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Background Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies. Methods We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. Results HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors. Conclusions Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

Simple Summary Functionality of the cellular DNA damage response (DDR) network affects risk for developing cancer, and the DDR is also a target of cancer therapies. Thus, it is important that we have reliable laboratory methods for determining the activity of this network. We describe the development and analytical validation of a targeted mass spectrometry-based 51-plex assay (DDR-2) for measuring proteins and post-translational modifications related to the DDR. The findings demonstrate identification of potential novel pharmacodynamic biomarkers. Abstract The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

Summary: A 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 PLX4720. 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. Motivation: A 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 by 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.

RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute’s RAS Initiative has a focus on understanding pathways and discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network., Design Type(s)disease analysis objectiveMeasurement Type(s)immunocapture • immunoprecipitation • antibody • in situ immunoassay • ex situ immunoassayTechnology Type(s)western blot analysis • liquid chromatography-tandem mass spectrometry • immuno-MRM • immunohistochemistry • RPPA protein profiling assayFactor Type(s)Sample Characteristic(s)Homo sapiens • MCF-10A cell • BxPC-3 cell • NCI cell • breast • ovary • colon • lung • A549 cell • NCI-H1792 cell • HeLa cell • HEK293 Machine-accessible metadata file describing the reported data (ISA-Tab format)

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.

Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents.

Although high-grade serous ovarian cancers (HGSOC) are highly chemosensitive with an 85% initial response rate to platinum-based chemotherapy, 15% of patients are “exceptional nonresponders,” with platinum-refractory tumors that remain stable or progress during treatment. Unfortunately, we have no predictive biomarkers to identify refractory patients up front, and they receive futile chemotherapy through which most patients become too ill to be eligible for clinical trials. Hence, no progress has been made in treating these deadly tumors. The goal of this study is to identify mechanisms of platinum refractoriness to: i) predict refractory HGSOCs up front and ii) identify potential new drug targets in refractory disease to point to desperately needed new therapeutic approaches. Of note, 80-90% of patients who are initially platinum responsive will relapse and develop platinum-resistant disease, and it is possible that findings in platinum-refractory tumors might also provide insights into platinum-resistant tumors. Our NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC)-funded approach combines genomic and proteomic (“proteogenomic”) analyses of both preclinical models (0, 8, and 24 hours post-platinum exposure) and treatment-naïve human tumors. For preclinical models, we studied a well-characterized collection of patient-derived xenograft (PDX) models (10 sensitive, 10 refractory), as well as intrapatient HGSOC cell line pairs derived from patients before and after the development of platinum resistance. For the PDX models, proteogenomic profiling included RNASeq, WES, global proteomics, and phosphoproteomics at all 3 time points (0, 8, 24 hours). For cell line models (3 sensitive, 3 resistant), proteogenomic profiling was performed at all 3 timepoints (0, 8, 24 hours), and experiments were performed in complete biologic triplicate. Analyses included RNASeq, WES, global proteomics, phosphoproteomics, ubiquitin proteome, acetylated proteome, and pTyr. A large collection of 275 human HGSOCs (an approximate equal balance of platinum sensitive and refractory tumors) is currently undergoing proteomic profiling, and genomic profiles (WGS, RNASeq) will be performed on a subset. In parallel, we have performed a comprehensive review of 31 years of published work on platinum responses of human cancers, identifying ~700 genes implicated in the response and scoring each gene with respect to strength of the published evidence. Using a Bayesian approach, we are integrating the curated candidates from the literature with our empirical proteogenomic datasets to identify a candidate signature for detecting platinum-refractory disease prior to chemotherapy. We are also performing gene-regulatory network analysis to identify potential drivers of chemo response. NextGen, targeted, multiplex, multiple reaction monitoring mass spectrometry-based assays are being developed to quantify proteins in the signature for validation studies, using independent patient cohorts. This abstract is also being presented as Poster A62. Citation Format: Jacob J. Kennedy, Shrabanti Chowdhury, Sara R. Savage, Xiaonan Hou, Catherine J. Huntoon, Richard G. Ivey, Qing Yu, Chenwei Lin, Dongqing Huang, Lei Zhao, Uliana J. Voytovich, Regine M. Schoenherr, Zahra Shire, Steven J. Skates, Jeffrey R. Whiteaker, Andrew N. Hoofnagle, Samuel C. Mok, Bing Zhang, Larry M. Karnitz, S. John Weroha, Steven P. Gygi, Scott H. Kaufmann, Pei Wang, Michael J. Birrer, Amanda G. Paulovich. Proteogenomic approach to identify mechanisms of platinum refractoriness in high-grade serous ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR02.

