title: Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins.
abstract: Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.
authors: Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA. Electronic address: Brooke.Kaiser@pnnl.gov.
authors: Brooke L
authors: BL
authors: Deatherage Kaiser
authors: Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
authors: David S
authors: DS
authors: Wunschel
authors: Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
authors: Michael A
authors: MA
authors: Sydor
authors: Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
authors: Marvin G
authors: MG
authors: Warner
authors: Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
authors: Karen L
authors: KL
authors: Wahl
authors: Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
authors: Janine R
authors: JR
authors: Hutchison
mesh_terms: D001408:Bacillus anthracis / Q000737:chemistry* / Q000187:drug effects; D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D013171:Spores, Bacterial / Q000737:chemistry* / Q000187:drug effects; D014238:Trichloroacetic Acid / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D014238:Trichloroacetic Acid
keywords: Bacillus anthracis spores; Protein extraction; Proteomics; Trichloroacetic acid