A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.

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Background: The Ity/Lsh/Bcg gene on mouse chromosome 1 regulates priming/activation of macrophages for antimicrobial and tumouricidal activity. A candidate gene expressed in macrophages has been identified by positional cloning and full-length sequence analysis, and encodes the Natural resistance-associated macrophage protein (Nramp). In this study, we have tested the hy-

Nramp1 (natural resistance-associated macrophage protein) was positionally cloned as the defective biallelic locus in inbred mouse strains associated with uncontrolled proliferation of obligate intracellular macrophage pathogens. The causative defect was described as G169D within membrane spanning domain 4 of a transporter. The biochemical activity of Nramp1 is implied from sequence conservation with Nramp2. Nramp2 encodes a divalent cation transporter and is the carrier of a defect in models of microcytic anaemia, associated with impaired intestinal iron uptake. Iron sequestration has been proposed as an antimicrobial mechanism. Therefore, such an activity for Nramp1 is consistent with model systems. Here we showed that Nramp1 directs iron transport within the macrophage. We describe stable, high-level Nramp1G169 allele-derived polypeptide expression in Balb/c Nramp1D169 RAW264.7 cells. Transfectants express levels, comparable to those in Nramp1G169-resistant macrophages, of a 90-100x103 MW Nramp1 polypeptide. Expression of the Nramp1 polypeptide correlates with lower cellular iron loads and a reduced chelatable iron pool following challenge with iron: nitrilotriacetate. Pulse chase experiments support an enhanced iron flux in expressing cells. These data are supported using the fluorescent iron probe calcein. In Nramp1G169-expressing cells we observed an increased iron flux into the cytoplasm from a calcein-inaccessible cellular location. These data suggest Nramp1, in resting macrophage cells, mobilizes iron, from an intracellular vesicle, which is destined for cell secretion. We propose that under these conditions Nramp1 plays a role in a salvage pathway of iron recycling.

Nramp1 (natural resistance-associated macrophage protein) controls innate immunity and encodes a transporter of unknown function. Here we describe an antibody to Nramp1 displaying immunoreactivity towards a mature heavily glycosylated polypeptide of 90^100 kDa and a precursor form of 45 kDa in macrophages. Ectopic expression of the Nramp1 cDNA in COS-1 cells demonstrates that Nramp1 modulates cellular iron levels following loading with low molecular weight iron chelates. Surprisingly, Nramp1 does not enhance iron uptake, but expression is associated with reduced cellular iron loads. We propose Nramp1 may play a role in a salvage pathway of iron recycling.z 1998 Federation of European Biochemical Societies.

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