Halogenation activity of mature MPO and recombinant pro-MPO.

<p>Initial rates of NADH oxidation were determined with various concentrations of A) bromide or B) chloride, 20 nM mature MPO or recombinant pro-MPO and 100 μM NADH in 50 mM phosphate buffer (pH 7.4) at 21°C. The reaction was started by adding 50 μM H₂O₂. The kinetics of the reaction of myeloperoxidase-generated hypobromous and hypochlorous acids with NADH were monitored by measuring the bromohydrin and chlorohydrin product at 275 nm. Data are representative of two or more experiments.</p

Increased detection of pro-MPO in plasma from patients with myocardial infarction (MI) compared to healthy controls.

<p>Plasma was purified by affinity chromatography with MPO-specific antibodies, subjected to digestion with trypsin and analyzed for the presence of pro-MPO and mature MPO-specific peptides by SRM-based LC-MS/MS. Samples that showed a peak are denoted by “+”, those that don’t by “-”.</p

Detection of mature MPO and pro-MPO in plasma using affinity-purification and immunblot analysis.

<p><b>A)</b> Plasma was spiked with MPO standard (10 nM) and HL60 cell lysate (containing 10 nM MPO) and subjected to affinity purification. Purified samples along with MPO, pro-MPO standards and HL-60 lysates were separated by 10% SDS/PAGE, transferred to PVDF and probed with MPO-specific antibody. <b>B)</b> Neutrophils (5x10⁶ cells/ml) were added back into plasma and then stimulated with CytB and FMLP for 30 min at 37°C. Neutrophils were centrifuged and cell free plasma MPO was subjected to affinity purification and analyzed as described in A. After the ECL fluorescence of blots was developed, a photograph of the blot showing the molecular weight markers was taken and aligned with the fluorescence image as indicated by the black line.</p

LC-MS/MS analysis of mutual, pro-MPO- and MPO- specific peptides.

<p><b>A)</b> Location of tryptic peptides used for LC-MS/MS analysis within the MPO sequence. The <i>N-</i>terminal signal peptide is shown in dark grey, the pro-peptide in bold light grey and the sequence of mature MPO in bold black. Highlighted by the black and grey boxes are tryptic peptides specific to pro-MPO or present in both mature MPO and pro-MPO, respectively. Twenty-five μg of recombinant pro-MPO or mature MPO were digested with trypsin and analyzed by LC-MS/MS. <b>B), D) +F)</b> Extracted ion chromatograms for SRM transitions specific for the mutual, pro-MPO and mature MPO-specific peptides (MS1/MS2 551.8/272.2, 1000.9/1033.5 and 648.8/935.5, respectively, MS1 = m/z for the doubly charged precursor species and MS2 = m/z for the singly charged y–ion fragment, c–carbamidomethyl-cysteine). <b>C), E) +G)</b> CID-MS/MS spectra confirming the sequence of the respective peptide. Representative chromatograms and spectra are shown.</p

LC-MS/MS detection of pro-MPO in affinity-purified plasma separated by SDS/PAGE.

<p>Plasma was purified by affinity chromatography with an MPO-specific antibody and subjected to separation by SDS/PAGE. Bands with the molecular weight of pro-MPO (90 kDa) were subjected to in-gel tryptic digestion and analyzed by LC-MS/MS using SRM for a pro-MPO-specific peptide (1000.9->1033.5). <b>(A)</b> Extracted ion chromatogram for the SRM transition for the pro-MPO specific peptide. <b>(B)</b> CID-MS/MS spectrum confirming the sequence for the pro-MPO-specific peptide SSGcAYQDVGVTcPEQDK. Representative chromatograms and spectra are shown.</p