Quantifying the many immunomodulatory proteins in the “cancer-immunity cycle” is critical for both patient selection and development of novel immunotherapies. Conventional technologies to measure proteins (IHC, ELISA, Western blot, flow cytometry) are often semiquantitative, not multiplexable, feature poor specificity, and are difficult to develop. Due to these limitations, investigators have turned to mRNA-based measurements to infer protein expression of immunomodulatory factors. However, as has been unequivocally demonstrated for human breast, colorectal, and ovarian cancers (Pubmed IDs: 27251275, 25043054, 27372738), RNA levels can be poor predictors of protein levels or activity. Thus, direct quantification of proteins is desirable. As part of the National Cancer Institute’s Cancer Moonshot, we are developing >300 multiplex assays for quantifying immunomodulatory proteins in tissue and plasma using a next-gen technology for protein quantification, based on targeted multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS complements traditional immunoassays by enabling highly specific, precise, harmonizable quantification of proteins in biospecimens, even at high multiplex levels. As proof of concept, we will present a novel multiplex assay based on next-gen MRM-MS for quantification of 47 immunomodulatory proteins in plasma and/or tumor tissue. The technology uses immunoaffinity enrichment coupled with MRM-MS (immuno-MRM assay). Assay bioanalytical validation was conducted in accordance with industry standards for use in FFPE tissue and plasma matrices and included determination of limits of detection, linear range of response, intraday and interday repeatability, stability of analytes in sample processing, and characterization of reproducibility of endogenous analyte measurement. The mean linear range was over three orders of magnitude with median intra- and interday CVs less than 10%. The characterized assays are being applied to a panel of tumor specimens from a variety of cancer types to document detection and determine minimum sample requirements. Additional assay panels are under development to provide measurements of up to 350 immunomodulatory proteins. Citation Format: Jeffrey R. Whiteaker, Lei Zhao, Richard G. Ivey, Julia Voytovich, Regine M. Schoenherr, Dongqing Huang, Jacob J. Kennedy, Amanda G. Paulovich. Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B8.

A lack of analytically robust and multiplexed assays has hampered studies of the large, branched phosphosignaling network responsive to DNA damage. To address this need, we developed and fully analytically characterized a 62-plex assay quantifying protein expression and post-translational modification (phosphorylation and ubiquitination) after induction of DNA damage. The linear range was over 3 orders of magnitude, the median inter-assay variability was 10% CV and the vast majority (∼85%) of assays were stable after extended storage. The multiplexed assay was applied in proof-of-principle studies to quantify signaling after exposure to genotoxic stress (ionizing radiation and 4-nitroquinoline 1-oxide) in immortalized cell lines and primary human cells. The effects of genomic variants and pharmacologic kinase inhibition (ATM/ATR) were profiled using the assay. This study demonstrates the utility of a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to studies on inter-individual variation in the radiation response.

