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Enhanced Sample Preparation for Proteomic Analysis Utilizing Reversible Biotinylation and Polymer Based Protein Engineering
Author(s) -
Lucas Amber,
Cummings Chad,
Russell Alan,
Minden Jonathan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.821.1
Subject(s) - biotinylation , proteome , biotin , avidin , chromatography , chemistry , proteomics , sample preparation , protein precipitation , precipitation , protein purification , centrifugation , biochemistry , mass spectrometry , physics , meteorology , gene
Proteome sample preparation for gel‐based and mass spectrometry‐based studies has remained relatively unchanged for many years. The goal of these sample preparation schemes is to remove contaminants such as lipids, DNA, RNA, and carbohydrates from cell or tissue homogenates. The vast majority of current methods rely on protein precipitation followed by centrifugation and resolubilizing the proteins. Unfortunately none of these techniques provide an unbiased sampling of the tentire proteome. Regardless of the method (ammonium acetate precipitation, TCA/acetone precipitation, PEG precipitation, etc.), not all proteins will be precipitated and not all will be resolubilized. We have developed an unbiased method for proteome sample preparation based on protein capture, not precipitation. We recently showed that Biotin‐CDM can be used as a tool to reversibly tag the entire proteome for purification.1 Biotin‐CDM reacts with lysine residues present on the surface of proteins at elevated pH to covalently biotinylate proteins. Biotinylated proteins are bound to Avidin beads and then all non‐protein contaminants are washed away. The biotin tag is then reversed by lowering the pH to release the proteins from the Avidin beads, resulting in pur, unmodified, and unbiased proteome sample. Because an excess of Biotin‐CDM is required to tag all proteins, we utilized polymer based protein engineering to develope a novel method that separates free Biotin‐CDM from biotinylated proteins. Thus, we have developed a method to prepare proteome samples in an unbiased manner that could be used for any organism.