Protein phosphorylation analysis in archival clinical cancer samples by shotgun and targeted proteomics approaches.
Author(s): Gámez-Pozo A, Sánchez-Navarro I, Calvo E, Díaz E, Miguel-Martín M, López R, Agulló T, Camafeita E, Espinosa E, López JA, Nistal M, Vara JÁ
Publication: Mol Biosyst, 2011, Vol. 7, Page 2368-74
PubMed ID: 21617801 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of protein extraction method and formalin-fixation and paraffin-embedding on the detection of phosphorylated proteins.
Conclusion of Paper
The protein yields and phosphorylation patterns from formalin-fixed paraffin-embedded (FFPE) specimens were dependent on the protein extraction method used. Comparable levels of proteins and phosphoproteins were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) in frozen and FFPE specimens.
Studies
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Study Purpose
The purpose of this study was to determine the effects of protein extraction method on protein yield and phosphorylation using FFPE specimens. Protein was extracted from 3 7 µm sections of three different xylene deparaffinized FFPE specimens.
Summary of Findings:
The protein yield from FFPE specimens was highest when the SDS method was used for extraction and lowest when the ammonium bicarbonate (ABC)/acetonitrile (ACN) method was used. Staining with Pro-Q diamond revealed differences in the phosphorylation patterns between FFPE specimens extracted using 2%SDS and Qproteome, with higher levels of phosphorylation found after extraction with Qproteome.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Colorimetric assay Protein 1D/2D gels Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method FFPE PES (Agilent)
Qproteome (QIAgen)
SDS
Guanidine hydrochloride (GdnHCl)
ABC/ACN
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Study Purpose
The purpose of this study was to compare phosphorylation of FFPE and frozen specimens. Protein was extracted using guanidine hydrochloride (GdnHCl) from 3 7 µm sections of three non-small cell lung cancer (NCSLC) and 2 renal cell carcinoma (RCC) FFPE biopsy specimens and from 3 NCSLC frozen biopsy specimens.
Summary of Findings:
When FFPE specimens were used instead of frozen, comparable, but slightly fewer, numbers of unique proteins (151 versus 156) and phosphoproteins (49 versus 56) were identified. Of these, 28 phosphoproteins were identified in both FFPE and frozen specimens. In general, cellular compartments were equally represented using FFPE and frozen specimens, although using FFPE specimens, membrane-related proteins were slightly under-represented. The authors report that preservation method had no effect on the detection of phosphoproteins. Selected reaction monitoring was comparable when using frozen and FFPE specimens.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Peptide LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Preaquisition Diagnosis/ patient condition NSCLC
RCC