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

CE-microreactor-CE-MS-MS for protein analysis.

Thesis (Ph. D.)--University of Washington, 2006.Capillary electrophoresis-mass spectrometry (CE-MS) provides a powerful system that combines the speed and automation capabilities of CE with the detection and identification capabilities of MS. Integrating on-line enzymatic microreactor digestion with a CE-MS system reduces sample handling and allows fast digestions of small protein samples.In this thesis work, an on-line CE-microreactor-CE-MS-MS instrument is developed that can separate proteins in a first dimension, digest them inside a pepsin-immobilized microreactor, separate peptides in a second dimension, and detect the peptides with MS. The development begins from the MS end by coupling capillary free zone electrophoresis (CZE) to a linear ion trap mass spectrometer with a sheath flow interface, and works its way forward to the protein separation.Two CE-MS interfaces are tested and a sheath flow configuration is chosen for further instrument development because it provides robust electrosprays and does not cause band broadening. The CZE separation is optimized using bulk solution digested cytochrome C. Peptide-capillary wall interactions are reduced by coating capillaries with poly(vinyl alcohol) and by using high ionic strength buffers. The information dependent acquisition (IDA) capability of the mass spectrometer is used to obtain peptide fragment mass spectra that are submitted to a protein database search engine to identify cytochrome C.Pepsin-immobilized monolithic capillary microreactors are prepared and their homogeneity and digestion efficiency are investigated in both off-line and on-line experiments with CZE-ESI-MS. On-line coupling is achieved with a Plexiglas-glass interface. The microreactors are rather heterogeneous, yet cytochrome C was still successfully digested within 2 min. Hydroquinone (and p-benzoquinone) is added to buffers to alleviate CE current instabilities due to the electrolysis of water.The instrument is modified to a 2D CE-UV-microreactor-CE-ESI-MS-MS system by adding a protein separation capillary in front of the peptide separation. The protein separation capillary end that is coupled to the peptide separation dimension has an ∼ 1 cm microreactor integrated in it. A cytochrome C and myoglobin mixture is separated and identified using this 2D system. Only myoglobin is detected in a 6 protein sample, possibly because the other proteins contain disulfide bonds and/or are pepsin-resistant

Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues

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 (<i>R</i>² = 0.94) and immuno-MRM (<i>R</i>² = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens