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J Biomol Tech. 2010 September; 21(3 Suppl): S54.
PMCID: PMC2918089

Label-free Differential Proteomics Using a Combination of MS Profiling and Targeted MS/MS

J. Meza
The Jackson Laboratory, Agilent Technologies, Bar Harbor, ME, United States



Differential proteomic analysis is an essential tool in the effort to elucidate the biochemical mechanisms that underlie phenotypes in mouse models of human diseases. There are many methods for differential proteomic analysis of complex samples, such as mouse tissue lysates, including two dimensional gel electrophoresis, iTRAQ and label-free methods. The Association for Biomolecular Resource Facilities proteomic research group study from 2007 (ABRF PRG07) evaluated multiple laboratories and methodologies for performing differential proteomics. Overall, label-free mass spectrometry (MS)-based methodologies were the most sensitive and most accurate for identifying and quantifying differentially expressed/spiked proteins. In an effort to evaluate a label-free method for (MS) based differential proteomic analysis, a combination of MS profiling and targeted MS/MS experiments have been used to identify tryptic peptides derived three proteins that were differentially spiked into the Sigma Proteomics Dynamic Range Standard Set (UPS2). LC/MS analyses were done on a microfluidic-based nanoflow LC coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer. The MS profiling data was processed using differential analysis software. Features that were differentially-spiked were subjected to targeted MS/MS and the peptides were identified using database search software. Our results illustrate the effectiveness of this approach, as all three unknown differentially-spiked proteins were successfully identified and quantitated. Beyond the QTOF itself, this study demonstrates the importance of both reproducible high resolution HPLC and appropriate software as being critical components of the described label-free method.

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