Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care

Targeted mass spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP Kinase, and AKT signaling.

SummaryA primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX-4720. These assays replace over 60 Western blots with quantitative mass spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism of action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov.MotivationA lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 Western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor

Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells

<div><p>The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation−mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous <i>hHGF</i> knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.</p></div

Novel antibodies detect upregulation of EMT transcription factor expression coinciding with reduced E-cadherin expression in a NSCLC xenograft model with constitutive HGF signaling.

<p>SCID or homozygous <i>hHGF</i> knock-in mice were implanted with H596 NSCLC tumors, and tumors were harvested 33 days after implantation. FFPE tissue sections were assessed by immunofluorescence microscopy after staining with DAPI (blue) or with fluorescence-conjugated anti-rabbit antibodies following application of primary antibodies to E-cadherin (green) and EMT TFs (red), including Snail <b>(A-C)</b>, Slug <b>(D-F)</b>, and Sox9 <b>(G-I)</b>. <b>(A, D, G)</b> Representative 20X magnification images for each group (SCID or <i>hHGF</i> knock-in). White arrows indicate examples of regions rich in hHGF-secreting mouse stromal cells, which are not recognized by the anti−human E-cadherin, Sox9, Slug, or Snail antibodies. White circles indicate the regions shown at higher magnification in panels <b>(C)</b>, <b>(F)</b>, and <b>(I)</b>, respectively. <b>(B, E, H)</b> Quantitation of tumor cell nuclear area positive for Snail <b>(B)</b> and Sox9 <b>(H)</b> and tumor area positive for Slug <b>(E)</b> was performed using Definiens software. Means ± standard deviations are shown (black bars). Each point represents the EMT TF expression value for a single tumor core; points with the same symbol indicate cores harvested from the same animal. Significant differences in EMT TF expression between SCID and homozygous <i>hHGF</i> knock-in mice are indicated (*<i>P</i> < 0.05, ***<i>P</i> < 0.001, N.S.: not significant; <i>n</i> = 3–6 animals per group). <b>(C, F, I)</b> High-magnification (60X) images of the circled regions in panels <b>(A)</b>, <b>(D)</b>, and <b>(G)</b>, respectively.</p

Western blot and immunofluorescence microscopy characterization of novel anti-GSC and anti-Sox9 antibodies in target protein−overexpressing cell lines and NAMEC8 xenograft models.

<p>Data are shown for GSC 1–5 <b>(A-C)</b> and Sox9 15-4 <b>(D-F)</b>. <b>(A</b>, <b>B</b>, <b>D</b> and <b>E)</b> <i>In vitro</i> target protein detection by EMT TF antibodies was assessed by comparisons of wild-type and constitutive target protein−overexpressing cell lines. <b>(A</b> and <b>D)</b> Western blot detection of GSC and Sox9, respectively. <b>(B</b> and <b>E)</b> 20X magnification immunofluorescence images show staining with DAPI (blue) and with the antibody of interest and fluorescence-conjugated anti-rabbit secondary antibody (green). <b>(C</b> and <b>F)</b> <i>In vivo</i> target protein detection by EMT TF antibodies was assessed by immunofluorescence microscopy analysis (60X magnification) of tumor tissue from NAMEC8 xenograft models (gold, EMT TF antibody; blue, DAPI; red, vimentin).</p