Branson sonifier 250

Octyl glucopyranoside, OG, detergent was dissolved in 100 mM NH₄HCO₃ (pH 8) for cell lysis and the OG concentration was 4.5% (w/v). The tissue samples from the LCM were re-suspended in 5 μL of the lysis buffer by placing the lysis buffer directly onto the adhesive cap. The lysis buffer was then pipetted up and down several times, and the sample in the lysis buffer was transferred from the cap to the bottom of the Eppendorf tube via a quick centrifugation. The samples were further processed by two different approaches, Fig. 1. In one approach, we employed an on-ice sonication for 20 minutes (Branson Sonifier 250, VWR Scientific, Batavia, IL) for cell lysis and protein extraction. We prepared three brain sections using the sonication approach. In the other approach, another four samples originating from the same brain slice as the sonication method went through a repeated freeze/thaw protocol; the samples were placed into liquid nitrogen and thawed at 37 °C for 2 minutes for a total of 6 rounds of the freeze/thaw. The seven samples were marked in Fig. 2. All the samples were then spun down. Each sample was diluted by a factor of two with water to get a sample containing 50 mM NH₄HCO₃ (pH 8) and about 2% OG for CZE-MS/MS analysis.

Zebrafish brain samples were kindly provided by Professor Jose Cibelli’s group at the Department of Animal Science of Michigan State University. The whole protocol related to the zebrafish were performed in compliance with relevant laws or guidelines, and the protocol followed guidelines defined by the Institutional Animal Care and Use Committee of Michigan State University. Using zebrafish for scientific research has been approved by the Institutional Animal Care and Use Committee of Michigan State University. Male zebrafish brains were lysed in 8 M urea and 100 mM NH₄HCO₃ (pH 8.0) containing complete protease inhibitor cocktail and PhosSTOP (EASYpacks) from Roche (Indianapolis, IN). Homogenization with a Homogenizer 150 from Fisher Scientific (Pittsburgh, PA) and sonication with a Branson Sonifier 250 from VWR Scientific (Batavia, IL) were performed on ice for protein extraction. Samples were then centrifuged at 15,000 x g for 10 minutes to effectively separate lipids and cell debris from the proteins. The supernatant was collected for further preparation. The protein concentration was determined using a BCA assay. 0.7 mg of zebrafish brain proteins were reduced with DTT (1 M, 2 μL/mg of proteins) at 37 ºC for 30 minutes, alkylated with IAA (1 M, 5 μL/mg of proteins) at room temperature for 20 minutes in dark, and quenched with DTT (1M, 2 μL) before SEC-CZE-MS/MS analyses.