De Ceuleneer, Marlies

Deforce, Dieter

Tilleman, Kelly

Van Steendam, Katleen

English

Ghent University Academic Bibliography

Specific modification of citrullinated peptides facilitates their detection  De Ceuleneer, M; Tilleman, K; Van Steendam, K; Deforce, D  Laboratory for Pharmaceutical Biotechnology, Ghent University, Belgium  Introduction  Citrullinated proteins are modified proteins in which an arginine is enzymatically converted to a citrulline. Antibodies against citrullinated proteins (anti-CPP) are highly specific for Rheumatoid Arthritis (RA) and can be detected very early in the course of the disease, even before clinical onset. Since it is becoming clear that citrullinated epitopes instead of the mere presence of citrulline may be relevant for the induction of ACPA, it is of great importance to characterize the exact position of this citrulline in the citrullinated proteins. The purpose of this study was to specifically modify citrullinated peptides in such a way that makes them discernible from non-citrullinated peptides and to identify them by liquid chromatography coupled to mass spectrometry (LC-MS).  Method  Citrullinated and non-citrullinated synthetic peptides were modified at 37°C with 2,3-butanedione (BD) in TFA and were analyzed by nanoLC-MS and by infusion tandem mass spectrometry using a QqTOF instrument (Waters).  Next, these peptides were spiked in a Cytochrome C-digest after which the complete mixture was modified, followed by LC-MS analysis. Recombinant human fibrinogen was citrullinated in vitro by Peptidyl Arginine Deiminase 4 (PAD4) from rabbit skeletal muscle. After tryptic digest, peptides were fractionated on RP-LC and fractions were divided in two, one to be modified and one as reference sample. After modification, peptides were analysed by LC-MS.     Results  Modification with BD was specific for synthetic citrullinated peptides and produced a mass shift of 50Da (m/z = 25 Da for doubly charged ions). Complete modification was established after 16h with 50mM BD/30 µl TFA. Based on MSMS-spectra, the butanedione-adduct was shown to be covalently bound to the citrulline residue of the peptide and the amino acid sequence of the modified peptide could still be established at a concentration of 25pmol/µl. When synthetic citrullinated peptides were spiked into a cytochrome C-digest, complete modification of the citrullinated peptide remained possible in a 1/100 dilution (160fmol citrullinated peptide/16pmol cytochrome C-digest).  Besides the additional mass, modified citrullinated peptides also showed a significant shift in retention time (p<0.0001). Peptides of the cytochrome C-digest that were not citrullinated were not affected by this shift in retention time or by the additional mass characteristic.  To test for the presence of citrullinated peptides in a whole protein, we digested 10µg of in vitro citrullinated recombinant human fibrinogen and modified the peptides after fractionation. Results are shown in figure 1; a citrullinated peptide could be identified and its presence was confirmed by modification.      Figure 1A: Peptide ESSHHPGIAEFPSRGK should have a calculated mass of 608.1[3+]. However, in the unmodified sample, a shift of +0.33Da can be observed, indicative for the 1Da mass shift from arginine to citrulline; Figure 1B: In the modified sample, a peptide with m/z 625.2 [3+] can be found. The difference in m/z between both peptides mentioned is 16.6Da. Since the peptides are triply charged, this corresponds to a 50Da mass shift, also indicative for a modified citrullinated residue. The observed shift in Rt amounts to 11mins (from 22.3 mins in the non-modified sample to 33.9mins in the modified sample); Figure 1C: Modification is not quantitative, as can be seen here by the presence of a residual amount of unmodified peptide in the modified sample; Figure 1D: No trace of the m/z 625.2 [3+] can be found in the unmodified sample, proving that this peptide is indeed a product of modification rather than a peptide resulting from the digest.    Conclusions Specific modification of citrullinated proteins with 2,3-butanedione causes a 50Da-mass shift and a significantly longer retention  of the modified citrullinated peptides on nanoLC-MS.  Non-citrullinated peptides remained unaffected in mass and retention time.  When applied to an in vitro citrullinated protein, our method could identify a citrullinated peptide in the mixture of tryptic peptides. Therefore, it should be possible to find citrullinated peptides in in vivo citrullinated proteins, found in the joint of patients with Rheumatoid Arthritis. This might give valuable insights into the immunology of this autoimmune disorder.    A B C D 

Specific modification of protein-bound citrulline residues facilitates their detection

Specific modification of protein-bound citrulline residues facilitates their detection   De Ceuleneer M., Tilleman K., Van Steendam K., Deforce D. Laboratory of Pharmaceutical Biotechnology, Ghent University  Although auto-antibodies against citrullinated proteins are well-known for diagnosis of rheumatoid arthritis, the exact position of citrulline in these proteins, important to evaluate the conformation of the epitopes, remains elusive.  The purpose of this study was to specifically modify citrullinated peptides to make them discernable from non-citrullinated peptides and to identify them by liquid chromatography coupled to mass spectrometry (LC-MS). Methods Citrullinated and non-citrullinated synthetic peptides were modified at 37°C with 2,3-butanedione (BD) in TFA and were analysed by nanoLC-MS and by infusion tandem MS.  Next, these peptides were spiked in a Cytochrome C-digest, after which the complete mixture was modified, followed by LC-MS analysis.    Peptides were also purified from mixtures by immunoprecipitation (IP) with anti-citrulline antibody bound to protein A-agarose beads and subsequently modified and identified. Results Modification with BD was specific for citrullinated peptides and produced a mass shift of 50Da. Complete modification was established after 16h with 50mM BD/30 µl TFA. Based on MSMS-spectra, the butanedione-adduct was proved to be covalently bound to the citrulline residue of the peptide and the amino acid sequence of the modified peptide could still be established at a concentration of 25pmol/µl. When synthetic citrullinated peptides were spiked into a cytochrome C-digest, complete modification of the citrullinated peptide remained possible in a 1/100 dilution (160fmol citrullinated peptide/16pmol cytochrome C-digest), even though the reaction did result in significant reduction in ionization capacity (p<0.01). Modified citrullinated peptides also showed a significant shift in retention time (p<0.0001). Non-citrullinated peptides of the cytochrome C-digest were not affected. In order to obtain comparatively higher ion counts of the modified citrullinated peptides, an IP experiment was conducted. 2 nmol of citrullinated peptide could be purified from a mixture with its non-citrullinated counterpart by IP with an anti-citrulline antibody. Afterwards, modification of the purified citrullinated peptides resulted in the identification of the modified peptide by the specific additional mass shift and  RT shift. Moreover a gain in ion count was observed; the proportion of citrullinated peptide as opposed to non-citrullinated peptide detected increased five-fold after IP (p< 0.05).  Conclusion Specific modification of citrullinated proteins with 2,3-butanedione causes a 50Da-mass shift and a significantly longer retention  of the modified citrullinated peptides on nanoLC-MS.  Non-citrullinated peptides remained unaffected in mass and retention time. Also, peptides could be selectively purified from mixtures by immunoprecipitation. This opens possibilities for the identification of citrullinated peptides in complex mixtures and to identify the in vivo citrullinated status of proteins in the inflamed joint.   

https://biblio.ugent.be/publication/1019941/file/1025387

Specific modification of protein-bound citrulline residues facilitates their detection

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Ghent University Academic Bibliography

Ghent University Academic Bibliography