Nano rp hplc system

Degradation of CCL4 and CCL4 P8A by IDE were performed at 37°C at a 50:1 chemokine/IDE molar ratio. After the indicated time, the reaction was stopped by the addition of an equal volume of stop buffer (200 mM EDTA and 0.2% trifluoroacetic acid). Reaction products were treated with 10 mM tris(2-carboxyethyl)phosphine (TCEP) for 15 min at room temperature and then desalted over a C18 ZipTip (Millipore) prior to MALDI-TOF mass spectrometry. For liquid chromatography-electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry in conjunction with the collision induced dissociation in the gas phase for tandem mass spectrometry data acquisition, enzyme reactions were carried out as described above in the presence of 0.8 mg/ml heparin. After the reactions were stopped and further treated with TCEP, samples (12μl) were injected into a nano RP-HPLC system (Dionex), with a C8 analytical column (Agilent). Peptides were eluted from the nano column with a linear gradient of 5-95% acetonitrile in 0.1% formic acid and sprayed into a LTQ-FT tandem MS instrument (Thermo Scientific). Spectra acquisition and analysis was performed as described [7 (link)]. MS and tandem MS/MS data were analyzed with web-based analysis tools FindPept, Mascot, and MassMatrix [54 (link)] and are summarized in Table S2.