Abstract
Background:Investigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases.Methods:Adjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS.Results:We report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, ‘systemic lupus erythematosus’, ‘interferon-α’, ‘TGF-β’, and ‘extracellular matrix’, were reproducibly quantified between FFPE and FFTs. However, 12%−29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including ‘inflammatory response’ and ‘innate immune system’ were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys.Conclusion:This is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.
Highlights
Fresh frozen tissue (FFT), when available, is the gold standard for clinical proteomics
In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys
This is the first report of lupus nephritis kidney proteomics using FFPE tissue
Summary
Fresh frozen tissue (FFT), when available, is the gold standard for clinical proteomics. Advances in mass spectrometry (MS), especially improved protein digestion and direct quantification techniques, have made proteome analyses feasible for complex tissues. These advances include the ability to access the proteome in formalin-fixed, paraffin-embedded (FFPE) tissues that are a readily available treasure trove of information that can be harnessed via hospital tissue banks. Since the use of formalin for fixation crosslinks certain amino acids and the process of paraffin embedding might result in the loss of proteins, there is a possibility of introducing an inherent sample bias to proteomics studies using FFPE tissue [5]. Proteomic studies have been conducted using FFPE tissues, studies comparing FFPE and FFTs in inflammatory diseases or evaluating for inflammatory pathways are lacking.
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