Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts; §Eli

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.

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While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V, and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell.

Background:Researchers typically search for disease markers using a "targeted" approach in which a hypothesis about the disease mechanism is tested and experimental results either confirm or disprove the involvement of a particular gene or protein in the disease. Recently, there has been interest in developing disease diagnostics based on unbiased quantification of differences in global patterns of protein and peptide masses, typically in blood from individuals with and without disease. We combined a suite of methods and technologies, including novel sample preparation based on carrier-protein capture and biomarker enrichment, highresolution mass spectrometry, a unique cohort of wellcharacterized persons with and without Alzheimer disease (AD), and powerful bioinformatic analysis, that add statistical and procedural robustness to biomarker discovery from blood. Methods: Carrier-protein-bound peptides were isolated from serum samples by affinity chromatography, and peptide mass spectra were acquired by a matrixassisted laser desorption/ionization (MALDI) orthogo-

Background: Most cases of ovarian cancer are detected at later stages when the 5-year survival is ϳ15%, but 5-year survival approaches 90% when the cancer is detected early (stage I). To use mass spectrometry (MS) of serum proteins for early detection, a seamless workflow is needed that provides an opportunity for rapid profiling along with direct identification of the underpinning ions. Methods: We used carrier protein-bound affinity enrichment of serum samples directly coupled with MALDI orthagonal TOF MS profiling to rapidly search for potential ion signatures that contained discriminatory power. These ions were subsequently directly subjected to tandem MS for sequence identification. Results: We discovered several biomarker panels that enabled differentiation of stage I ovarian cancer from unaffected (age-matched) patients with no evidence of

In this study, we sought to explore the merit of proteomic profiling strategies in patients with cancer before and during radiotherapy in an effort to discover clinical biomarkers of radiation exposure. Patients with a diagnosis of cancer provided informed consent for enrollment on a study permitting the collection of serum immediately before and during a course of radiation therapy. High-resolution surfaceenhanced laser desorption and ionization-time of flight (SELDI-TOF) mass spectrometry (MS) was used to generate high-throughput proteomic profiles of unfractionated serum samples using an immobilized metal ion-affinity chromatography nickel-affinity chip surface. Resultant proteomic profiles were analyzed for unique biomarker signatures using supervised classification techniques. MS-based protein identification was then done on pooled sera in an effort to begin to identify specific protein fragments that are altered with radiation exposure. Sixty-eight patients with a wide range of diagnoses and radiation treatment plans provided serum samples both before and during ionizing radiation exposure. Computer-based analyses of the SELDI protein spectra could distinguish unexposed from radiation-exposed patient samples with 91% to 100% sensitivity and 97% to 100% specificity using various classifier models. The method also showed an ability to distinguish high from low dose-volume levels of exposure with a sensitivity of 83% to 100% and specificity of 91% to 100%. Using direct identity techniques of albuminbound peptides, known to underpin the SELDI-TOF fingerprints, 23 protein fragments/peptides were uniquely detected in the radiation exposure group, including an interleukin-6 precursor protein. The composition of proteins in serum seems to change with ionizing radiation exposure. Proteomic analysis for the discovery of clinical biomarkers of radiation exposure warrants further study. (Cancer Res 2006; 66(3): 1844-50)

Mass spectrometry profiling of human serum generated a robust classifier composed of seven low-molecular-weight ions that yielded a highly sensitive and specific diagnostic procedure for the discrimination of women with stage 1 breast cancer compared with women without breast cancer in this research study set.