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

Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues.

Author(s): Rossouw SC, Bendou H, Blignaut RJ, Bell L, Rigby J, Christoffels A

Publication: Pathol Oncol Res, 2021, Vol. 27, Page 622855

PubMed ID: 34257588 PubMed Review Paper? No

Purpose of Paper

This paper evaluated the effects of storage of formalin-fixed, paraffin-embedded (FFPE) blocks at room temperature for 1, 5, or 10 years and protein purification by three different methods on the protein concentration, the number of peptides and proteins identified, protein integrity, and the reproducibility of label-free LC-MS/MS results using FFPE colorectal carcinoma specimens.

Conclusion of Paper

Protein yields differed significantly among FFPE blocks stored at room temperature for different durations (p<0.0001), the highest protein yields were observed among blocks stored for 1 y (average yield = 3.82 mg/ml protein), followed by 5 y (2.46 mg/ml protein), then 10 y (1.65 mg/ml protein).  

All identified proteins from FFPE colorectal specimens fell within a similar molecular weight range (40-60 kDa), displayed similar levels of hydropathicity, had a similar frequency of gene ontology terms, and had a similar percentage of peptides with methionine oxidation regardless of storage duration or purification method.  When samples from all FFPE block storage time points were considered,  protein purification with detergent removal plates (DRP) resulted in the “highest percentage of uniquely identified peptides” and higher mean numbers of identified peptides and proteins than protein purification by acetone precipitation and formic acid resolubilization (APFAR) or Single-Pot Solid-Phase-enhanced Sample Preparation with hydrophilic interaction liquid chromatography and magnetic bead-based digestion (SP3/HILIC), although differences between purification methods were observed for specific FFPE block storage durations.  A statistical analysis of purification methods that considered FFPE block age determined that in FFPE blocks stored for 1 y, DRP resulted in a significantly different (higher) number of “validated peptide identifications” relative to the APFAR and SP3/HILIC purification methods (p< 0.0001) and a significantly different “distribution of validated peptide identifications” from the APFAR purification method (p=0.0002).  Among FFPE blocks stored for 10 y, the DRP purification method differed significantly from the APFAR method (p=0.0299) in “validated peptide identifications”.  The authors concluded that the efficiency of trypsin digestion was not affected by FFPE block storage duration.  The authors also concluded that while protein purification with DRP was superior to the other methods evaluated in terms of efficiency and reproducibility, the APFAR and SP3/HILIC purification methods displayed consistency across FFPE block storage durations. 

Studies

  1. Study Purpose

    This retrospective study evaluated the protein concentration, the number of peptides and proteins identified, protein integrity, and the reproducibility of label-free LC-MS/MS results in formalin-fixed, paraffin-embedded (FFPE) colorectal carcinoma specimens that were stored as blocks at room temperature for 1, 5, or 10 years prior to protein extraction and purification by three different methods.  Surgically resected low- or high-grade colorectal carcinoma specimens from 17 patients were used to represent each FFPE storage time point (1, 5, or 10 y); specimens were not case-matched. Additional details on fixation and processing were not provided. For each specimen, a total tumor area of 25 mm3 was manually microdissected from 25 µm-thick sections, deparaffinized in xylene, and washed before protein extraction using an SDS-based protocol. Protein extracts were stored at -80°C until protein yields could be determined with a BCA Protein Assay Kit. Protein extracts then underwent protein purification by one of three gel-free methods prior to LC-MS/MS analysis with a quadrupole-Orbitrap mass spectrometer: acetone precipitation and formic acid resolubilization, APFAR), (HILIC), Single-Pot Solid-Phase-enhanced Sample Preparation with hydrophilic interaction liquid chromatography and magnetic bead-based digestion (SP3/HILIC), and detergent removal plates (DRP).

    Summary of Findings:

    Protein yield was adversely and significantly affected by the duration of FFPE block storage at room temperature (p<0.0001), as FFPE blocks stored for shorter durations had higher protein yields.  The difference in the distribution of protein yields was also significant when FFPE blocks stored for 1 y (average yield = 3.82 mg/ml protein) were compared to those stored for 5 y (2.46 mg/ml protein) or 10 y (1.65 mg/ml protein), whereas blocks stored for 5 and 10 y did not differ significantly from each other.  

    When samples from all FFPE block storage time points were considered protein purification with DRP yielded the highest mean numbers of identified peptides and proteins, although differences between purification methods did occur among specific FFPE block storage durations.  A statistical analysis of purification methods that considered FFPE block age determined that in FFPE blocks stored for 1 y, DRP resulted in a significantly different (higher) number of “validated peptide identifications” relative to the APFAR and SP3/HILIC purification methods (p< 0.0001), although these methods did not differ from each other. The DRP purification method also differed from the APFAR method in the “distribution of validated peptide identifications” (p=0.0002) among FFPE blocks stored for 1 y (differences between the other purification methods were not significant). No significant effect of protein purification method was observed among FFPE blocks stored for 5 years.  Among FFPE blocks stored for 10 y, the DRP purification method differed significantly from the APFAR method (p=0.0299) in “validated peptide identifications” (differences between other purification methods were not significant). The APFAR purification method displayed the greatest overlap in the peptides identified across samples and storage durations (46.5%), followed by SP3/HILIC (45.4%) and DP3 (43%) methods. Conversely, the “highest percentage of uniquely identified peptides” occurred when protein extracted from FFPE blocks stored for 1 y was purified by the DRP method.  The molecular weight range of identified proteins (40-60 kDa) and peptides (>1000 Da and <2000 Da) were similar regardless of storage duration.  Further, the authors state that the levels of hydropathicity were similar across FFPE block storage time points and purification methods. Regarding reproducibility, PCA plots revealed clustering by FFPE block age for all purification methods. However, the DRP method had the lowest variance (10.73%) followed by the SP3/HILIC method (13.68%) and then the APFAR method (14.57%). Gene ontology (GO) annotation indicated that all FFPE block storage durations and purification methods had a similar frequency of GO terms, demonstrating an approximately equivalent usability, although statistical comparisons determined that slight but significant enrichment was present when protein from FFPE blocks that were stored for 1 y was purified by the DRP or APFAR method (p<0.05).  The authors concluded that the efficiency of trypsin digestion was not affected by FFPE block storage duration (the percentage of peptides that did not have any missed cleaves was ≥85% for samples that were purified by APFAR and DRP, and ≥80% for those purified by the SP3/HILIC method for all FFPE block storage durations). The percentage of peptides with methionine oxidation did not differ among FFPE block storage durations or between protein purification methods.  The authors concluded that while protein purification with DRP was superior to the other methods evaluated in terms of efficiency and reproducibility, the APFAR and SP3/HILIC purification methods displayed consistency across FFPE block storage durations. 

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Protein LC-MS or LC-MS/MS
    Peptide LC-MS or LC-MS/MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte purification DRP
    APFAR
    SP3/HILIC
    Storage Storage duration 1 y
    5 y
    10 y

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