NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Comparison of matched formalin-fixed paraffin embedded and fresh frozen meningioma tissue reveals bias in proteomic profiles.

Author(s): Schoffman H, Levin Y, Itzhaki-Alfia A, Tselekovits L, Gonen L, Vainer GW, Hout-Siloni G, Barshack I, Cohen ZR, Margalit N, Shahar T

Publication: Proteomics, 2022, Vol. 22, Page e2200085

PubMed ID: 36098096 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to compare the results of global, bottom-up proteomic LC-MS/MS-based analysis of formalin-fixed paraffin-embedded (FFPE) meningioma specimens with case-matched snap-frozen specimens and assess the influence of storage duration (5-13 y).

Conclusion of Paper

Global proteomic analysis did not reveal any statistically significant differences between FFPE and snap-frozen meningioma specimens (p=0.053), as similar numbers of protein groups and matched peptides were identified in case-matched FFPE and snap-frozen specimens. Substantial and in some cases significant differences were observed between FFPE and snap-frozen specimens in the number and type of chemical and protein modifications observed. Formylation, methylation, and lysine residue modifications were more prevalent among FFPE than snap-frozen specimens, whereas phosphorylation and iron (Fe) modifications were more prevalent among snap-frozen than FFPE specimens. The number of missed cleavage sites was also higher among FFPE than snap-frozen specimens. Unsupervised cluster analysis of the proteome revealed that samples clustered by preservation method, not patient, and correlation coefficients were higher when FFPE samples from different patients were compared than when samples from the same patient preserved by different methods were compared. Further, the mean molecular weight of the proteins identified in FFPE and snap-frozen specimens differed significantly (33.5 versus 80.6 KDa, respectively; p<0.0001), a pattern that was validated in a separate published dataset.  The authors attributed the differences that were observed between matched FFPE and snap-frozen specimens that were analyzed by Gorilla gene ontology enrichment analysis (overrepresentation of ribosomal genes among FFPE specimens) to being an artifact of fixation or extraction rather than one of biological origin.

Studies

  1. Study Purpose

    The purpose of this paper was to compare the results of global, bottom-up proteomic LC-MS/MS-based analysis of FFPE meningioma specimens with case-matched snap-frozen specimens and assess the influence of storage duration (5-13 y). In total, 20 intracranial meningioma benign tumors (grade I) were procured during surgery, divided in half, and preserved by snap-freezing in liquid nitrogen and by formalin fixation and paraffin embedding between the years of 2006 and 2014. Snap-frozen specimens were stored in liquid nitrogen; additional details on preservation and storage were not provided. For each FFPE specimen, protein was isolated from ten 8 µm-thick FFPE sections using Tris-SDS and sonication and then stored at -80°C until further processing. For snap-frozen specimens, tissue was minced on dry ice, incubated in a digestion buffer that contained SDS, and sonicated; the resultant supernatant was stored at -80°C until further processing.  Protein concentration was quantified by the BCA assay. For each sample, 20 µg of protein was loaded into an S-Trap microcolumn, digested with trypsin, eluted, and stored at -80°C until analysis.  Peptides were separated with a T3 HSS nano-column and analyzed on a quadrupole orbitrap mass spectrometer.  MetaMorpheus software was used to process raw data. Proteins represented by fewer than two peptides were not considered for analysis.  Statistical significance was set at an FDR q value < 0.05.

    Summary of Findings:

    Global proteomic analysis did not reveal any statistically significant differences between FFPE and snap-frozen meningioma specimens (p=0.053). Similar numbers of protein groups (mean 5,378 versus 5,338) and matched peptides (56,186 versus 55,132) were identified in case-matched FFPE and snap-frozen specimens, respectively. Storage duration was not correlated with the number of matched proteins in either FFPE (r2=0.21) or snap-frozen (r2=0.13) specimens.  FFPE and snap-frozen meningioma tumor specimens did differ significantly from one another in the proportion of modified “Peptide Spectrum Matches (PSMs)” (23% versus 8%, respectively; P<0.0001). Formylation and methylation (including di- and tr-methylation) modifications were more prominent among FFPE specimens (representing 17% and 36%, respectively, of the modifications identified) than snap-frozen specimens (0.7% and 3.1%, respectively).  Conversely, phosphorylation (~10% versus 2%) and Fe (7 versus 1%) modifications were more prominent in snap-frozen than FFPE specimens (p<0.001).  There was a 14-fold increase in modified lysine residues among FFPE specimens relative to snap-frozen specimens, whereas arginine modifications did not differ between the two preservation methods. The number of peptides with a missed cleavage site was also 10% higher in FFPE than snap-frozen specimens, with at least one missed cleavage site present in 30% of the peptides identified in FFPE specimens.  Case-matched FFPE and snap-frozen specimens clustered by preservation method, not patient, in non-supervised cluster analysis of the proteome, and the correlation coefficient was higher when samples of the same preservation method but from different patients were compared (mean r2=0.70) than when samples from the same patient but preserved by different methods were compared (mean r2=0.59).  Further analysis revealed that 716 proteins were overrepresented (intensity was higher) in all snap-frozen specimens and 1,150 proteins were overrepresented in all FFPE specimens, of which 265 and 58 proteins demonstrated a ≥2-fold difference. Additional investigation revealed that low molecular weight proteins were overrepresented in FFPE specimens, whereas high molecular weight proteins were overrepresented in snap-frozen specimens.  This translated to a significant difference in the mean molecular weight of the proteins identified in FFPE and snap-frozen specimens (33.5 versus 80.6 KDa, respectively; p<0.0001).  The authors validated these findings in a published dataset that included FFPE and snap-frozen urothelial bladder cancer specimens. The authors attributed the differences that were observed between matched FFPE and snap-frozen specimens that were analyzed by Gorilla gene ontology enrichment analysis (overrepresentation of ribosomal genes among FFPE specimens) to being an artifact of fixation or extraction rather than one of biological origin.

    Biospecimens
    Preservative Types
    • Frozen
    • Formalin
    Diagnoses:
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    Peptide LC-MS or LC-MS/MS
    Protein LC-MS or LC-MS/MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 5-13 y
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    Snap frozen

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