comparing to standard BSA. The kinase assay was performed following the New England Biolab protocol. Briefly, 400 ng GST- LRRK2 fragment protein was incubated with different concen- tration of PKA catalytic subunit protein at 30uC for 20 min. Or GST-LRRK2 fragment protein was incubated with PKA catalytic subunit protein and different concentration of H89 at 30uC for 20 min. The reaction was stopped by adding 66 SDS-PAGE sample buffer, followed by boiling the samples for 5 min. Supporting Information Figure S1 Identification of LRRK2 S935 phosphoryla- tion by MALDI QqTOF and ion trap mass spectrome- ters. MALDI QqTOF mass spectrum of tryptic digested peptides extracted from the affinity purified mouse brain LRRK2 (band shown in Figure 5A). Two tryptic peptides containing the S935 phosphorylation site are labeled A* and B*. The corresponding unphosphorylated peptides are labeled A and B. (TIF) Figure S2 Identification of LRRK2 S935 phosphoryla- tion by MALDI QqTOF and ion trap mass spectrome- ters. (A) MALDI ion trap MS/MS spectrum of the phosphor- ylated peptide HSNpSLGPVFDHEDLLR. (B) MALDI ion trap MS/MS spectrum of the phosphorylated peptide HSNpSLGP- VFDHEDLLRR. Both MS/MS spectra show dominant peaks due to characteristic loss of H3PO4 (98 Da). For both (A) and (B), insets show zoomed-in regions of the spectra. Phosphorylated- S935-containing fragment ions are labeled in red. (TIF) Figure S3 Analysis of phosphorylation sites in LRRK2 by nano-HPLC/velos LTQ Orbitrap mass spectrometer. MS/MS spectra of phosphorylated peptides at serine 910 (SNpSISVGEVYR) (A), serine 912 (LVKRKSNSIpSVGEVY) (B) in purified LRRK2 protein, respectively. In this study, we digested the purified LRRK2 protein in-gel using trypsin or chymotrypsin. The MS/MS data were analyzed by the Mascot algorithm to identify the protein and its posttranslational modifications. The candidate peptides bearing the serine phos- phorylation were further examined manually as previously described [45]. The phosphorylated peptide can be identified by a mass shift of 79.96633 Da at serine/threonine/tyrosine residues. (TIF) Figure S4 Analysis of phosphorylation sites in LRRK2 by nano-HPLC/velos LTQ Orbitrap mass spectrometer. MS/MS spectra of phosphorylated peptides at serine 935 (HSNpSLGPVFDHEDLLR) (A), and serine 973 (QSDpSSSSLA- SER) (B) in purified LRRK2 protein, respectively. In this study, we digested the purified LRRK2 protein in-gel using trypsin or chymotrypsin. The MS/MS data were analyzed by the Mascot algorithm to identify the protein and its posttranslational modifications. The candidate peptides bearing the serine phos- References 1. Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, et al. (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 44: 595–600. 2. Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, et al. (2004) Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44: 601–607. 3. Li X, Tan YC, Poulose S, Olanow CW, Huang XY, et al. (2007) Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson’s disease R1441C/G mutants. J Neurochem 103: 238–247. Phosphorylation and 14-3-3 Binding of LRRK2 and PD phorylation were further examined manually as previously described [45]. The phosphorylated peptide can be identified by a mass shift of 79.96633 Da at serine/threonine/tyrosine residues. (TIF) Figure S5 Confirmation of LRRK2 S935 phosphoryla- tion by a combination of alkaline phosphatase treatment and mass spectrometry. (A) Coomassie blue stain gel showing affinity purified FLAG-LRRK2 and its interacting proteins from FLAG-LRRK2 BAC transgenic mouse brain and lung, with and without alkaline phosphatase (AP) treatment. LRRK2 and AP bands are labeled by arrows. AP treatment was performed by incubating anti-FLAG immunoprecipitation eluent in AP (Roche, 2 U/mL) at 37uC for 1 h. (B) MALDI QqTOF mass spectra of LRRK2 tryptic digested peptides extracted from the gel bands shown in (A). The monoisotopic peaks of the unphosphorylated and phosphorylated LRRK2 tryptic peptide 932[HSNSLG- PVFDHEDLLR]947 are highlighted in yellow. (C) MS3 tandem mass spectrum confirming phosphorylated LRRK2 tryptic peptide 932[HSNpSLGPVFDHEDLLR]947, using a MALDI ion trap mass spectrometer. (TIF) Figure S6 Identification of 14-3-3 isoforms by MALDI QqTOF and ion trap mass spectrometry. (A) MALDI QqTOF mass spectrum of tryptic digested peptides extracted from the affinity purified mouse brain 14-3-3 (band shown in Figure 5A). (B) MALDI ion trap MS/MS spectra of unique tryptic peptides of 14-3-3c. (TIF) Figure S7 Identification of 14-3-3 isoforms by MALDI QqTOF and ion trap mass spectrometry. MALDI ion trap MS/MS spectra of unique tryptic peptides of 14-3-3e (A), 14-3-3g (B). (TIF) Figure S8 Identification of 14-3-3 isoforms by MALDI QqTOF and ion trap mass spectrometry. MALDI ion trap MS/MS spectra of unique tryptic peptides of 14-3-3f (A), and 14- 3-3h/t (B). (TIF) Acknowledgments We thank The Yue lab members for critical reading of the manuscript and comments. Author Contributions Conceived and designed the experiments: XL QJW BTC ZY. Performed the experiments: XL QJW NP SL. Analyzed the data: XL QJW YZ ZY BTC SL. Contributed reagents/materials/analysis tools: XL NP YZ BTC. Wrote the paper: XL YZ ZY. 4. West AB, Moore DJ, Biskup S, Bugayenko A, Smith WW, et al. (2005) Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci U S A 102: 16842–16847. 5. Smith WW, Pei Z, Jiang H, Dawson VL, Dawson TM, et al. (2006) Kinase activity of mutant LRRK2 mediates neuronal toxicity. Nat Neurosci 9: 1231–1233. 6. Guo L, Gandhi PN, Wang W, Petersen RB, Wilson-Delfosse AL, et al. (2007) The Parkinson’s disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity. Exp Cell Res 313: 3658–3670. PLoS ONE | www.plosone.org 12 March 2011 | Volume 6 | Issue 3 | e17153