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Abstract:
The efficient analysis of secretomes is important to study the
mechanisms of bacterial secretion. However, secretome analysis of
bacteria that rely on rich media for optimal secretion via modern
quantitative shotgun proteomics workflows is often hampered by the
higher degree of sample impurities. This may be a reason for the low
number of quantitative secretome investigations in such cases. We
assessed the efficiency and amenability for rich media secretome
analysis of different workflows including precipitation, SP3, and a
combined, serial workflow. Using the model organism Pseudomonas
aeruginosa, we found that the combined TCA-SP3 strategy outperformed the
other tested methods on all monitored qualitative and quantitative
levels. This method proved to be most efficient in the recovery of
proteins secreted by the type III secretion system (T3SS), including all
known effector proteins and secretion machinery components. We monitored
the compositional changes of secretome samples over time, and observed a
strong increase in the secreted protein fraction by the T3SS 2 to 3 h
after T3SS induction. Our study conceptually illustrates how the
combination of TCA precipitation and SP3 results in orthogonality in
depleting sample impurities accompanied by improved chromatographic
peptide separation, and more efficient MS detection with improved
quantification parameters.
