PRISM, a Generic Large Scale Proteomic Investigation Strategy for Mammals*S

Abstract

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.

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

“…Raw files were converted to m/zXML using ReAdW and searched using a locally installed version of X!TANDEM (version 2010.12.01.1) against a mouse or human UniProt database (version 2011_03, containing 16,351 mouse or 20,227 human protein sequences). To estimate and minimize our false-positive rate, the protein sequence database also contained every protein sequence in its reversed amino-acid orientation (target-decoy strategy) as recently described 60 . Search parameters were as follows: Parent ion D-mass of 4 Da, fragment mass error of 0.4 Da and complete carbaminomethyl modification of cysteine by iodoacetic acid (IAA) and a potential oxidation of cysteine.…”

Multidimensional protein identification technology (MudPIT): Technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue

“…A fully-automated 20 h long 12-step multi-cycle MudPIT procedure was set up as described previously [16].°Briefly,°an°HPLC°quaternary pump was interfaced with an LCQ DECA XP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). A 100-m i.d.…”

“…Individual samples were loaded manually onto separate columns using a pressure vessel. Chromatography solvent conditions°were°exactly°as°described°earlier° [16].…”

“…A comprehensive, nonbiased description of the proteome or set of expressed proteins in healthy and diseased cardiac tissue could provide a breakthrough in understanding of the pathogenesis of heart disease by furthering knowledge of unknown critical disease pathways, leading to novel diagnostic and therapeutic targets. Nevertheless, the complexity and markedly skewed composition of the cardiac muscle proteome represents a considerable experimental challenge and effective sample fractionation methods are required in order to detect low abundance proteins [16]. Historically, 2D-gel electrophoresis has provided a useful method for highresolution separation of complex protein samples, including cardiac tissue [13].…”

“…However, gel-based proteomic techniques are generally biased towards detection of high abundance housekeeping enzymes, with reduced detection of low abundance proteins, membrane proteins, and proteins with extremes in isoelectric point and molecular weight [17,18], limitations that are further compounded by the need to analyze many individual gel spots. To circumvent these problems, several groups have developed gel-free protein expression profiling strategies coupling high-efficiency liquid chromatography separation procedures with automated tandem mass spectrometry, allowing for large-scale 'shotgun' sequencing of complex mixtures [16]. The archetypal approach, termed MudPIT (for Multidimensional Protein Identification Technology) [19], pioneered in the laboratory of John Yates, III, has proven to be a remarkably effective and robust methodology for investigating global changes in protein expression as a function of development and disease [20 -22].…”