present review, based on papers released on PubMed and MEDLINE up to July 2017, we point to update the knowledge on NETs, from their structure to their roles in infectious diseases as well as in cardiovascular diseases, autoimmunity, metabolic disorders and cancer, with a look to future perspectives and therapeutic opportunities.

An in silico analysis of primary and secondary structure specificity determinants for human peptidylarginine deiminase types 2 and 4.

Human peptidylarginine deiminases (hPADs) are a family of five calcium-dependent enzymes that facilitate citrullination, which is the post-translational modification of peptidyl arginine to peptidyl citrulline. The isozymes hPAD2 and hPAD4 have been implicated in the development and progression of several autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. To better characterize the primary and secondary structure determinants of citrullination specificity, we mined the literature for protein sequences susceptible to citrullination by hPAD2 or hPAD4. First, protein secondary structure classification (α-helix, ß-sheet, or coil) was predicted using the PSIPRED software. Next, we used motif-x and pLogo to extract and visualize statistically significant motifs within each data set. Within the data sets of peptides predicted to lie in coil regions, both hPAD2 and hPAD4 appear to favor citrullination of glycine-containing motifs, while distinct hydrophobic motifs were identified for hPAD2 citrullination sites predicted to reside within α-helical and ß-sheet regions. Additionally, we identified potential substrate overlap between coil region citrullination and arginine methylation. Together, these results confirm the importance and offer some insight into the role of secondary structure elements for citrullination specificity, and provide biological context for the existing hPAD specificity and arginine post-translational modification literature.

Probing the Roles of Calcium-Binding Sites during the Folding of Human Peptidylarginine Deiminase 4.

Our recent studies of peptidylarginine deiminase 4 (PAD4) demonstrate that its non-catalytic Ca2+-binding sites play a crucial role in the assembly of the correct geometry of the enzyme. Here, we examined the folding mechanism of PAD4 and the role of Ca2+ ions in the folding pathway. Multiple mutations were introduced into the calcium-binding sites, and these mutants were termed the Ca1_site, Ca2_site, Ca3_site, Ca4_site and Ca5_site mutants. Our data indicate that during the unfolding process, the PAD4 dimer first dissociates into monomers, and the monomers then undergo a three-state denaturation process via an intermediate state formation. In addition, Ca2+ ions assist in stabilizing the folding intermediate, particularly through binding to the Ca3_site and Ca4_site to ensure the correct and active conformation of PAD4. The binding of calcium ions to the Ca1_site and Ca2_site is directly involved in the catalytic action of the enzyme. Finally, this study proposes a model for the folding of PAD4. The nascent polypeptide chains of PAD4 are first folded into monomeric intermediate states, then continue to fold into monomers, and ultimately assemble into a functional and dimeric PAD4 enzyme, and cellular Ca2+ ions may be the critical factor governing the interchange.

Molecular Interplay between the Dimer Interface and the Substrate-Binding Site of Human Peptidylarginine Deiminase 4.

Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should be a dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis.

Identification of novel PAD4 inhibitors based on a pharmacophore model derived from transition state coordinates.

1.4 Protein arginine deiminases 4 (PAD4) is an attractive target for the development of novel and selective inhibitors of Rheumatoid Arthritis (RA). F-amidine is known as mechanism-based inhibitor targeting PAD4 and used as inactivators by covalently modifying the active site Cys645. To identify novel structural inhibitors of PAD4, we investigated the flexibility of protein on basis of the transition state geometry of PAD4 inhibited by F-amidine from our previous QM/MM calculation. And a pharmacophore model was generated containing four features (ADHH) using five representative structures from molecular dynamic (MD) simulation on basis of the transition state geometry of PAD4 inhibited by F-amidine. We performed virtual screening using the pharmacophore model and molecular docking methods, resulting in the discovery of two molecules with KD (dissociation equilibrium constant) values of 112µM and 218µΜ against PAD4 through Surface Plasmon Resonance (SPR) experiments. These two molecules could potentially serve as PAD4 inhibitors.

Evolution of a mass spectrometry-grade protease with PTM-directed specificity.

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (kcat/KM = 6.9 × 105 M-1⋅s-1). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.