Dataset Information

Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones.

ABSTRACT: Characterization of histone post-translational modifications (PTMs) is still challenging, and robust histone sample preparation is essential for convincing evaluation of PTMs by mass spectrometry. An effective protocol for extracting plant histone proteins must also avoid excessive co-extraction of the numerous potential interfering compounds, including those related to secondary metabolism. Currently, the co-existence of histone marks is addressed mostly by shotgun proteomic analysis following chemical derivatization of histone lysine residues. Here, we report a straightforward approach for plant histone sample preparation for mass spectrometry, based on filter-aided sample preparation coupled with histone propionylation. The approach offers savings in sample handling and preparation time, enables removal of interfering compounds from the sample, and does not require either precipitation or dialysis of histone extract. We show the comparison of two protocol variants for derivatization of histone proteins, in-solution propionylation in the vial and propionylation on the filter unit. For both protocols, we obtained identical abundances of post-translationally modified histone peptides. Although shorter time is required for histone protein labeling on the filter unit, in-solution derivatization slightly outweighed filter-based variant by lower data variability. Nevertheless, both protocol variants appear to be efficient and convenient approach for preparation of plant histones for mass spectrometric analysis.

SUBMITTER: Ledvinova D

PROVIDER: S-EPMC6156276 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones.

Ledvinová Dominika D Mikulášek Kamil K Kuchaříková Hana H Brabencová Sylva S Fojtová Miloslava M Zdráhal Zbyněk Z Lochmanová Gabriela G

Frontiers in plant science 20180919

Characterization of histone post-translational modifications (PTMs) is still challenging, and robust histone sample preparation is essential for convincing evaluation of PTMs by mass spectrometry. An effective protocol for extracting plant histone proteins must also avoid excessive co-extraction of the numerous potential interfering compounds, including those related to secondary metabolism. Currently, the co-existence of histone marks is addressed mostly by shotgun proteomic analysis following ...[more]

PMID: 30283482

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Project description:Due to its outstanding throughput and analytical resolution, gel-free LC-based shotgun proteomics represents the gold standard of proteome analysis. Thereby, the efficiency of sample preparation dramatically affects the correctness and reliability of protein quantification. Thus, the steps of protein isolation, solubilization, and proteolysis represent the principal bottleneck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by the application of detergents. However, these compounds ultimately compromise reverse-phase chromatographic separation and disrupt electrospray ionization. Filter-aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols remain to be confirmed. Thereby, the most important gap is the absence of the data on the linearity of underlying protein quantification methods for plant matrices. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (Pisum sativum) seed and Arabidopsis thaliana leaf proteomes by the shotgun approach. For this aim, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Furthermore, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. The spiking experiments revealed high sensitivities (LODs of up to 4 fmol) for spiked BSA and LDRs of at least 0.6 × 102. Thereby, phenol extraction yielded slightly better recoveries, whereas the detergent-based method showed better linearity. Thus, our results indicate the very good applicability of FASP to quantitative plant proteomics with only limited impact of the protein isolation technique on the method's overall performance.

Dataset Information

Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones.

Publications

Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones.

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