Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Targeted proteomics has become increasingly
popular recently because
of its ability to precisely quantify selected proteins in complex
cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap
Velos Pro mass spectrometer in targeted parallel reaction monitoring
(PRM) despite its unconventional dual ion trap configuration. We evaluated
absolute specificity (>99%) and sensitivity (100 amol on column
in
1 μg of total cellular extract) using full and mass range scans
as survey scans together with data-dependent (DDA) and targeted MS/MS
acquisition. The instrument duty cycle was a critical parameter limiting
sensitivity, necessitating peptide retention time scheduling. We assessed
synthetic peptide and recombinant peptide standards to predict or
experimentally determine target peptide retention times. We applied
optimized PRM to protein degradation in signaling regulation, an area
that is receiving increased attention in plant physiology. We quantified
relative abundance of selected proteins in plants that are mutant
for enzymatic components of the N-end rule degradation (NERD) pathway
such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two
E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated
proteins, which might represent degradation targets. We also targeted
FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible
for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation
of plant immunity by degradation of the receptor.