Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has become a valuable tool to address a broad range of questions in many areas of biomedical research. One such application allows spectra to be obtained directly from intact tissues, termed "profiling" (low resolution) and "imaging" (high resolution). In light of the fact that MALDI tissue profiling allows over a thousand peptides and proteins to be rapidly detected from a variety of tissues, its application to disease processes is of special interest. Matrix-assisted laser desorption/ionization (MALDI)1 mass spectrometry (MS) from intact tissues has had a major impact on the study of molecular biology and biochemistry by permitting sensitive, rapid, and molecularly specific analyses of peptides and proteins. This technology provides information on molecular weights with high mass accuracy and protein modifications such as phosphorylation and acetylation and allows for the identification of proteins by peptide sequencing combined with protein data base searches. Although a more traditional application of MALDI MS combines separation technology such as two-dimensional gels for analysis of relatively pure proteins obtained from a complex biological sample, the required procedures are lengthy. Most importantly, because these protocols involve tissue mincing and extraction, spatial localization and relative concentrations of a given protein are lost.Tissue profiling by MALDI MS can be performed on intact tissue sections for the determination of the spatial distribution of compounds and their relative expression levels without the need for molecularly specific exogenous compounds such as antibody-based reagents. Simple sample preparation protocols allow for fast and reliable analysis, typically resulting in the detection of over a thousand ion signals in the mass range of 2

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin ␤4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time-and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.

The mannose receptor is expressed on mature macrophages and immature dendritic cells, and functions to mediate phagocytosis of pathogens and capture of Ags for delivery to MHC class II-containing intracellular compartments. It has been previously reported that HIV-1-infected macrophages have reduced functions associated with the mannose receptor, including impaired Pneumocystis carinii phagocytosis and mannosylated albumin uptake. Several HIV-1-derived proteins including the Tat protein have been shown to transcriptionally repress host cell genes. The present study was undertaken to define the role of the HIV-1-derived protein Tat in HIV-mediated mannose receptor down-regulation. Cotransfection of the human macrophage cell line U937 with a Tat expression vector and a mannose receptor promoter-luciferase reporter construct resulted in down-regulation of mannose receptor promoter activity. This repression was targeted to the basal promoter. Expression of either one- or two-exon Tat resulted in decreased promoter activity. The addition of the transactivation response element (TAR) sequence enhanced the Tat-mediated repression. Down-regulation was also seen when transfected cells were treated with exogenously added Tat protein. These results are consistent with a mechanism whereby Tat reduces mannose receptor promoter activity by interfering with the host transcriptional initiation machinery, potentially resulting in decreased levels of surface mannose receptor available for Ag or pathogen capture.