Limits...
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.

Show MeSH

Related in: MedlinePlus

Log2 ratios for average normalized peak areas from FFPEversusfrozen tissues. MRM quantitation was done by the LRP method. Peptidesare ordered on the x-axis in order of decreasinglog2 ratios (y-axis values). Equivalent intensitiesyield a value of zero. Log2 ratios less than zero indicate higherpeak areas from peptides derived from frozen tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3368395&req=5

fig3: Log2 ratios for average normalized peak areas from FFPEversusfrozen tissues. MRM quantitation was done by the LRP method. Peptidesare ordered on the x-axis in order of decreasinglog2 ratios (y-axis values). Equivalent intensitiesyield a value of zero. Log2 ratios less than zero indicate higherpeak areas from peptides derived from frozen tissue.

Mentions: We also consideredthe effect of fixation on the magnitude of thepeak areas observed during MRM analysis, a more direct measurementof relative peptide abundance. Of the 114 peptides monitored, 70%showed a significant difference in average normalized peak area betweenfixed and frozen samples (two tailed t test, p < 0.05, Supporting InformationFigure 1). Figure 3 plots the log2 ratioof LRP-normalized peak areas for peptides from FFPE to frozen tissues.These data demonstrate that peptides from FFPE tissue generally yieldlower normalized average peak areas than peptides from frozen tissue,given the predominance of log2 (FFPE:frozen) peptide intensity valuesbelow zero in Figure 3. This result furtherillustrates the significant impact of fixation on the quantitationof proteins in tissue.


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)

Log2 ratios for average normalized peak areas from FFPEversusfrozen tissues. MRM quantitation was done by the LRP method. Peptidesare ordered on the x-axis in order of decreasinglog2 ratios (y-axis values). Equivalent intensitiesyield a value of zero. Log2 ratios less than zero indicate higherpeak areas from peptides derived from frozen tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Log2 ratios for average normalized peak areas from FFPEversusfrozen tissues. MRM quantitation was done by the LRP method. Peptidesare ordered on the x-axis in order of decreasinglog2 ratios (y-axis values). Equivalent intensitiesyield a value of zero. Log2 ratios less than zero indicate higherpeak areas from peptides derived from frozen tissue.
Mentions: We also consideredthe effect of fixation on the magnitude of thepeak areas observed during MRM analysis, a more direct measurementof relative peptide abundance. Of the 114 peptides monitored, 70%showed a significant difference in average normalized peak area betweenfixed and frozen samples (two tailed t test, p < 0.05, Supporting InformationFigure 1). Figure 3 plots the log2 ratioof LRP-normalized peak areas for peptides from FFPE to frozen tissues.These data demonstrate that peptides from FFPE tissue generally yieldlower normalized average peak areas than peptides from frozen tissue,given the predominance of log2 (FFPE:frozen) peptide intensity valuesbelow zero in Figure 3. This result furtherillustrates the significant impact of fixation on the quantitationof proteins in tissue.

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