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Pressure-assisted sample preparation for proteomic analysis.

ABSTRACT: Pressure-assisted digestion of proteins, also known as pressure cycling technology (PCT), using a Barocycler NEP 2320 was compared with the conventional method using atmospheric pressure. Our objective was to demonstrate that PCT provides more controlled enzymatic digestion of proteins than prolonged digestion at atmospheric pressure ranging from 18 to 24 h. More controlled digestion would be beneficial for studies of highly posttranslationally modified protein such as histones. For the comparison of these two techniques, recombinant and native histone H4 were used as model proteins. PCT was optimized for pressure and time, and it was found to be most effective at 15 kpsi for 120 min of incubation. In conclusion, the PCT method was found to be much faster than using atmospheric pressure. PCT was also found to allow for unambiguous control of digestion parameters and to provide a high yield of sequence coverage compared with atmospheric pressure.

SUBMITTER: Olszowy PP

PROVIDER: S-EPMC3761939 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Pressure-assisted sample preparation for proteomic analysis.

Olszowy Pawel P PP Burns Ariel A Ciborowski Pawel S PS

Analytical biochemistry 20130329 1

Pressure-assisted digestion of proteins, also known as pressure cycling technology (PCT), using a Barocycler NEP 2320 was compared with the conventional method using atmospheric pressure. Our objective was to demonstrate that PCT provides more controlled enzymatic digestion of proteins than prolonged digestion at atmospheric pressure ranging from 18 to 24 h. More controlled digestion would be beneficial for studies of highly posttranslationally modified protein such as histones. For the comparis ...[more]

PMID: 23545193

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Project description:BackgroundSample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC-MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC-MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation.ResultsThe proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12-38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent.ConclusionsWe provide detailed protocols for efficient plant protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile.

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Pressure-assisted sample preparation for proteomic analysis.

Publications

Pressure-assisted sample preparation for proteomic analysis.

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