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Precision of multiple reaction monitoring mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue.

Sprung RW, Martinez MA, Carpenter KL, Ham AJ, Washington MK, Arteaga CL, Sanders ME, Liebler DC - J. Proteome Res. (2012)

Bottom Line: We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues.The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues.The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens.

View Article: PubMed Central - PubMed

Affiliation: Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, and Departments of ‡Biochemistry, §Pathology, and ¶Medicine, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.

ABSTRACT
We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18-20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens.

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MRM chromatograms for the HER2 intracellular domain peptideELVSEFSRfrom analyses of FFPE xenograft specimens derived from the HER2-overexpressingcell line BT474 (left) and the non-HER2-expressing cell line Sum159(right). MRM quantitation was by stable isotope dilution. MonitoredMRM transitions are depicted for the unlabeled, endogenous peptide,and the isotope-labeled internal standards (insets). The intensityscales (y-axis) are identical in the two sets ofplots.
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fig4: MRM chromatograms for the HER2 intracellular domain peptideELVSEFSRfrom analyses of FFPE xenograft specimens derived from the HER2-overexpressingcell line BT474 (left) and the non-HER2-expressing cell line Sum159(right). MRM quantitation was by stable isotope dilution. MonitoredMRM transitions are depicted for the unlabeled, endogenous peptide,and the isotope-labeled internal standards (insets). The intensityscales (y-axis) are identical in the two sets ofplots.

Mentions: We then analyzed FFPE and frozenxenograft tissue samples fromhuman BT474 and Sum159 (HER2-negative) breast cancer cell lines. Whereassignals from HER2 peptides were readily discernible in BT474 xenografts,HER2 peptide signals were absent in Sum159 xenografts, consistentwith their low level of HER2 expression (Figure 4). Estimates of HER2 receptor numbers from quantification of theDPPFCVAR peptide were 60% higher than from quantification of the ELVSEFSRpeptide. The DPPFCVAR peptide is derived from the ectodomain of theHER2 receptor, whereas ELVSEFSR is in the intracellular activationdomain of the receptor. This result could be the result of proteolyticshedding of the receptor ectodomain. Indeed, BT474 xenografts havebeen reported to shed the HER2 extracellular domain into the circulation.18


Precision of multiple reaction monitoring mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue.

Sprung RW, Martinez MA, Carpenter KL, Ham AJ, Washington MK, Arteaga CL, Sanders ME, Liebler DC - J. Proteome Res. (2012)

MRM chromatograms for the HER2 intracellular domain peptideELVSEFSRfrom analyses of FFPE xenograft specimens derived from the HER2-overexpressingcell line BT474 (left) and the non-HER2-expressing cell line Sum159(right). MRM quantitation was by stable isotope dilution. MonitoredMRM transitions are depicted for the unlabeled, endogenous peptide,and the isotope-labeled internal standards (insets). The intensityscales (y-axis) are identical in the two sets ofplots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368395&req=5

fig4: MRM chromatograms for the HER2 intracellular domain peptideELVSEFSRfrom analyses of FFPE xenograft specimens derived from the HER2-overexpressingcell line BT474 (left) and the non-HER2-expressing cell line Sum159(right). MRM quantitation was by stable isotope dilution. MonitoredMRM transitions are depicted for the unlabeled, endogenous peptide,and the isotope-labeled internal standards (insets). The intensityscales (y-axis) are identical in the two sets ofplots.
Mentions: We then analyzed FFPE and frozenxenograft tissue samples fromhuman BT474 and Sum159 (HER2-negative) breast cancer cell lines. Whereassignals from HER2 peptides were readily discernible in BT474 xenografts,HER2 peptide signals were absent in Sum159 xenografts, consistentwith their low level of HER2 expression (Figure 4). Estimates of HER2 receptor numbers from quantification of theDPPFCVAR peptide were 60% higher than from quantification of the ELVSEFSRpeptide. The DPPFCVAR peptide is derived from the ectodomain of theHER2 receptor, whereas ELVSEFSR is in the intracellular activationdomain of the receptor. This result could be the result of proteolyticshedding of the receptor ectodomain. Indeed, BT474 xenografts havebeen reported to shed the HER2 extracellular domain into the circulation.18

Bottom Line: We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues.The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues.The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens.

View Article: PubMed Central - PubMed

Affiliation: Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, and Departments of ‡Biochemistry, §Pathology, and ¶Medicine, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.

ABSTRACT
We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18-20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens.

Show MeSH
Related in: MedlinePlus