An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags

Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis

Summary of 2D-GE Differential Protein Spots.

<p>Venn diagram depicting gel spots that were identified at each time point. Numbers noted in parenthesis are the total number spots with differential intensity identified at that specific time point. Within each time point the breakdown of spots is provided in which the gel intensity suggested either increased (up arrow) or decreased (down arrow) expression relative to the control. A total of 109 spots were differentially expressed. In week one, 22 were unique to this time point whereas 8 were unique to week three and 37 were unique to week five. From the MS analysis, a total of 77 unique proteins were identified in both the mutant and control (paired) gel spots. The protein identifications included 49 increased and 22 decreased.</p

Protein Characterization.

<p>Distribution of differentially expressed proteins by cellular location (A), and biological process (B).</p