6 research outputs found
Additional file 1: Table S1. of Comparative expression profiling reveals a role of the root apoplast in local phosphate response
ATH1 dataset. Shown is the relative average expression value of all probe sets (B-G) and the linear fold change of all pairwise comparions (L-AC). (XLSX 9833 kb
Assessment of Label-Free Quantification in Discovery Proteomics and Impact of Technological Factors and Natural Variability of Protein Abundance
We
evaluated the state of label-free discovery proteomics focusing
especially on technological contributions and contributions of naturally
occurring differences in protein abundance to the intersample variability
in protein abundance estimates in this highly peptide-centric technology.
First, the performance of popular quantitative proteomics software,
Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked
using their default parameters and some modified settings. Beyond
this, the intersample variability in protein abundance estimates was
decomposed into variability introduced by the entire technology itself
and variable protein amounts inherent to individual plants of the <i>Arabidopsis thaliana</i> Col-0 accession. The technical component
was considerably higher than the biological intersample variability,
suggesting an effect on the degree and validity of reported biological
changes in protein abundance. Surprisingly, the biological variability,
protein abundance estimates, and protein fold changes were recorded
differently by the software used to quantify the proteins, warranting
caution in the comparison of discovery proteomics results. As expected,
∼99% of the proteome was invariant in the isogenic plants in
the absence of environmental factors; however, few proteins showed
substantial quantitative variability. This naturally occurring variation
between individual organisms can have an impact on the causality of
reported protein fold changes
Assessment of Label-Free Quantification in Discovery Proteomics and Impact of Technological Factors and Natural Variability of Protein Abundance
We
evaluated the state of label-free discovery proteomics focusing
especially on technological contributions and contributions of naturally
occurring differences in protein abundance to the intersample variability
in protein abundance estimates in this highly peptide-centric technology.
First, the performance of popular quantitative proteomics software,
Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked
using their default parameters and some modified settings. Beyond
this, the intersample variability in protein abundance estimates was
decomposed into variability introduced by the entire technology itself
and variable protein amounts inherent to individual plants of the <i>Arabidopsis thaliana</i> Col-0 accession. The technical component
was considerably higher than the biological intersample variability,
suggesting an effect on the degree and validity of reported biological
changes in protein abundance. Surprisingly, the biological variability,
protein abundance estimates, and protein fold changes were recorded
differently by the software used to quantify the proteins, warranting
caution in the comparison of discovery proteomics results. As expected,
∼99% of the proteome was invariant in the isogenic plants in
the absence of environmental factors; however, few proteins showed
substantial quantitative variability. This naturally occurring variation
between individual organisms can have an impact on the causality of
reported protein fold changes
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
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
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