Project Summary Many diseases are caused by a large number of combined effects, from genetic predisposition, to lifestyle and environmental factors; which makes understanding, treating, and curing these multifactorial diseases still a considerable challenge. Some of the unsolved questions can only be addressed in human tissue specimens. The availability of clinically annotated human tissue samples from banks and clinical repositories provides a golden opportunity to unravel cellular pathways that may lead to viable therapies. The investigation of numerous cell types and subtypes within their native tissue context calls for technologies to enable the analysis of single cells or small cell subpopulations from tissue. Current available technologies do not simultaneously fulfill the requisites of cellular resolution, minimum sample preparation, minimized sample loss, sample native state preservation, high sensitivity and specificity, and high throughput. As a result, to date, there is no commercial method extracting and analyzing the minute amounts of protein from a few cells procured from tissue through laser capture microdissection. We propose to develop a robust, quantitative, and sensitive assay, designated Laser-Capture Microdissection combined with Microfluidic Mass Spectrometry (LCM-MIMAS), to overcome the bottlenecks of current technology for quantifying peptides and proteins in single cells or small cellular ensembles that combines the power of LCM (cell selectivity and spatial organization), MALDI-TOF mass spectrometry (high mass accuracy, high speed data acquisition), and immunocapture (for high targeted sensitivity). For proof-of-principle, we will detect the peptide hormones insulin, glucagon, and somatostatin in cells from archived human pancreatic tissue (Aim 1), and amyloid beta (A?) in cells from archived human brain (Aim 2). We have chosen these two models because both have a high cellular complexity, are representative of two multifactorial diseases - diabetes and Alzheimer?s disease - for which there is no cure and where new technologies to assess molecular pathways could lead to novel or improved therapeutic approaches. Successful LCM-MIMAS will allow protein quantification with unprecedented detail on a vast array of biological systems within small cellular ensembles (1-100 cells) including neurodegenerative diseases, but also cancer, cardiovascular disease, diabetes, microorganism-host interactions, and many other diseases.