Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

Author(s): Fu Z, Yan K, Rosenberg A, Jin Z, Crain B, Athas G, Heide RS, Howard T, Everett AD, Herrington D, Van Eyk JE

Publication: Proteomics Clin Appl, 2013, Vol. 7, Page 217-24

PubMed ID: 23339088 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if prolonged formalin fixation and storage of formalin-fixed paraffin-embedded (FFPE) aorta specimens affects protein yield or the number of peptides identified by mass spectrometry. Aortas collected during autopsy were either fixed in 10% buffered formalin for 24 h at 4°C and stored as paraffin blocks  for 15 years (2 specimens) or fixed and stored in 10% buffered formalin at 4°C for 3 months or 15 years (6 specimens). Three snap-frozen aorta specimens stored at -80°C for 3 months were also used for comparison. The efficacy of heat and pressure during protein extraction was also evaluated in formalin-fixed and FFPE specimens. Protein lysates were stored at -80°C prior to protein quantification and analysis by mass spectrometry and the multiple reaction monitoring (MRM) assay. Statistical significance was not assessed.

   Summary of Findings:

   Protein yield, represented as the percentage of the protein recovered from snap-frozen specimens (100%), was the greatest when specimens stored in formalin for prolonged periods were extracted under high heat and pressure (95°C at 40,000 psi) compared to extraction using heat alone or at room temperature when specimens were stored for 3 months (73.8%, 46.2%, 16.1%, respectively) or 15 years (69.3%, 50.0%, 4.7%, respectively). Protein recovery was also improved for specimens stored as FFPE blocks for 15 years when extracted under high heat and pressure (88.7%) compared to heat alone (63.7%). With the exception of specimens stored in formalin for 15 years (mean: 96 spectra), the number of protein spectra were similar among snap-frozen specimens (mean: 8003 spectra), those stored in formalin for 3 months (mean: 5923 spectra), and FFPE specimens stored for 15 years (mean: 6754 spectra) when proteins were extracted using high heat and pressure. The number of identifiable proteins were also similar among snap-frozen specimens , those stored in formalin for 3 months, and FFPE specimens stored for 15 years  when proteins were extracted using high heat and pressure (mean: 238, 225, and 213; respectively), while specimens stored in formalin for 15 years had a much lower number of proteins identified (mean: 20). Protein extraction without pressure either at room temperature or at 95°C resulted in much lower numbers of spectra and identifiable proteins for specimens stored in formalin or as FFPE blocks. The protein periostin was quantifiable by MRM assay in specimens stored for up to 15 years as FFPE blocks and those stored in formalin for 3 months, but not in specimens stored in formalin for 15 years.

   Biospecimens

   • Tissue - Aorta

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Autopsy
   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	MS/MS
   Protein	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	3 mon 15 y
   Biospecimen Preservation	Time in fixative	24 h 3 mon 15 y

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