Fig 1.
Workflow to rapidly develop SRM targeted proteomic methods from shotgun proteomic data.
(a) Identification of proteins from either in-house shotgun DDA acquisition using an LC-QToF or data downloaded from public MS data repositories; (b) construction of a proteome spectral library from raw data containing retention times for a set of host-specific reference peptides and peptides of interest; (c) prediction of targeted peptide retention time based on the spectral library and measured retention times of reference peptides in a new chromatography gradient; (d) predicted RT of targeted peptide used without further methods optimization in a rapid SRM method.
Fig 2.
Comparison of the accuracy of peptide retention time prediction for different RT windows from proteomic libraries constructed from in-house and public MS data.
The percentage of peptides that were detected of a selection of five hundred (500) peptides from (a) an in-house E. coli library; (b) an in-house S. cerevisiae library; and (c) a S. cerevisiae library downloaded from the Chorus Project for chromatographic gradients of 120 minutes (Circle), 20 minutes (Triangle), and 2 minutes (Diamond).
Table 1.
Summary of discovery proteomic-based spectral libraries of microbes commonly used in biotechnology research and development.
Fig 3.
Examples of rapid MRM target method development for quantifying pathway proteins in R. toruloides (top), P. putida (middle), and C. glutamicum (bottom). Peptides of pathway proteins selected from spectral libraries generated via in-house shotgun proteomics or online databases.
Fig 4.
(A) Central carbon pathways (glycolysis, lysine degradation, aromatic monomer degradation pathways, and tricarboxylic acid (TCA) cycle) in P. putida; (B-E) comparison of the relative protein abundances of P. putida grown on 10 mM of glucose, p-coumarate, and 5-aminovalerate carbon sources in MOPS media. The error bar shows the standard deviation of measured peak area of three biological replicates. Statistical significance of p-coumarate and 5-aminovalerate against glucose were calculated by moderated t-test with the limma package in R, and resulting p-values were adjusted using the Benjamini-Hochberg (BH) method. *, **, and *** indicate adjusted P < 0.05, 0.01 and 0.001, respectively.