Multidimensional Protein Identification Technology (MudPIT): Technical Overview of a Profiling Method Optimized for the Comprehensive Proteomic Investigation of Normal and Diseased Heart Tissue

• Published in: Journal of the American Society for Mass Spectrometry Vol. 16; no. 8; pp. 1207 - 1220
• Main Authors: Kislinger, Thomas, Gramolini, Anthony O., MacLennan, David H., Emili, Andrew
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2005; Springer Nature B.V
• Subjects: Abundance; Animals; Cardiomyopathies - physiopathology; Data mining; Fractionation; Heart; Heart - physiology; Mass spectrometry; Mass Spectrometry - instrumentation; Mass Spectrometry - methods; Mice; Pattern recognition; Proteins; Proteomics - instrumentation; Proteomics - methods; Regulators
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2005.02.015

An optimized analytical expression profiling strategy based on gel-free multidimensional protein identification technology (MudPIT) is reported for the systematic investigation of biochemical (mal)-adaptations associated with healthy and diseased heart tissue. Enhanced shotgun proteomic detection coverage and improved biological inference is achieved by pre-fractionation of excised mouse cardiac muscle into subcellular components, with each organellar fraction investigated exhaustively using multiple repeat MudPIT analyses. Functional-enrichment, high-confidence identification, and relative quantification of hundreds of organelle- and tissue-specific proteins are achieved readily, including detection of low abundance transcriptional regulators, signaling factors, and proteins linked to cardiac disease. Important technical issues relating to data validation, including minimization of artifacts stemming from biased under-sampling and spurious false discovery, together with suggestions for further fine-tuning of sample preparation, are discussed. A framework for follow-up bioinformatic examination, pattern recognition, and data mining is also presented in the context of a stringent application of MudPIT for probing fundamental aspects of heart muscle physiology as well as the discovery of perturbations associated with heart failure.