We have developed a systematic analytical approach, termed PRISM (Proteomic Investigation Strategy for Mammals), that permits routine, large scale protein expression profiling of mammalian cells and tissues. PRISM combines subcellular fractionation, multidimensional liquid chromatography-tandem mass spectrometry-based protein shotgun sequencing, and two newly developed computer algorithms, STATQUEST and GOClust, as a means to rapidly identify, annotate, and categorize thousands of expressed mammalian proteins. The application of PRISM to adult mouse lung and liver resulted in the high confidence identification of over 2,100 unique proteins including more than 100 integral membrane proteins, 400 nuclear proteins, and 500 uncharacterized proteins, the largest proteome study carried out to date on this important model organism. Automated clustering of the identified proteins into Gene Ontology annotation groups allowed for streamlined analysis of the large data set, revealing interesting and physiologically relevant patterns of tissue and organelle specificity. PRISM therefore offers an effective platform for in-depth investigation of complex mammalian proteomes.Molecular & Cellular Proteomics 2:96 -106, 2003.

Mouse-derived C2C12 myoblasts serve as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocyte formation comprehensively, we used large scale gel-free tandem mass spectrometry to monitor global proteome alterations throughout a time course analysis of the myogenic C2C12 differentiation program. The relative abundance of ϳ1,800 high confidence proteins was tracked across multiple time points using capillary scale multidimensional liquid chromatography coupled to high throughput shotgun sequencing. Hierarchical clustering of the resulting profiles revealed differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across the early, mid, and late stages of differentiation. Several hundred previously uncharacterized proteins were likewise detected in a stage-specific manner, suggesting novel roles in myogenesis and/or muscle function. These proteomic data are complementary to recent microarraybased studies of gene expression patterns in developing myotubes and provide a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.

Polymer microbeads are witnessing renewed interest for performing biomolecule recognition assays with distinct advantages over planar microarray technology. In this study, DNA hybridization assays are performed on the surfaces of 1-microm-diameter, synthetically modified polystyrene microbeads. The microbead surfaces contain varying amounts of poly(acrylic acid) as a source of carboxylate groups to which a DNA capture strand may bind. Through a series of controlled experiments in which the microbead carboxylate density and DNA:surface area ratios are systematically altered, we find that the density of carboxylate groups on the microbead surface may be the most important parameter affecting not only the total number of DNA strands that may bind to the microbead surface but, surprisingly, also the efficiency of DNA hybridization with complementary strands. These studies are aimed directly at understanding the physical interactions between DNA strands and an anionic microbead surface.

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