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A Robust Protocol for Protein Extraction and Digestion

  • Michelle Atallah
  • Mark R. Flory
  • Parag MallickEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)

Abstract

Proteins play a key role in all aspects of cellular homeostasis. Proteomics, the large-scale study of proteins, provides in-depth data on protein properties, including abundances and post-translational modification states, and as such provides a rich avenue for the investigation of biological and disease processes. While proteomic tools such as mass spectrometry have enabled exquisitely sensitive sample analysis, sample preparation remains a critical unstandardized variable that can have a significant impact on downstream data readouts. Consistency in sample preparation and handling is therefore paramount in the collection and analysis of proteomic data.

Here we describe methods for performing protein extraction from cell culture or tissues, digesting the isolated protein into peptides via in-solution enzymatic digest, and peptide cleanup with final preparations for analysis via liquid chromatography-mass spectrometry. These protocols have been optimized and standardized for maximum consistency and maintenance of sample integrity.

Key words

Proteomics Protein extraction Acetone precipitation Enzymatic solution digest Liquid chromatography-mass spectrometry 

References

  1. 1.
    Mallick P, Kuster B (2010) Proteomics: a pragmatic perspective. Nat Biotechnol 28: 695–709CrossRefPubMedGoogle Scholar
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    Kachuk C, Stephen K, Doucette A (2015) Comparison of sodium dodecyl sulfate depletion techniques for proteome analysis by mass spectrometry. J Chromatogr A 1418:158–166CrossRefPubMedGoogle Scholar
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    Wiśniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Michelle Atallah
    • 1
  • Mark R. Flory
    • 1
  • Parag Mallick
    • 1
    Email author
  1. 1.Canary Center at Stanford for Cancer Early DetectionStanford UniversityPalo AltoUSA

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