Urine is without doubt one of the engaging sources for early and delicate biomarker discovery since it could possibly accumulate and mirror adjustments within the human physique whereas being collected non-invasively.
Nonetheless, evaluation of the urine proteome presents challenges because of its large dynamic vary, spanning roughly 10 orders of magnitude in protein concentrations.
The presence of high-abundance proteins in urine can overshadow potential illness biomarkers, making their identification tough. Fractionation and depletion methods are generally employed earlier than performing mass spectrometry (MS) evaluation with a view to improve the detection and identification of those elusive biomarkers.
As a promising various, urine fractionation by ultracentrifugation (UC) will be employed to deplete high-abundance proteins, permitting for the isolation of exosomes with excessive purity whereas retaining many of the high-abundance proteins within the supernatant. Nonetheless, the robust centrifugal shearing power throughout UC can unavoidably disrupt the intact construction of exosomes, ensuing within the isolation of solely hint quantities from a small urine quantity.
With all the prevailing limitations in thoughts, a group of researchers in China proposed a novel solid-phase alkylation (SPA)-based pattern preparation technique for low-loss, anti-interference processing of sub-microgram proteomic samples.
Our technique combines UC fractionation, solid-phase extraction (SPA) pattern preparation, and liquid chromatography-mass spectrometry (LC-MS) to allow complete proteome profiling of urine, often known as CPU, or complete proteome profiling of urine. This facile technique resulted within the identification of a complete of 1,659 proteins utilizing a brief LC gradient of roughly 1 hour, which is 2.3 occasions greater than the 730 proteins recognized from uncooked urine with out fractionation.”
Huiming Yuan, Research Corresponding Creator and Professor, Dalian Institute of Chemical Physics, Chinese language Academy of Sciences
Notably, compared to current urine pattern preparation strategies, CPU presents important benefits. Not solely does it drastically scale back the evaluation time by 3-4 occasions, nevertheless it additionally enhances the identification protection of the urine proteome by 130%-160%.
“The tactic was additional employed together with label-free quantification to conduct comparative proteome evaluation of urine from each IgA nephropathy (IgAN) sufferers and wholesome donors,” shared lead creator Xinxin Liu, a technician at Dalian Institute of Chemical Physics, Chinese language Academy of Sciences. “In consequence, 227 differentially expressed proteins have been recognized, shedding mild on potential biomarkers related to IgAN.”
A number of members of the solute provider household 22 (SLC22) have been discovered to be up-regulated in IgAN sufferers, suggesting a possible hyperlink to the disruption of renal metabolic operate in these people.
The group reported their research within the KeAi journal Urine.
“Our outcomes demonstrated that our developed technique holds promise as a invaluable software for the invention of disease-related biomarkers in urine” Liu concluded.
Liu, X., et al. (2023) A facile technique for complete proteome evaluation of urine utilizing ultracentrifugation fractionation, solid-phase alkylation based mostly pattern preparation and liquid chromatography-mass spectrometry. Urine. doi.org/10.1016/j.urine.2023.05.002.