4800 maldi tof tof mass spectrometer

Protamine- and histone-NPM2 complexes, prepared as described in Section 2.5, were extensively dialyzed against 100 mM NaCl, 10 mM PIPES (pH 7.3), and 0.1 mM EDTA buffer for 4 h at 4 °C. A minimum concentration of 30 μg of protein was used for each CXMS experiment. Two samples, NPM2+histone octamers (NPM2-histone run) and NPM2+protamines (NPM2-protamine run) were subjected to CXMS in two independent runs. The pH of each mixture was adjusted to 8.0-8.5 by the addition of 0.2 M Na₂HPO_4, and proteins were crosslinked with 0.05 mM cyanurobiotindipriopyonylsuccinimide (CBDPS) (Creative Molecules Inc.) for 30 min at 25 °C [17] . Reactions were quenched with ammonium bicarbonate at a final concentration of 30 mM, and cross-linked proteins were then digested with sequencing-grade trypsin (Promega, Madison, WI) overnight at 37 °C at a 10:1 substrate:enzyme ratio. Resulting peptide mixtures were affinity-purified using monomeric avidin-agarose beads (Thermo Scientific, Rockford, IL) and analysed using nano-LC-MALDI-MS/MS via the Eksigent® 1D nano-LC system (AB/Sciex, Dublin, CA), Dionex® Probot spotter (Thermo Scientific, Rockford, IL) and the 4800 MALDI-TOF/TOF mass spectrometer (AB/Sciex, Foster City, CA). Data was analyzed with the DXMSMS Match program [22] of ICC-CLASS [23] .

mAb150-immunoprecipitated proteins were resolved in 12.5% SDS-PAGE gel stained with mass-spectroscopy compatible Coomassie stain (Pierce, CA, USA). Individual protein bands (based on molecular weight) were excised, digested, desalted and purified for acquisition of spectra a 4800 MALDI-TOF/ TOF mass spectrometer (AB Sciex, Framingham, MA) and protein identification performed using MASCOT (version 2.1; http://www.martixscience.com) on search engine against the SwissProt database as described earlier [16] (link).