Proteomics under pressure: development of essential sample preparation techniques in proteomics using ultrahigh hydrostatic pressure.
The minimization of preanalytical variables in sample preparation is imperative for successful discovery-driven and translational research involving large-scale biomolecular profiling. Here, we demonstrate a novel technique using high hydrostatic pressure in addition to several chaotropes and solvents to maximize efficiency of both cell lysis and enzymatic digestion while minimizing the time, manual involvement in sample processing, and preanalytical variability introduced prior to mass spectrometry-based proteomic analysis. The digestion techniques were evaluated and optimized for in-solution, in-gel, and on-membrane applications using protein standards and cell lysates. The lysis techniques were evaluated using human HepG2 cells. Our results demonstrate that the use of elevated pressure and organic solvents can achieve superior protein recovery of organelle-, complex-, and especially membrane-associated proteins, meanwhile obtaining more than a 20-fold increase in throughput with improved reproducibility. This study introduces the concept of ultrahigh-performance sample preparation platforms for targeted characterization of proteome subsets in biological systems.
HSPH Proteomics Resource, Department of Genetics and Complex Diseases, Harvard School of Public Health, Harvard University, 655 Huntington Avenue, SPH-1 Room 409, Boston, Massachusetts 02115, United States.
Emily
E
Freeman
Alexander R
AR
Ivanov
D056945:Hep G2 Cells; D006801:Humans; D006874:Hydrostatic Pressure; D008565:Membrane Proteins / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000737:chemistry; D012995:Solubility; D012997:Solvents / Q000737:chemistry
D008565:Membrane Proteins; D020543:Proteome; D012997:Solvents; D010447:Peptide Hydrolases