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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.

We hypothesized that one mechanism underlying the association between obstructive sleep apnea (OSA) and Alzheimer Disease is OSA leading to decreased slow wave activity, increased synaptic activity, decreased glymphatic clearance, and increased amyloid-β. Polysomnography and lumbar puncture were performed in OSA and control groups. Slow wave activity negatively correlated with cerebrospinal fluid amyloid-β-40 among controls and was decreased in the OSA group. Unexpectedly, amyloid-β-40 was decreased in the OSA group. Other neuronally-derived proteins, but not total protein, were also decreased in the OSA group, suggesting that OSA may affect the interaction between interstitial and cerebrospinal fluid.

The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1-3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8 -10 or the nearly identical CCPs 15-17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins onefourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

We have analyzed a soluble form of the glycoprotein (G) obtained from vesicular stomatitis virus (VSV) by treatment of intact virions with cathepsin D. This form lacks the carboxy-terminal and membrane-spanning domains and thus is analogous to the previously described secreted form of G, Gs. The molecular weight of the cathepsin D produced G, G(Cath D), measured by sedimentation equilibrium in the analytical ultracentrifuge is 57 600, indicating that it is a monomer. Intact G protein extracted from virions by octyl beta-D-glucoside also is monomeric, based on sedimentation equilibrium analysis. These results suggest that G may be monomeric in virions. The Stokes radii (Rs) of the two forms of G were obtained from their migration in nondenaturing polyacrylamide gradient gels. The Rs of G(Cath D) in the absence of nonionic detergent was 37 A; in the presence of nonionic detergent, it increased to 55 A. The Rs of detergent-extracted intact G was 63 A in nonionic detergent. From the molecular weight and Rs of G(Cath D), we calculated a sedimentation coefficient of 3.8 S; the value determined by centrifugation in a sucrose gradient was 3.7 S. Viruses such as VSV fuse with cell membranes at low pH [White, J., Matlin, K., & Helenius, A. (1981) J. Cell Biol. 89, 674-679]. We have used the fluorescent probe cis,trans,trans,cis-9,11,13,15-parinaric acid (cis-PnA) to detect a reversible conformational change in G(Cath D) when the protein was exposed to an acidic environment close to pH 5. cis-PnA binds to hydrophobic regions of protein, causing a quenching of the intrinsic tryptophan fluorescence and an increase in the fluorescence of the probe.