In-Depth Quantitative Proteomics Analysis of the Pseudomonas aeruginosa Secretome

Secreted proteins play vital roles in bacterial communication, metabolism, and virulence. However, analysis of the bacterial secretome can be challenging, especially if bacteria require rich media for growth or secretion. In this protocol, we describe an efficient and sensitive method to analyze the secretome by shotgun proteomics, using a combination of trichloroacetic acid (TCA) precipitation and single-pot solid-phase-enhanced sample preparation (SP3) for the preparation of the samples. The method was used to identify and quantify proteins secreted by wildtype Pseudomonas aeruginosa PAO1, highlighting its applicability for proteins secreted in limited amounts and in rich media.