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A rapid protein sample preparation method based on organic-aqueous microwave irradiation technique

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Abstract

Fast and efficient sample preparation methods are a prerequisite for protein identification in bottom-up proteomics. Here, an innovative microwave irradiation sample preparation method was developed based on an optimized organic-aqueous solvent system for protein identification. Specifically, protein solutions containing high-concentration acetonitrile were subjected to 5 min microwave irradiation. After cooling down, trypsin was added and the digestion was performed with 30 s microwave irradiation, and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A shortened processing time of only 5.5 min is needed with this method (more than 12 h is necessary in the traditional overnight protein sample preparation). Moreover, due to the absence of urea and other chaotropic reagents, the digests can be readily identified by MALDI-TOF MS. When an assessment of this method was performed by digesting a model protein BSA, 69% ± 3% sequence coverage corresponding to 47 ± 3 peptides was obtained, which shows better protein identification than that from the standard overnight protein sample preparation method (51% ± 2% sequence coverage and 23 ± 1 peptides). Another model protein a-casein was used for the analysis of protein phosphorylation with the newly developed method that yielded 4 phosphopeptides with 8 phosphorylation sites, whereas 3 phosphopeptides with 2 phosphorylation sites were obtained from the traditional overnight approach. Moreover, the organic-aqueous microwave irradiation method provides effective digestion for proteins down to fmol.

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Author notes

  1. ShuaiBin Wu

    Present address: College of Chemistry and Chemical Engineering, Hubei Normal University, Huangshi, 35002, China

Authors and Affiliations

  1. Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    ShuaiBin Wu, KaiGuang Yang, Zhen Liang, LiHua Zhang & YuKui Zhang

  2. China National Tobacco Quality Supervision and Test Center, Zhengzhou, 450001, China

    ShuaiBin Wu & HongFei Zhang

  3. Department of Biological Chemistry, the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    JunFeng Ma

Authors
  1. ShuaiBin Wu
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  2. HongFei Zhang
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  3. KaiGuang Yang
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Correspondence to ShuaiBin Wu.

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Wu, S., Zhang, H., Yang, K. et al. A rapid protein sample preparation method based on organic-aqueous microwave irradiation technique. Sci. China Chem. 58, 526–531 (2015). https://doi.org/10.1007/s11426-014-5163-2

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  • DOI: https://doi.org/10.1007/s11426-014-5163-2

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