Voyager de str

The dried peptides were mixed with saturated matrix solution (a-cyano-4-hydroxycinnamic acid in 60% acetonitrile-0.1% trifluoroacetic acid) and analyzed with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) (Voyager-DE STR; Applied Biosystems, Inc.) or electrospray ionization quadrupole-time of flight tandem mass spectrometry (ESI-q-TOF MS/MS, Micromass/Waters Corp.). Using the Micromass ProteinLynx Global Server (PLGS) 2.1 data processing software, the data output was shown as a single MASCOT-searchable peak list (.pkl) file. The peak list files were used to query the SwissProt database using MASCOT (global search engine). All reported assignments were verified by automatic and manual interpretation of spectra from Mascot in a blinded mode.

The molecular weight of each MA methylester was determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry on a Voyager DE-STR workstation (Applied Biosystems, Foster City, CA) using 2.4-dihydroxybenzoic acid (2.5-DHB) as a matrix, as reported previously [26 (link)]. The samples were analyzed in the reflection mode with an accelerating voltage operating in the negative ion mode of 25kV. Next, 2.5-DHB at the concentration of 10 mg/ml was mixed with 1.0 ml of the matrix solution. The sample mixture was applied to the sample plate as a 1.0-ml droplet. The samples were then allowed to crystallize at room temperature. The MALDI-TOF mass spectra in the positive mode were acquired as described by Fujita et al. [26 (link)].

To screen for binding partners of Tce1 with GST pull-down^{20 (link),43 (link)}, 0.5 mg purified GST-Tce1 protein was incubated with 100 μl pre-washed glutathione beads for 2 h at 4 °C, then mixed with cleared cell lysates collected from 100 ml of Yptb culture for another 4 h. After incubation, the beads were collected and washed three times with PBS containing 300 mM NaCl, and three times with PBS containing 500 mM NaCl. Proteins binding on the beads were boiled with SDS sample buffer, resolved by SDS–PAGE and visualized by silver staining (Bio-Rad). Individual protein bands on the gel were excised, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization/mass spectrometry (Voyager-DE STR, Applied Biosystems, Waltham, MA). To analyze protein interactions, purified GST fusion protein was mixed with 6×His fusion protein in PBS on a rotator for 2 h at 4 °C, and GST or an irrelevant protein CheY (BTH_II2365 in Burkholderia thailandensis) fused to GST were used as negative controls. After adding 40 μl of pre-washed glutathione beads slurry, binding was allowed to proceed for another 2 h at 4 °C. The beads were then washed five times with TEN buffer (100 mM Tris-Cl, 10 mM EDTA, 500 mM NaCl, pH 8.0). Retained proteins were resolved by SDS–PAGE and visualized by western blot.

Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-ToF) mass spectrometry analysis (Voyager-DE-STR; Applied Biosystems, Carlsbad, CA, USA) was performed in linear mode using a standard Nitrogen laser. Recrystallized alpha-cyano-4-hydroxycinnamic 31 (link) (Sigma-Aldrich, Steinheim, Germany) was used as MALDI matrix. Samples of Rpr (0.3 μg/μl) alone or reacted with Thsp, respectively, were each diluted 1:1 with formic acid (‘High Purity Grade’, Sigma-Aldrich). One microlitre of each sample was spotted on to a standard stainless steel MALDI target followed by the addition of 2 μl matrix solution. The spotted solution was gently mixed with the tip of a pipette for better mixing and crystallization. Mass spectra were acquired using an acceleration voltage of 20 kV and an extraction delay of 200–1200 nsec. For each spot, spectra from 500 laser shots were summed. All measurements were performed in replicate.