Affinity purification of transgenic mouse brain samples Affinity purification of FLAG-LRRK2 and its interacting proteins was carried out as described in [42], with slight modification. In brief, brain extracts were obtained from non- transgenic and FLAG-LRRK2 mice (P30) by homogenizing 2 brains with a motor-driven homogenizer (speed 2.5, 12 strokes) in 3 ml buffer containing 0.32 M sucrose, 1 mM NaHCO3, 20 mM HEPEs/pH 7.4, 1 mM MgCl2, 0.25 mM CaCl2, EDTA-free protease inhibitor cocktail, 200 mg/mL PMSF, pepstatin 4 mg/ mL and DNase I. The tissue extracts were centrifuge at 1,500 g for 5 min and the pellets were homogenized again in 1 ml buffer for 8 strokes and centrifuged again. Supernatants from the two homogenization steps were pooled, centrifuged at 750 g for 10 min twice. The collected supernatants were diluted with equal volumes of 26 pull-out buffer (16 pull-out buffer containing 20 mM HEPEs/pH 7.4, 1 mM MgCl2, EDTA-free protease inhibitor cocktail, 200 mg/mL PMSF, 4 mg/mL pepstatin, 0.1% triton X-100 and 150 mM NaCl). Sample equivalent to half brain was incubated with FALG M2 beads (,10 ml bed volume) for 2 hours at 4uC. After washed 5 times with 16pullout buffer, beads were eluted with 250 ml elution buffer (0.5 mM EDTA and 0.5 M NH3?H2O) at room temperature. In-gel digestion of purified LRRK2 protein with trypsin and chymotrypsin The purified LRRK2 was resolved in SDS-PAGE and visualized by colloidal Coomassie blue staining. The protein band of interest were excised, in-gel digested after reduction and alkylation, and extracted using the clean protocol previously described [43]. Nano-HPLC/mass spectrometric analysis The proteolytic peptides derived from LRRK2 were analyzed in Velos LTQ Orbitrap mass spectrometer (Thermo Fisher Scien- tific, Waltham, MA). The peptides were separated in a home- made capillary HPLC column (110 mm length675 mm internal diameter, 5 mm particle size, 100 A˚ pore diameter) with Jupiter C12 resin (Phenomenex, St. Torrance, CA) using a gradient from 2% to 30% solvent B in solvent A (mobile phase A: 0% ACN in 0.1% formic acid; and mobile phase B: 100% ACN in 0.1% formic acid) for 50 min. The eluted peptides were directly electrosprayed into the mass spectrometer using a nanospray source. The spray voltage was set to 2.1 kV and the temperature of the heated capillary was set to 300uC. The MS was operated in the data-dependent mode to automatically switch between Orbitrap- MS and Velos LTQ-MS/MS acquisition. Survey full scan MS spectra (from m/z 350–1500) were acquired in the Orbitrap with resolution R = 60,000 at m/z = 400. The 20 most intense ions were sequentially isolated for fragmentation in the linear ion trap using collision-induced dissociation (normalized collision energy = 35%, activation Q = 0.250 and activation time = 10 ms) in the Velos LTQ. Maximal filling times were 30 ms for the full scans and 25 ms for the MS/MS scans. Precursor ion charge state screening was enabled, and singly and unassigned charge states were rejected. A lock-mass ion from ambient air (m/z 445.120024) was used for internal calibration of all full scan measurements with the Orbitrap detector [43,44]. The following parameters were specified in MS/MS analysis: dynamic exclusion (36 seconds); the repeat count (2) and the exclusion window (+3 and 21.5 Da). Protein sequencing alignment Mass spectra collected by MALDI-QqTOF and MALDI-ion trap mass spectrometers were analyzed by the computer search engines Phosphorylation and 14-3-3 Binding of LRRK2 and PD ProFound (http://prowl.rockefeller.edu/prowl-cgi/profound.exe), Xproteo (http://www.xproteo.com) and GPM (http://prowl.rock efeller.edu/tandem/thegpm_tandem.html) using the NCBI non- redundant mouse protein database. All MS/MS spectra were searched against the IPI-mouse data base (v3.74) protein sequence database (56860 sequences) using Mascot (v2.1). The specific parameters for protein sequence database searching included serine, threonine and tyrosine phosphorylation; cysteine carbamidomethy- lation and methionine oxidation as variable modifications. Other parameters used in data analysis were: four allowed missing cleavages; mass error of 10 ppm for precursor ions and 0.5 Da for fragment ions. Charge states of +2, and +3 were considered for parent ions. If more than one spectrum was assigned to a peptide, only the spectrum with the highest Mascot score was selected for manual analysis. All peptides identified with peptide scores of mascot .20 were manually examined using rules described previously [45]. Immunoprecipitation Immunoprecipitation was described as previously with modifi- cation [3]. LRRK2 transgenic brain was homogenized with homogenization buffer (50 mM Tris HCl at pH 7.5, 5% Glycerol, 1 mM NaHCO3, 0.25 mM CaCl2, 1 mM MgCl2, 1 mM PMSF, 10 mg/mL pepstatin, 20 mM beta-Glycerol phosphate, 1 mM Na Vanadate, 50 mM NaF, 10 mM Pyrophosphate, 0.01%Triton X- 100 and mini complete protease inhibitor cocktail), and incubated at 4uC on rotator for 30 min. Transfected HEK-293T cells were harvested and suspended in cell lyses buffer (50 mM Tris HCl at pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 mg/mL pepstatin, 20 mM beta-Glycerol phosphate, 1 mM Na Vanadate, 50 mM NaF, 10 mM Pyrophosphate, 1%Triton X-100 and mini complete protease inhibitor cocktail). The cell lysate or homogenized brain lysate were clarified at 12,000 g for 10 min at 4uC, the 36FLAG- LRRK2 protein was purified using anti-FLAG Affinity Gel according to the manual. For Myc-14-3-3 immunoprecipitation, the HEK-293T cell lysate was precleared using agarose gel for 1 hour at 4uC, and then anti-Myc antibody was added and incubated for overnight. The beads were washed with modified RIPA buffer (50 mM Tris HCl at pH 7.5, 300 mM NaCl, 10 mM MgCl2, 1 mM PMSF, 10 mg/mL pepstatin, 0.5% Sodium Deoxycholate, 0.1% Sodium Dodecyl Sulfate). The immunopre- cipitated protein was eluted by using 16 sample buffer. GST-pull down assay GST-14-3-3 isoform protein was expressed in bacteria with 100 mM IPTG induction for 4 hours at 37uC, and purified following the manufacture’s instruction (GE Healthcare). The protein concentration was measured on SDS-PAGE and Coo- massie Staining comparing to Standard BSA. 5 mg of each GST- 14-3-3 isoform protein was incubated with 100 mg FLAG-LRRK2 transfected HEK-293T cell lyses supernatant for 4 hours at 4uC and washed with cell lyses buffer. The bound protein was eluted with 16 SDS-PAGE sample buffer. For the phosphopeptide competition assay, 2 mg of brain lysate from FLAG-LRRK2 Wt transgenic mice was incubated with different concentration of non- phosphopeptide or phophopeptide (S935) for 1 hour. Then GST- 14-3-3c beads were added and incubated for 4 hours. The beads were then washed and the bound protein was eluted with 16 sample buffer. PKA kinase assay GST-LRRK2 fragment (800–1000aa) was expressed in bacteria with 50 mM IPTG induction at RT for overnight and purified following the manufacture’s instruction. The protein concentra- tion was measured on SDS-PAGE and Coomassie Staining and PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17153