The Fanconi anemia pathway is an important coordinator of DNA repair pathways and is particularly relevant to repair of DNA inter-strand crosslinks. Central to the pathway is monoubiquitination of FANCD2, requiring the function of multiple proteins in an upstream Fanconi core complex. We present development and analytical characterization of a novel assay for quantification of unmodified and monoubiquitinated FANCD2 proteoforms, based on peptide immunoaffinity enrichment and targeted multiple reaction monitoring mass spectrometry (immuno-MRM). The immuno-MRM assay is analytically characterized using fit-for-purpose method validation. The assay linear range is >3 orders of magnitude with total repeatability

Peptide immunoaffinity enrichment coupled with targeted mass spectrometry is a quantitative approach for the robust and reproducible quantification of peptide analytes. The approach is capable of multiplexed quantification of peptides, including post-translational modifications such as phosphorylation. Anti-peptide antibodies are used to enrich analytes and heavy stable isotope-labeled standards. The enriched peptides are directly measured by multiple reaction monitoring (MRM), a well characterized quantitative mass spectrometry-based method. Quantification is performed by measuring the analyte (light) peptide response relative to the heavy standard, which is spiked at a known concentration. Here, we describe the methodology for multiplexed measurement of phosphorylated peptides on the ATM kinase and their non-modified peptide analogs in cellular lysates. The method provides quantitative measurements of phospho-signaling and can be extended to a number of other phosphopeptides and sample types.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates immune responses. Previous studies in this laboratory have found that AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice suppresses the anti-cholera toxin (CT) IgA response, but fecal antibody levels recover at least two weeks earlier than serum levels after repeated immunization. Studies by others have shown that TCDD exposure increases splenic myeloid-derived suppressor cell (MDSC) numbers in mice, and MDSC have been associated with enhanced IgA responses. In this study, we hypothesized that TCDD exposure would increase MDSC numbers in CT-immunized mice. Female C57Bl/6 mice were administered peanut oil (vehicle) or TCDD (40 μg/Kg) orally on day 0, and each animal was orally immunized with cholera toxin (CT, 10 μg/Kg) on days 1 and 8. On day 14, CT-specific serum IgA levels (ELISA) were significantly suppressed by TCDD exposure to 21.6% of control (P < 0.05), but fecal IgA levels were not significantly different. Total spleen cell numbers were significantly reduced by TCDD exposure to 72.9% of control (P < 0.05), but neither the percent nor number of MDSC (CD11b+ Gr1+, by flow cytometry) per spleen was significantly altered: 2.21% ± 0.48% and 1.91% ± 0.23% (450,000 ± 98,000 and 290,000 ± 34,000) for control and TCDD-treated, respectively (mean ± SEM). These results suggest that AhR activation differentially impacts antigen-specific IgA responses between serum and feces in cholera toxin-immunized mice, but not the number of MDSCs.

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) utilizes antibodies to enrich peptides from complex matrices for quantitation by stable isotope dilution mass spectrometry. SISCAPA improves sensitivity and limits the sample handling required for plasma-based analysis. Thus far, SISCAPA assays have been performed using polyclonal antibodies, yet monoclonal antibodies are an attractive alternative since they provide exquisite specificity, a renewable resource, and the potential for isolation of clones with very high affinities (10−9 M or better). The selection of a good monoclonal antibody out of hundreds-to-thousands of clones presents a challenge, since the screening assay should ideally be in the format of the final SISCAPA assay, but performing the assays manually is labor- and time-intensive. In this manuscript, we demonstrate that monoclonal antibodies can be used in SISCAPA assays, and we describe an automated high-throughput SISCAPA method that makes screening of large numbers of hybridomas feasible while conserving time and resources.

We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome c and myoglobin generated Mascot MOWSE scores of 107 for cytochrome c and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively.

BACKGROUNDFor 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.CONTENTThe 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.

Multiple approaches for simplifying the serum proteome have been described. These techniques are generally developed across different laboratories, samples, mass spectrometry platforms, and analysis tools. Hence, comparing the available schemes is impossible from the existing literature because of confounding variables. We describe a head-to-head comparison of several serum fractionation schemes, including N-linked glycopeptide enrichment, cysteinyl-peptide enrichment, magnetic bead separation (C3, C8, and WCX), size fractionation, protein A/G depletion, and immunoaffinity column depletion of abundant serum proteins. Each technique was compared to results obtained from unfractionated human serum. The results show immunoaffinity subtraction is the most effective means for simplifying the serum proteome while maintaining reasonable sample throughput. The reported dataset is publicly available and provides a standard against which emergent technologies can be compared and evaluated for their contribution to serum-based biomarker discovery.

