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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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(A) Quantification of HER2 receptor protein in frozen (red bars)and FFPE (blue bars) BT474 xenograft tissues. HER2 protein was quantifiedbased on MRM of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of the receptor. MRM quantitationwas by stable isotope dilution. Plotted values are mean ± SDfor 3 process replicates of one frozen and one FFPE tumor. (B) Quantificationof HER2 receptor protein in 5 human HER2-positive and 5 HER2 negativehuman FFPE breast tumor tissues. MRM quantitation was by stable isotopedilution analysis of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of HER2. Plotted values are mean± SD for 3 process replicates of each specimen.
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fig5: (A) Quantification of HER2 receptor protein in frozen (red bars)and FFPE (blue bars) BT474 xenograft tissues. HER2 protein was quantifiedbased on MRM of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of the receptor. MRM quantitationwas by stable isotope dilution. Plotted values are mean ± SDfor 3 process replicates of one frozen and one FFPE tumor. (B) Quantificationof HER2 receptor protein in 5 human HER2-positive and 5 HER2 negativehuman FFPE breast tumor tissues. MRM quantitation was by stable isotopedilution analysis of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of HER2. Plotted values are mean± SD for 3 process replicates of each specimen.

Mentions: To explore the application of MRM for analysesof HER2 in FFPEhuman breast tumor tissues, we analyzed 5 HER2-overexpressing breastcancer tissues and 5 HER2-negative breast cancers, which had beenclassified based on previous IHC analysis in the Vanderbilt clinicalimmunohistochemistry laboratory. MRM analyses of these specimens (Figure 5B) demonstrate a clear difference in signal intensityfor HER2 peptides between the HER2+ and HER2 negative tumors and indicatea wide range of biological variability in receptor expression levels.Quantification of receptor levels was performed by stable isotopedilution. Assuming a yield of 200 pg protein from an average cell,we estimated HER2 receptor levels ranging from 110 000 to 468 000receptors per cell in the HER2 positive cancers and fewer than 14 000receptors per cell in the HER2 negative cancers. Because of the higherlimit of quantification for the DPPFCVAR peptide, it was not possibleto use this peptide to quantify receptor levels in all specimens.In tissues most highly overexpressing HER2, quantification of receptorlevels based on DPPFCVAR yielded the same result as ELVSEFSR quantification,suggesting no evidence of receptor ectodomain shedding in the tissuesanalyzed.


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)

(A) Quantification of HER2 receptor protein in frozen (red bars)and FFPE (blue bars) BT474 xenograft tissues. HER2 protein was quantifiedbased on MRM of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of the receptor. MRM quantitationwas by stable isotope dilution. Plotted values are mean ± SDfor 3 process replicates of one frozen and one FFPE tumor. (B) Quantificationof HER2 receptor protein in 5 human HER2-positive and 5 HER2 negativehuman FFPE breast tumor tissues. MRM quantitation was by stable isotopedilution analysis of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of HER2. Plotted values are mean± SD for 3 process replicates of each specimen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: (A) Quantification of HER2 receptor protein in frozen (red bars)and FFPE (blue bars) BT474 xenograft tissues. HER2 protein was quantifiedbased on MRM of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of the receptor. MRM quantitationwas by stable isotope dilution. Plotted values are mean ± SDfor 3 process replicates of one frozen and one FFPE tumor. (B) Quantificationof HER2 receptor protein in 5 human HER2-positive and 5 HER2 negativehuman FFPE breast tumor tissues. MRM quantitation was by stable isotopedilution analysis of peptides representing the extracellular (DPPFCVAR)and intracellular (ELVSEFSR) domains of HER2. Plotted values are mean± SD for 3 process replicates of each specimen.
Mentions: To explore the application of MRM for analysesof HER2 in FFPEhuman breast tumor tissues, we analyzed 5 HER2-overexpressing breastcancer tissues and 5 HER2-negative breast cancers, which had beenclassified based on previous IHC analysis in the Vanderbilt clinicalimmunohistochemistry laboratory. MRM analyses of these specimens (Figure 5B) demonstrate a clear difference in signal intensityfor HER2 peptides between the HER2+ and HER2 negative tumors and indicatea wide range of biological variability in receptor expression levels.Quantification of receptor levels was performed by stable isotopedilution. Assuming a yield of 200 pg protein from an average cell,we estimated HER2 receptor levels ranging from 110 000 to 468 000receptors per cell in the HER2 positive cancers and fewer than 14 000receptors per cell in the HER2 negative cancers. Because of the higherlimit of quantification for the DPPFCVAR peptide, it was not possibleto use this peptide to quantify receptor levels in all specimens.In tissues most highly overexpressing HER2, quantification of receptorlevels based on DPPFCVAR yielded the same result as ELVSEFSR quantification,suggesting no evidence of receptor ectodomain shedding in the tissuesanalyzed.

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