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

Abstract

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. (J Am Soc Mass Spectrom 2005, 16, 1207-1220

Nuclear protein extraction from frozen porcine myocardium

“…Next, we tested several fractionation protocols based on differential centrifugation at different sucrose densities [8,13]. In our hands, these laborious protocols gave variable degrees of B23 and total protein recovery in the nuclear fraction (data not shown).…”

Strategies for Protein Separation

“…Though, greater sensitivity leads to much higher variability in peptide detection at each fractionation step from run to run, demanding a greater number of technical replicas, which compromises the overall throughput. Several groups have evaluated the number of replicates necessary to observe a particular percentage of the proteins in a sample (Liu et al, 2004;Slebos et al, 2008;Kislinger et al, 2005). On the other hand, Tabb et al (2009) concluded that a standardized platforms results a high degree of repeatability and reproducibility (around 70-80%) in protein identifications.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…Sethuraman et al [118] simultaneously identified and quantified oxidant-sensitive cysteine thiols from a rabbit heart membrane fraction treated with H 2 O 2 using isotope-coded affinity tag (ICAT) reagent [119], which specifically labels the thiol groups of free cysteine. Furthermore, dedicated MS-based methods have been developed to study oxidations at other specific residues (reviewed in [120]), and advancements in the fields of MudPIT [121] and "top-down" MS [122] greatly enhanced the ability to thoroughly interrogate protein modifications.…”