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SILIA-Based 4C Quantitative PTM Proteomics

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Plant Phosphoproteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2358))

Abstract

To absolutely and relatively quantitate the alteration of a posttranslationally modified (PTM) proteome in response to a specific internal or external signal, a 15N-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 either 14N/15N-coded tissues or the 14N/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 the 14N/15N isotope-coded PTM peptide ions and to perform the statistical evaluation of the quantitation results. The Student t-test values of ratios of quantifiable 14N/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.

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Acknowledgments

This work was supported by grants, 31370315, 31570187, 31870231, from National Science Foundation of China and grants, 16101114, 16103817, 16103615, 16100318, 16101819, AOE/M-403-16, from RGC of Hong Kong as well as grants, VPRGO17SC07PG, FP704, IRS18SC17, IRS19SC15, IRS20SC15, SBI18SC04, SJTU19SC03, CRP01, from the HKUST.

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Authors and Affiliations

  1. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, SAR, China

    Emily Oi Ying Wong & Ning Li

  2. Shenzhen Research Institute, The Hong Kong University of Science and Technology, Hong Kong, SAR, China

    Emily Oi Ying Wong & Ning Li

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  1. Emily Oi Ying Wong
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  2. Ning Li
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Correspondence to Ning Li .

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Editors and Affiliations

  1. State Key Laboratory of Conservation and Utilization of Bio-Resources in Yunnan and Center for Life Science, School of Life Sciences, Yunnan University, Kunming, China

    Xu Na Wu

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Wong, E.O.Y., Li, N. (2021). SILIA-Based 4C Quantitative PTM Proteomics. In: Wu, X.N. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 2358. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1625-3_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1625-3_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1624-6

  • Online ISBN: 978-1-0716-1625-3

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