Fig. 1.

Biochemical fractionation and sample preparation. A, 300–350 mg of white matter was dissected from three human postmortem prefrontal cortexes. Three mouse brains, minus olfactory bulbs and cerebellum, were obtained. These tissues were subject to biochemical fractionation to obtain total homogenate (Hom), synaptosomal (Syn) and intermediate membrane fractions as well as preparations enriched for vesicular (Ves), Parasynaptic (Para), and postsynaptic density (PSD) membranes. A crude membrane fraction was prepared from [¹³C₆] lysine labeled (>97%) mouse brain tissue for use as a stable isotope labeled internal standard proteome ([¹³C₆]brain ISTD). B, human subject demographic information. C, two proteomes were prepared for LC-MS/MS: 1) 30 μg of the [¹³C₆]brain ISTD and 2) 30 μg of fraction H, from human subject 1, mixed with 15 μg of the [¹³C₆]brain ISTD. The proteomes were incubated at 95 °C for 20 min with 1× LDS loading buffer, separated on a 1.5 mm 4–12% Bis-Tris Gel (Invitrogen), cut into five fractions, diced into 2 mm cubes, reduced alkylated, digested with trypsin. Peptides were recovered from the gel cubes in 50/50 H₂O/ACN with 3% formic acid, desalted and filtered. D, the biochemical fractions were mixed with the [¹³C₆]brain ISTD at a 2:1 ratio by mass and prepared for LC-SRM/MS. Specifically, 30 μg of the total homogenate, synaptosomal, intermediate membrane and postsynaptic density preparations were prepared with 15 μg ISTD and 6 μg of the parasynaptic membrane preparation was prepared with 3 μg [¹³C₆]brain ISTD .