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

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Suppression of nitric oxide generation by antioxidants in alveolar macrophage-derived cell line MH-S

Preincubation of alveolar macrophages (AM) with bacterial endotoxin &I’S) enhances macrophage resistance to intracellular replication of pathogens and potentiates the activating effect of interferon-gamma (IFN-gamma). Such immunologic activation also induces the expression of nitric oxide synthase (iNOS) to generate reactive nitric oxide (NO) which contributes to the antimicrobial activity against pathogens. With the use of MH-S cell line derived from murine AM, we have examined the stimulatory effects of LPS (0.001 mg/ml) and IFN-gamma (100 U/ml) on extracellular NO production. The NO level in the culture medium was l&fold higher than basal value in 18 hours. In the p-ce of N(G)-methyl-L-arginine (L-NMMA, 1 mM), the amount of NO generated was suppressed to 19.3%, indicating that the in- in NO was the consequence of expression of iNOS. S-Methylisothiourea (SMT), a highly selective inhibitor of iNOS, was found to be very effective with an IC, of 0.01 mM concentration. Pyrrolidine dithiocarbamate (PDTC) and diethyldithiocarbamate (DETC) were both strong inhibitors of inducible NO production with IC, of 0.04 and 0.05 mM, respectively. Other antioxidank, e.g. N-acetylcysteine, also exhibit inhibitory effects. but comparatively weaker (IC, = 4 mM). To investigate the role of poly-ADP-ribosylation in iNO! induction, poly_(ADP-ribose) poly-merase (PARP) inhibitors, e.g. 3_aminobenzamide, were examined for their effects on NO production. It was found that all PARP inhibitors are weak inhibitors with IC,, in the range of 10-25 mM. In conclusion, the established transformed murine AM MH-S cell line can be useful in the study of cytokines and oxidative stress in microbicidal defenses of alveolar macrophages

Hotspot KRAS exon 2 mutations in CD166 positive colorectal cancer and colorectal adenoma cells

2017-2018 > Academic research: refereed > Publication in refereed journal201809 bcmaVersion of RecordPublishe

The effects of cyclooxygenase-2 inhibitors in galactosamine-lipopolysaccharide induced acute liver injury in mice

Accurate in-situ measurement of part dimensions during fabrication is of much interest to the manufacturing industry for process automation [1]. This work addresses one such application, with a specific goal to make precise on-line thickness measurements on thin metal parts of rotation: hemispherical shells 100 to 200 mm in diameter. Current manufacturing practice prevents monitoring the thickness before final inspection is performed at a separate metrology station. With the shells held in place by a vacuum chuck in a turning center, part access is restricted to one side for in-process monitoring, suggesting the ultrasonic technique for the measurement