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

J Proteome Res

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.

Published: June 2012

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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368395PMC
http://dx.doi.org/10.1021/pr300130tDOI Listing

Publication Analysis

Top Keywords

ffpe frozen
24
ffpe
12
ffpe tissue
12
frozen tissues
12
multiple reaction
8
reaction monitoring
8
formalin-fixed paraffin-embedded
8
frozen tissue
8
peptides ffpe
8
c-terminal peptides
8

Similar Publications

Formalin-fixed paraffin-embedded tissue (FFPET), which is the most widely used pathology archive, usually has low-quality DNA and RNA due to extensive nucleic acid crosslinking. RNA fluorescence in situ hybridization (RNA-FISH) has been increasingly utilized in research and clinical settings to diagnose disease pathology. In this study, the effect of RNA degradation over archival time on RNA-FISH signals in FFPET and fresh frozen tissue (FFT) was systematically assessed.

View Article and Find Full Text PDF

Characterizing the expression of novel targets in normal and diseased tissues is a fundamental component of a target validation data package. Often these targets are presented to the pathology team for assessment with bulk or single-cell RNAseq data and limited to no spatial tissue expression data. hybridization to detect mRNA (RNAscope) is a valuable tool to (1) identify cells that may express the target protein and to corroborate protein expression during immunohistochemical (IHC) assay development or (2) to use as surrogate for single-cell expression IHC when antibodies are not available.

View Article and Find Full Text PDF

Robust collection and processing for label-free single voxel proteomics.

Nat Commun

January 2025

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA.

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures tissue heterogeneity, precluding proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single microscale tissue voxels and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics.

View Article and Find Full Text PDF

Low Bacterial Biomass in Human Pancreatic Cancer and Adjacent Normal Tissue.

Int J Mol Sci

December 2024

Department of Medicine, Vagelos College of Physicians & Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA.

The gut microbiome plays an important role in the carcinogenesis of luminal gastrointestinal malignancies and response to antineoplastic therapy. Preclinical studies have suggested a role of intratumoral gammaproteobacteria in mediating response to gemcitabine-based chemotherapy in pancreatic ductal adenocarcinoma (PDAC). To our knowledge, this is the first study to evaluate the impact of the PDAC microbiome on chemotherapy response using samples from human pancreatic tumor resections.

View Article and Find Full Text PDF

Studies have shown that the human microbiome influences the response to systemic immunotherapy. However, only scarce data exist on the impact of the urinary microbiome on the response rates of bladder cancer (BC) to local instillation therapy. We launched the prospective SILENT-EMPIRE study in 2022 to address this question.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!