Precision of multiple reaction monitoring mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue.
Author(s): Sprung RW, Martinez MA, Carpenter KL, Ham AJ, Washington MK, Arteaga CL, Sanders ME, Liebler DC
Publication: J Proteome Res, 2012, Vol. 11, Page 3498-505
PubMed ID: 22530795 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of preservation method on levels of proteins identified by reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS).
Conclusion of Paper
The majority of peptides were identified in both formalin-fixed, paraffin-embedded (FFPE) and frozen specimens, with the remaining peptides generally found using only frozen specimens. Further, the majority of peptides were found to differ in levels between frozen and FFPE specimens, with lower levels found in FFPE specimens. For FFPE specimens, there was an overall bias against identification of peptides with a lysine at the c-terminal, but this was not found to be true when only a subset of peptides was examined. Human epidermal growth factor receptor 2 (HER-2) status could be properly assigned based on 25 HER-2 peptides using FFPE tissue.
Studies
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Study Purpose
The purpose of this study was to determine the effects of preservation method on levels of peptides identified by LC-MS/MS. The comparison between FFPE and frozen specimens was conducted using an unspecified number of renal cell carcinoma specimens. 5 HER2-positive and 5 HER2-negative FFPE breast tumors were used to investigate if LC-MS/MS could distinguish between tumor types in FFPE specimens.
Summary of Findings:
Of the 2165 proteins identified, 1971 (91%) were identified using both FFPE and frozen specimens, 183 (9%) were just identified using frozen specimens and 11 (0.5%) were identified using only FFPE specimens. For FFPE specimens, there was a bias against identification of peptides with a lysine at the c-terminal. However, the coefficient of variance (CV) of 52 peptides with a lysine or arginine at the c-terminal was comparable between FFPE and frozen specimens, and for FFPE specimens, the CV was similar for lysine and arginine c-terminal peptides. Of 114 selected peptides, 18 had a CV >25% (the limit of quantification, LOQ) in both frozen and FFPE specimens, 19 additional peptides were below the LOQ in FFPE specimens but not in frozen specimens, and 9 were below the LOQ in frozen specimens only. 58 peptides were above the LOQ in both frozen and FFPE specimens. Importantly, 70% of the 114 peptides had significantly different peak heights in FFPE and frozen specimens, the majority of which were due to lower levels in the FFPE specimen. HER-2 status could be properly assigned in all 10 breast carcinoma specimens analyzed based on 25 HER-2 peptides in FFPE tissue.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
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)
Snap frozen
LC-MS or LC-MS/MS Specific Targeted peptide/protein HER-2