This study was conducted
to optimize a targeted plant proteomics
approach from signature peptide selection and liquid chromatography
with tandem mass spectrometry (LC-MS/MS) analytical method development
and optimization to sample preparation method optimization. Three
typical protein extraction and precipitation methods, including trichloroacetic
acid (TCA)/acetone method, phenol method, and TCA/acetone/phenol method,
and two digestion methods, including trypsin digestion and LysC/trypsin
digestion, were evaluated for selected proteins related to the impact
of engineered nanomaterials (ENMs) on wheat (Triticum
aestivum) plant growth. In addition, we compared two
plant tissue homogenization methods: grinding freeze-dried tissue
and fresh tissue into a fine powder using a mortar and pestle aided
with liquid nitrogen. Wheat plants were grown under a 16 h photoperiod
(light intensity 150 μmol·m–2·s–1) for 4 weeks at 22 °C with a relative humidity
of 60% and were watered daily to maintain a 70–90% water content
in the soil. Processed samples were analyzed with an optimized LC-MS/MS
method. The concentration of selected signature peptides for the wheat
proteins of interest indicated that the phenol extraction method using
fresh plant tissue, coupled with trypsin digestion, was the best sample
preparation method for the targeted proteomics study. Overall, the
optimized approach yielded the highest total peptide concentration
(68,831 ng/g, 2.4 times the lowest concentration) as well as higher
signature peptide concentrations for most peptides (19 out of 28).
In addition, three of the signature peptides could only be detected
using the optimized approach. This study provides a workflow for optimizing
targeted proteomics studies.