SILIA-Based 4C Quantitative PTM Proteomics

To absolutely and relatively quantitate the alteration of a posttranslationally modified (PTM) proteome in response to a specific internal or external signal, a¹⁵N-stable isotope labeling in Arabidopsis (SILIA) protocol has been integrated into the 4C quantitative PTM proteomics, named as SILIA-based 4C quantitative PTM proteomics (S4Quap). The isotope metabolic labeling produces both forward (F) and reciprocal (R) mixings of either¹⁴N/¹⁵N-coded tissues or the¹⁴N/¹⁵N-coded total cellular proteins. Plant protein is isolated using a urea-based extraction buffer (UEB). The presence of 8 M urea, 2% polyvinylpolypyrrolidone (PVPP), and 5 mM ascorbic acid allows to instantly denature protein, remove the phenolic compounds, and curb the oxidation by free radicals once plant cells are broken. The total cellular proteins are routinely processed into peptides by trypsin. The PTM peptide yield of affinity enrichment and preparation is 0.1–0.2% in general. Ion exchange chromatographic fractionation prepares the PTM peptides for LC-MS/MS analysis. The collected mass spectrograms are subjected to a target-decoy sequence analysis using various search engines. The computational programs are subsequently applied to analyze the ratios of the extracted ion chromatogram (XIC) of the¹⁴N/¹⁵N isotope-coded PTM peptide ions and to perform the statistical evaluation of the quantitation results. The Student t-test values of ratios of quantifiable¹⁴N/¹⁵N-coded PTM peptides are normally corrected using a Benjamini-Hochberg (BH) multiple hypothesis test to select the significantly regulated PTM peptide groups (BH-FDR < 5%). Consequently, the highly selected prospect candidate(s) of PTM proteins are confirmed and validated using biochemical, molecular, cellular, and transgenic plant analysis.