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Methods Mol Biol. 2011;728:47-67. doi: 10.1007/978-1-61779-068-3_3.

In-depth analysis of a plasma or serum proteome using a 4D protein profiling method.

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  • 1Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.


Comprehensive proteomic analysis of human plasma or serum has been a major strategy used to identify biomarkers that serve as indicators of disease. However, such in-depth proteomic analyses are challenging due to the complexity and extremely large dynamic range of protein concentrations in plasma. Therefore, reduction in sample complexity through multidimensional pre-fractionation strategies is critical, particularly for the detection of low-abundance proteins that have the potential to be the most specific disease biomarkers. We describe here a 4D protein profiling method that we developed for comprehensive proteomic analyses of both plasma and serum. Our method consists of abundant protein depletion coupled with separation strategies - microscale solution isoelectrofocusing and 1D SDS-PAGE - followed by reversed-phase separation of tryptic peptides prior to LC-MS/MS. Using this profiling strategy, we routinely identify a large number of proteins over nine orders of magnitude, including a substantial number of proteins at the low ng/mL or lower levels from approximately 300 μL of plasma sample.

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