Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography.
Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.
Havugimana
Pierre C
PC
Banting and Best Department of Medical Research, Department of Medical Genetics and Microbiology, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1.
Wong
Peter
P
Emili
Andrew
A
D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007474:Ion Exchange; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods
D011506:Proteins