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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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Characteristics of the shotgun proteomic data set. Highqualitativeconcordance was observed for proteins identified in both FFPE andfrozen tissue digests. Peptides observed in FFPE tissue were biasedagainst lysine C-terminal peptides, indicated by a lower lysine toarginine peptide ratio. Error bars represent standard deviation from4 IEF replicates.
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fig1: Characteristics of the shotgun proteomic data set. Highqualitativeconcordance was observed for proteins identified in both FFPE andfrozen tissue digests. Peptides observed in FFPE tissue were biasedagainst lysine C-terminal peptides, indicated by a lower lysine toarginine peptide ratio. Error bars represent standard deviation from4 IEF replicates.

Mentions: Shotgun proteomeanalysis of fixed and frozen RCC tissue indicated a qualitative concordancein protein groups identified in both FFPE and frozen tissue (Figure 1A), as we reported previously for comparison offrozen and FFPE tissues.5 (Spectral countdata for the identified proteins is presented in Supporting Information Table S1; a full summary of the dataset is provided in the accompanying IDPicker report. Both are providedas Supporting Information.) Of 2165 totalprotein groups identified, 91% were found in both sample types. However,the resulting peptide identifications obtained from tryptic digestsof fixed tissue were biased against lysine C-terminal peptides (Figure 1B). Whereas in frozen tissue the ratio of lysineC-terminal peptides to arginine C-terminal peptides was 1.11, theratio in FFPE samples was reduced to 0.93. These observations arein agreement with our previous analyses of frozen and FFPE colon adenocarcinomatissue5 and are consistent with the knownreactivity of aldehydes toward primary amines, which preferentiallyconsumes lysine residues and thus affects the yield of lysine C-terminalpeptides. We thus asked whether the modification of lysine residueswould affect the quantitation of lysine C-terminal peptides in a trypticdigest of FFPE 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)

Characteristics of the shotgun proteomic data set. Highqualitativeconcordance was observed for proteins identified in both FFPE andfrozen tissue digests. Peptides observed in FFPE tissue were biasedagainst lysine C-terminal peptides, indicated by a lower lysine toarginine peptide ratio. Error bars represent standard deviation from4 IEF replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Characteristics of the shotgun proteomic data set. Highqualitativeconcordance was observed for proteins identified in both FFPE andfrozen tissue digests. Peptides observed in FFPE tissue were biasedagainst lysine C-terminal peptides, indicated by a lower lysine toarginine peptide ratio. Error bars represent standard deviation from4 IEF replicates.
Mentions: Shotgun proteomeanalysis of fixed and frozen RCC tissue indicated a qualitative concordancein protein groups identified in both FFPE and frozen tissue (Figure 1A), as we reported previously for comparison offrozen and FFPE tissues.5 (Spectral countdata for the identified proteins is presented in Supporting Information Table S1; a full summary of the dataset is provided in the accompanying IDPicker report. Both are providedas Supporting Information.) Of 2165 totalprotein groups identified, 91% were found in both sample types. However,the resulting peptide identifications obtained from tryptic digestsof fixed tissue were biased against lysine C-terminal peptides (Figure 1B). Whereas in frozen tissue the ratio of lysineC-terminal peptides to arginine C-terminal peptides was 1.11, theratio in FFPE samples was reduced to 0.93. These observations arein agreement with our previous analyses of frozen and FFPE colon adenocarcinomatissue5 and are consistent with the knownreactivity of aldehydes toward primary amines, which preferentiallyconsumes lysine residues and thus affects the yield of lysine C-terminalpeptides. We thus asked whether the modification of lysine residueswould affect the quantitation of lysine C-terminal peptides in a trypticdigest of FFPE 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