Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Figure Lengend Snippet: Workflow for large-scale quantitative phosphoproteomics of synaptosomes. Synaptosomal proteins were extracted by lysis of synaptosomes and acetone precipitation. The protein pellets were dried, resuspended in 1% RapiGest buffer and digested by trypsin. Peptides were labeled with heavy and light dimethyl labeling ( Boersema et al., 2009 ). To obtain pair-wise comparisons between the three conditions, samples were mixed with a ratio of 1:1. The mixed peptides were separated by strong cation exchange (SCX) chromatography, with the eluate being divided into 12 fractions. Phosphopeptides in each fraction were separately enriched by TiO 2 microbeads ( Larsen et al., 2005 ) and subjected for mass spectrometry analysis. DOI: http://dx.doi.org/10.7554/eLife.14530.005
Article Snippet: The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan).
Techniques: Lysis, Labeling, Chromatography, Mass Spectrometry