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Evaluating the effect of formalin fixation on mass spectrometry‐based proteomic profiling (984.1)
Author(s) -
Atwood James,
Neumann Drexel,
Dammer Eric,
Duong Duc,
Dunn Chelsea,
Seyfried Nicholas
Publication year - 2014
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.28.1_supplement.984.1
Subject(s) - proteome , homogenization (climate) , chromatography , chemistry , mass spectrometry , proteomics , fixation (population genetics) , biology , biochemistry , biodiversity , ecology , gene
Intro: Formalin fixation is the universal standard for preserving tissue samples. However, due to the nature of the formalin fixation process, protein extraction and recovery is typically compromised when compared to proteomic analysis of fresh tissues. Herein, we performed a comparative proteomic analysis between matched fresh and formalin fixed brain tissue samples in order to evaluate the degree of proteome degradation and to establish and optimized protocol for protein extraction from formalin fixed samples. Method: Fresh and free floating formalin fixed brain tissues were homogenized under non‐denaturing and denaturing conditions. Protein recoveries were quantified and the protein mixture was enzymatically digested. Resulting peptides were analyzed by quantitative LC‐MS/MS to compare total proteome coverage between tissue types and preparation methods. Results: While total protein recovery was comparable across both tissue types the homogenization buffer affected yields dramatically. Proteomic analysis revealed a significant decrease in total proteome coverage in the formalin fixed tissues with proteins of known high abundance being most affected.