Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and ␤-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National A rapidly evolving approach to protein quantitation is the targeted analysis of representative peptides by liquid chromatography-tandem mass spectrometry by multiple reaction monitoring (LC-MRM-MS) 1 analysis (1-3). In this approach, peptides are quantified by monitoring several MRM transitions for each peptide with either a triple quadrupole or a quadrupole-ion trap instrument. Stable isotope dilution (SID), in which labeled peptides are used as internal standards is considered the gold standard for rigorous quantitation by LC-MRM-MS (1, 4, 5). In contrast to antibody-based quantitation, where antibody availability and specificity are often limiting, LC-MRM-MS enables configuration of an assay for essentially any protein. In practice, this approach has proven sensitive enough to apply to challenging protein quantitation problems. For example, proteins can be quantified at singledigit copy numbers in cells (6) and in plasma at levels approaching ng/ml (7,8). With antibody-based enrichment, LC-MRM-MS can achieve even greater sensitivity (9 -12).

Liver microsomes are widely used to study xenobiotic metabolism in Vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)-butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB-but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.

Advances in proteomic analysis of human samples are driving critical aspects of biomarker discovery and the identification of molecular pathways involved in disease etiology. Toward that end, in this report we are the first to use a standardized shotgun proteomic analysis method for in-depth tissue protein profiling of the two major subtypes of nonsmall cell lung cancer and normal lung tissues. We identified 3621 proteins from the analysis of pooled human samples of squamous cell carcinoma, adenocarcinoma, and control specimens. In addition to proteins previously shown to be implicated in lung cancer, we have identified new pathways and multiple new differentially expressed proteins of potential interest as therapeutic targets or diagnostic biomarkers, including some that were not identified by transcriptome profiling. Up-regulation of these proteins was confirmed by multiple reaction monitoring mass spectrometry. A subset of these proteins was found to be detectable and differentially present in the peripheral blood of cases and matched controls. Label-free shotgun proteomic analysis allows definition of lung tumor proteomes, identification of biomarker candidates, and potential targets for therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015370, 916 -932, 2012.Lung cancer is one of the deadliest cancers, with ϳ200,000 newly diagnosed individuals and 160,000 deaths every year in the United States (1). Despite the most advanced treatments that modern medicine has to offer, the five-year survival rate remains less than 15%. Although a small subset of tumors have been found to be driven by single mutated oncogenes for which active, but still noncurative, therapies are available, the vast majority of patients have complex multifactorial disease with few effective therapeutic options. New early detection strategies and molecular therapeutic targets are urgently needed to improve patient survival.Genomic analysis has enabled us to measure the sequence, copy number, and expression changes of thousands of genes simultaneously, which can be used to discover transcripts specifically altered or expressed in tumor tissues (2-4). Although genomic studies have given important new insights into the mechanisms of carcinogenesis, therapeutic targets, and most practical biomarkers are their protein products, and the correlation between transcript sequence or level and protein function remains complex and poorly understood. Protein expression, in part, depends on transcript levels, but it is clear that significant translational and post-translational regulation of protein levels and function occurs, adding another level of complexity in the regulation of activity, especially in tumor cells (5, 6). It would be ideal to have a comprehensive understanding of the novel changes in protein expression levels and the modifications of proteins in cancer cells, but the technology to directly study proteomes has lagged behind that to assess genomes and transcriptomes. We and others have used matrix-assisted laser desorption and...

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A novel epoxy thermoset is synthesized with dynamic SiOSi bonds. The thermosetting epoxy resins possess excellent reprocessing, reshaping, and shape memory ability due to the special molecular structure. The material exhibits high tensile strength and a fast stress relaxation process. Surprisingly, it can achieve complete healing and keep stable mechanical property after multi‐processing.

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