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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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CVs for MRM measurements of 52 lysine C-terminal peptides and 58arginine C-terminal peptides in 5 serial sections of frozen and FFPERCC samples. MRM quantitation was done by the LRP method. Comparisonof median CVs for peptide measurements from FFPE and frozen tissuesindicated no significant effect of fixation on the precision of peptidemeasurement (Mann–Whitney U test).
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fig2: CVs for MRM measurements of 52 lysine C-terminal peptides and 58arginine C-terminal peptides in 5 serial sections of frozen and FFPERCC samples. MRM quantitation was done by the LRP method. Comparisonof median CVs for peptide measurements from FFPE and frozen tissuesindicated no significant effect of fixation on the precision of peptidemeasurement (Mann–Whitney U test).

Mentions: Giventhe possible effects of formaldehyde fixation chemistry onlysine C-terminal tryptic peptides, the precision of MRM measurementwas analyzed separately for lysine and arginine C-terminal peptides.The median CVs for the 52 lysine C-terminal peptides were not significantlydifferent between analyses of fixed (median CV = 0.195) or frozentissue (median CV = 0.190) using the Mann–Whitney U test (Figure 2). Median CVs for analyses of the 58 arginine C-terminalpeptides also indicated no significant difference between fixed (medianCV = 0.183) or frozen tissue (median CV = 0.180, Figure 2). This result suggests that there is no discernible effectof formaldehyde fixation chemistry that affects the reproducibilityof peptide MRM measurement. In addition, there was no significantdifference in median CV between lysine C-terminal peptides (medianCV = 0.195) and arginine C-terminal peptides (median CV = 0.183) fromFFPE tissue, suggesting that MRM measurements of FFPE tryptic peptidesdisplay similar technical variation, regardless of the C-terminalamino acid.


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)

CVs for MRM measurements of 52 lysine C-terminal peptides and 58arginine C-terminal peptides in 5 serial sections of frozen and FFPERCC samples. MRM quantitation was done by the LRP method. Comparisonof median CVs for peptide measurements from FFPE and frozen tissuesindicated no significant effect of fixation on the precision of peptidemeasurement (Mann–Whitney U test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: CVs for MRM measurements of 52 lysine C-terminal peptides and 58arginine C-terminal peptides in 5 serial sections of frozen and FFPERCC samples. MRM quantitation was done by the LRP method. Comparisonof median CVs for peptide measurements from FFPE and frozen tissuesindicated no significant effect of fixation on the precision of peptidemeasurement (Mann–Whitney U test).
Mentions: Giventhe possible effects of formaldehyde fixation chemistry onlysine C-terminal tryptic peptides, the precision of MRM measurementwas analyzed separately for lysine and arginine C-terminal peptides.The median CVs for the 52 lysine C-terminal peptides were not significantlydifferent between analyses of fixed (median CV = 0.195) or frozentissue (median CV = 0.190) using the Mann–Whitney U test (Figure 2). Median CVs for analyses of the 58 arginine C-terminalpeptides also indicated no significant difference between fixed (medianCV = 0.183) or frozen tissue (median CV = 0.180, Figure 2). This result suggests that there is no discernible effectof formaldehyde fixation chemistry that affects the reproducibilityof peptide MRM measurement. In addition, there was no significantdifference in median CV between lysine C-terminal peptides (medianCV = 0.195) and arginine C-terminal peptides (median CV = 0.183) fromFFPE tissue, suggesting that MRM measurements of FFPE tryptic peptidesdisplay similar technical variation, regardless of the C-terminalamino acid.

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