Chemically modified [Cit⁵]CXCL8 recovery from cell culture supernatants.

<p>Culture supernatants of unstimulated cells were spiked with 20 ng/ml [Cit⁵]CXCL8 before chemical modification and detection. Results represent the mean % (± SEM) for n = 11.</p

Chemical modification of the ureido group of a citrulline residue by 2,3-butanedione and antipyrine.

<p>First 2,3-butanedione (a) reacts with the ureido group of citrulline to form a reactive imidazolone derivate (M_r+50). The first part of the reaction is followed by a nucleophilic addition of antipyrine (b) (M_r+188) to the imidazolone ring. The full reaction results in a total mass shift of +238Da.</p

ELISA standard curves for citrullinated CXCL8 in various biological fluids.

<p>Standard curves for [Cit⁵]CXCL8 were generated in culture medium containing 10% FBS or in human plasma, serum or whole blood. The indicated concentrations of [Cit⁵]CXCL8 represent the initial concentrations spiked in the complex samples before chemical modification and dialysis. Panel A shows two independent standard curves for [Cit⁵]CXCL8, standard 1 (○) and 2 (•), in RPMI 1640 medium containing 10% FBS. For both standards the detection range is 1–50 ng/ml of [Cit⁵]CXCL8 in the original sample. In a second series of experiments (Panel B) standard curves for [Cit⁵]CXCL8 were generated in RPMI 1640 medium containing 10% FBS (•) or in human plasma (▪), serum (▴) or whole blood (♦) (average of two independent experiments).</p

Principle of chemical modification and detection of citrulline-containing proteins.

<p>Peptidylcitrullines are chemically modified with antipyrine and 2,3-butanedione at low pH, followed by dialysis against a buffer with neutral pH (Panel A) and chemically modified citrullinated proteins are detected by sandwich ELISA (Panel B). Coating antibodies are specific for the protein of interest and antibodies against modified citrulline residues and peroxidase labelled anti-rabbit antibodies are used for detection. Chemically modified citrulline (Cit) is marked with an asterisk.</p

Sequence and M_r of synthetic citrulline-containing peptides, before and after chemical modification.

a<p>Theoretical monoisotopic M_r.</p>b<p>Experimentally determined M_r.</p>c<p>Cit* indicates that this citrulline was chemically modified with antipyrine and 2,3-butanedione.</p

Ion trap mass spectra of purified synthetic peptides containing citrulline and chemically modified synthetic peptides.

<p>Peptides (YAGCitLLTK-NH₂ [monoisotopic M_r = 920.5], PIECitTKLY-NH₂ [monoisotopic M_r = 1018.6], and PIECitTYLK-NH₂ [monoisotopic M_r = 1018.6]) were synthesized based on fluorenyl methoxycarbonyl (Fmoc) chemistry, purified using RP-HPLC, chemically modified with antipyrine and 2,3-butanedione and repurified by RP-HPLC. The M_r of the purified synthetic peptides was determined by deconvolution of the multiple charged ions in the raw spectra. As expected the M_r of the chemically modified synthetic peptides differs 238 mass units from the corresponding unmodified peptide. Total ion chromatograms (TIC; black lines) and extracted ion chromatograms for the charged ions of the specific peptides (EIC; gray fill) are shown as inserts in the experimentally determined mass spectra for (A) YAGCitLLTK-NH₂ [M_r = 920.6], (B) YAGCit*LLTK-NH₂ [M_r = 1158.6], (C) PIECitTKLY-NH₂ [M_r = 1018.7], (D) PIECit*TKLY-NH₂ [M_r = 1256.8], (E) PIECitTYLK-NH₂ [M_r = 1018.7] and (F) PIECit*TYLK- NH₂ [M_r = 1256.8]. Citrullines (Cit) marked with an asterisk are chemically modified.</p

Specificity of the ELISA for chemically modified citrullinated CXCL8.

<p>Chemokines [CXCL8 (♦)], citrullinated chemokines [[Cit⁵]CXCL8 (▪) and [Cit⁹]CXCL5 (•)] and RPMI 1640 medium containing 2% FBS [control medium (x)] were chemically modified with antipyrine and 2,3-butanedione at low pH. Subsequently, samples were dialyzed against a buffer with neutral pH and modified citrullinated CXCL8 ([Cit⁵]CXCL8*) was detected by ELISA using anti-CXCL8 coating antibodies and detection antibodies against modified citrulline residues. Untreated samples (Panel A) and samples treated with antipyrine and 2,3-butanedione (Panel B) are represented as dotted and full lines respectively.</p

Susceptibility of CXCL8(-2-77) to proteolysis by plasmin.

<p>CXCL8(1-77) and CXCL8(-2-77) were incubated with plasmin and the degree of conversion was determined using electrospray ion trap mass spectrometry. Percentages of intact CXCL8 [either CXCL8(1-77) or CXCL8(-2-77)] (black histograms), CXCL8(6-77) (dark grey histograms) and CXCL8(9-77) (light grey histograms) generated upon incubation with plasmin are depicted in function of time (min).</p

Chemical synthesis of naturally occurring CXCL8 variants.

a<p>CXCL8(-2-77), CXCL8(1-77), CXCL8(2-77), CXCL8(3-77) and CXCL8(6-77) were chemically synthesized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023913#s2" target="_blank">Materials & Methods</a> section. After purification, the M_r of the proteins was checked by electrospray ion trap mass spectrometry and the NH₂-terminal amino acid sequence was confirmed by NH₂-terminal sequencing based on Edman degradation.</p

Binding properties of the CXCL8 isoforms.

<p>HEK293 cells transfected with CXCR1 (A) or CXCR2 (B) were incubated with increasing concentrations of unlabeled CXCL8(-2-77) (○), CXCL8(1-77) (♦), CXCL8(2-77) (▪), CXCL8(3-77) (▴) or CXCL8(6-77) (•), together with ¹²⁵I-labeled CXCL8(6-77). The mean remaining % of ¹²⁵I-CXCL8(6-77) binding (± SEM) is specified at the y-axis [n≥4 (1 to 300 nM); n = 2 (0,3 nM)]. Analysis of the interaction of the CXCL8 variants with heparin was performed using heparin binding plates. The indicated concentrations of CXCL8(-2-77) (○), CXCL8(1-77) (♦), CXCL8(2-77) (▪), CXCL8(3-77) (▴) and CXCL8(6-77) (•) were added to immobilized heparin, the binding equilibrium was achieved and bound CXCL8 was detected with labeled antibodies. The mean (from four to six independent experiments) percentage heparin binding [compared to 300 nM CXCL8(1-77)] (± SEM) for the individual CXCL8 forms is indicated at the y-axis. To detect statistically significant differences between the CXCL8 variants and CXCL8(1-77), the Mann-Whitney U test was carried out [,*,#,§, p<0.05; **,##,§§, p<0.01; for comparison of CXCL8(-2-77) (), CXCL8(2-77) (*), CXCL8(3-77) (#) and CXCL8(6-77) (§) with CXCL8(1-77)].</p