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Evaluation of Optimized Tube-Gel Methods of Sample Preparation for Large-Scale Plant Proteomics.

ABSTRACT: The so-called tube-gel method is a sample preparation protocol allowing for management of SDS for protein solubilization through in-gel protein trapping. Because of its simplicity, we assumed that once miniaturized, this method could become a standard for large scale experiments. We evaluated the performances of two variants of the miniaturized version of the tube-gel method based on different solubilization buffers (Tris-SDS or urea-SDS). To this end, we compared them to two other digestion methods: (i) liquid digestion after protein solubilization in the absence of SDS (liquid method) and (ii) filter-aided sample preparation (FASP). As large-scale experiments may require long term gel storage, we also examined to which extent gel aging affected the results of the proteomics analysis. We showed that both tube-gel and FASP methods extracted membrane proteins better than the liquid method, while the latter allowed the identification and quantification of a greater number of proteins. All methods were equivalent regarding quantitative stability. However, important differences were observed regarding post-translational modifications. In particular, methionine oxidation was higher with the tube-gel method than with the other methods. Based on these results, and considering time, simplicity, and cost aspects, we conclude that the miniaturized tube-gel method is suitable for sample preparation in the context of large-scale experiments.

SUBMITTER: Balliau T

PROVIDER: S-EPMC5874765 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Evaluation of Optimized Tube-Gel Methods of Sample Preparation for Large-Scale Plant Proteomics.

Balliau Thierry T Blein-Nicolas Mélisande M Zivy Michel M

Proteomes 20180130 1

The so-called tube-gel method is a sample preparation protocol allowing for management of SDS for protein solubilization through in-gel protein trapping. Because of its simplicity, we assumed that once miniaturized, this method could become a standard for large scale experiments. We evaluated the performances of two variants of the miniaturized version of the tube-gel method based on different solubilization buffers (Tris-SDS or urea-SDS). To this end, we compared them to two other digestion met ...[more]

PMID: 29385730

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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

Evaluation of Optimized Tube-Gel Methods of Sample Preparation for Large-Scale Plant Proteomics.

Publications

Evaluation of Optimized Tube-Gel Methods of Sample Preparation for Large-Scale Plant Proteomics.

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