Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography-tandem mass spectrometry shotgun proteomic analysis.

Author(s): Sprung RW Jr, Brock JW, Tanksley JP, Li M, Washington MK, Slebos RJ, Liebler DC

Publication: Mol Cell Proteomics, 2009, Vol. 8, Page 1988-98

PubMed ID: 19467989 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine how the estimation of protein concentration using the bicinchoninic acid assay is affected when formalin-fixed tissue is used.

   Summary of Findings:

   Equal slices of a cylindrical specimen were frozen or subjected to formalin fixation. The formalin-fixed specimens consistently gave protein concentration estimates that were 56% of those from frozen specimens.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   Protein	Colorimetric assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen Formalin (buffered)

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2. Study Purpose

   The purpose of this study was to determine the effects of long-term storage, increased duration of fixation, and the use of different tryptic digestion buffers on shotgun proteomic analysis by LC-MS/MS of FFPE tissue specimens.

   Summary of Findings:

   Shotgun proteomic analysis of frozen and FFPE tissue gave essentially the same results, however fewer lysine-terminal peptides were observed in fixed specimens when compared to frozen ones. Nevertheless, multidimensional LC-MS/MS with gel-based isoelectric focusing of peptides showed a 92% overlap in the number and identity of protein groups between frozen and fixed specimens. No differences in the number of proteins groups identified were observed between frozen and FFPE specimens when ammonium bicarbonate buffer was used alone for tryptic digestion. However, the number of protein groups identified was significantly reduced with the use of EDTA or Pyridoxamine buffers with FFPE tissue when compared to frozen tissue (p<0.05). Increasing the duration of fixation resulted in a reduction of observed spectra after 2 and 4 days of fixation, and fewer protein groups identified after 4 days of fixation. Storage of FFPE specimens for up to 10 years had no significant impact on the number of protein groups, or total, lysine-terminal, or arginine-terminal spectral counts when compared to FFPE specimens stored for only 1 year. There was a slight increase in oxidized-methionine-containing peptides with increased storage.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   Peptide	LC-MS or LC-MS/MS
   Protein	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Storage	Storage duration	1 y 3 y 5 y 10 y
   Analyte Extraction and Purification	Protein digestion	Ammonium bicarbonate buffer EDTA in ammonium bicarbonate buffer Pyridoxamine in ammonium bicarbonate buffer
   Biospecimen Preservation	Time in fixative	1 d 2 d 4 d

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