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.

A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.

Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately four orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.

Object classification at intersection scenarios is necessary in order to provide a general environment description. Objects are observed using a multilayer laserscanner. Significant features for object classification are identified and their extraction is described. Classification is performed using well-known techniques of statistical learning. Classification results of several neural networks are described and compared with classification performance of support vector machines.

Incomplete labeling of proteins by a derivatizing reagent usually results in the formation of a large number of products, which can produce unacceptable band broadening during electrophoretic analysis. In this paper, we report on the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) with the lysine residues of ovalbumin. Mass spectrometry was first used to determine the distribution in the number of labels attached to the protein. At room temperature, 3.6+/-1.9 labels were attached after 30 min. The reaction rate and number of labels increased at elevated temperatures. At 65 degrees C, 6+/-2.5 labels were attached after 5 min. The apparent activation energy for this reaction is estimated as 48+/-17 kJ/mol. Based on the mass spectrometry study, the labeling reaction was assumed to consist of two steps. In the first, the protein unfolds to make lysine residues accessible. In the second, the reagents react with the epsilon -amine of the lysine residues. To test this hypothesis, submicellar capillary electrophoresis and laser-induced fluorescence were used to characterize the reaction mixture. The apparent activation energy was measured for the labeling reaction; the apparent activation energy was 57+/-12 kJ/mol for reaction performed in the separation buffer. Denaturing agents were added to the reaction mixture. The addition of 2 M thiourea with 6 M urea to the reaction resulted in a modest decrease in the apparent activation energy to 42+/-2 kJ/mol. The addition of 2.5 M or higher concentration of ethanol decreased the apparent activation energy to 32+/-2 kJ/mol. We conclude that the apparent activation energy for protein labeling is dominated by denaturation of the protein, and that the addition of suitable denaturing reagents can eliminate this contribution to the reaction chemistry.

We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity.

Replication-deficient adenovirus expression vectors were used to introduce a recombinant DNA construct containing enhanced green fluorescent protein (EGFP) under control of a truncated, human heat shock promoter into human prostate cancer cells growing either exponentially or in plateau phase. This was done to measure controlled, heat shock-induced EGFP expression under conditions relevant to treating human cancers with heat-activated gene therapy. Both the temporal duration and magnitude of EGFP expression increased proportionately with stronger heat shocks (time at temperature) up to maximum values that were induced by 4 h at 41.0 degrees C or 2 h at 42.0 degrees C. Longer heat shocks at either temperature yielded no additional EGFP expression and ultimately reduced it. Maximal EGFP expression was induced in exponential cultures by heat shocks delivered 12-24 h after virus infection. Induction at progressively later postinfection times induced increasingly lower, peak EGFP expression. Maximal EGFP expression could not be induced until 48 h after infection of plateau phase cultures but could still be induced 180 h after virus infection. However, peak EGFP levels in plateau cultures were approximately 25-50% of those observed in identically induced exponential cultures. Ostensibly, the differences in expression from the heat shock promoter observed in exponential and plateau cultures were attributable to cell division diluting the vector within exponential cultures and the lower metabolic activity in serum-starved plateau cultures. For all experimental conditions, EGFP expression induced from the heat shock promoter was comparable with or higher than that from the constitutively active cytomegalovirus promoter over any 24-h period. The experimental results demonstrated that EGFP expression from the heat shock promoter was controllable in both exponential and plateau phase cultures and support the plausibility of using controlled heat shock activation of this promoter as a means of regulating both the spatial and temporal expression of therapeutic DNA constructs within human tissues. The ability to localize and regulate expression from the heat shock promoter may prove particularly advantageous for many cancer applications, especially if the therapeutic products are highly toxic, e.g., proteotoxins or cytokines. However, the results of this study suggest that differential growth conditions within tumors could markedly affect the expression of recombinant DNA under control of both inducible and constitutive promoters. Consequently, inducing schemes may need to be spatially adjusted to obtain the desired therapeutic results in all tumor domains using heat-activated gene therapy.