Taking advantage of the recently developed Filter Assisted Sample Preparation (FASP) method for sample preparation, we performed an in-depth analysis of phosphorylation sites in mouse brain. To maximize the number of detected phosphorylation sites, we fractionated proteins by size exclusion chromatography (SEC) or separated tryptic peptides on an anion exchanger (SAX) prior or after the TiO(2)-based phosphopeptide enrichment, respectively. SEC allowed analysis of minute tissue samples (1 mg total protein), and resulted in identification of more than 4000 sites in a single experiment, comprising eight fractions. SAX in a pipet tip format offered a convenient and rapid way to fractionate phosphopeptides and mapped more than 5000 sites in a single six fraction experiment. To enrich peptides containing phosphotyrosine residues, we describe a filter aided antibody capturing and elution (FACE) method that requires only the uncoupled instead of resin-immobilized capture reagent. In total, we identified 12,035 phosphorylation sites on 4579 brain proteins of which 8446 are novel. Gene Ontology annotation reveals that 23% of identified sites are located on plasma membrane proteins, including a large number of ion channels and transporters. Together with the glycosylation sites from a recent large-scale study, they can confirm or correct predicted membrane topologies of these proteins, as we show for the examples calcium channels and glutamate receptors.