Label Free Quantitative Proteomic Analysis of Age-Related Protein Expression Changes in Mouse Brains

For this analysis, four young (2-month-old) and four aged (24-month-old) mice were used. The mouse brains were quickly dissected and sampled according to the procedure below.

The proteomic analysis of PTS-soluble proteins was performed as follows:

Homogenization and Collection of the Soluble Fraction:
Mouse brain tissue is pulverized and then homogenized in PTS (Phase Transfer Surfactant) buffer. The homogenate is subsequently centrifuged at 15,000 × g for 15 minutes, and the resulting supernatant is collected as the "PTS-soluble fraction".

Protein Reduction, Alkylation, and Digestion:
The proteins within the collected supernatant (30 µg) are first reduced with DTT (dithiothreitol), followed by an alkylation step. The proteins are then digested with the enzyme trypsin to produce peptide fragments suitable for analysis.

Peptide Purification:
After the digested sample is treated with trifluoroacetic acid (TFA), the peptides are purified using a GL-tip SDB solid-phase extraction column.

Mass Spectrometry:
The purified peptides are subjected to label-free quantitative proteomic analysis using a Q-Exactive mass spectrometer.