The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MSbased proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates ϳ60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one-and two-dimensional LC-MS/MS revealed a 60 -80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of ϳ100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with >90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative.

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of A␤ protofibrils. Library screening yielded several molecules that stimulate A␤ aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts A␤(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37°C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-A␤ aggregates exhibit a low thioflavin T response. Like A␤ fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-A␤ aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous A␤ fibril formation reaction: Ϸ12 of the 39 A␤(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the A␤ molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in A␤ conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester A␤ in a form and͞or location that cannot engage the toxic pathway.amyloid ͉ chemical biology ͉ hydrogen exchange ͉ proline scanning