EDTA-functionalized magnetic nanoparticles: A suitable platform for the analysis of low abundance urinary proteins

Abstract

Urine is a highly attractive source of biological information and disease biomarkers, whose proteome characterization is ongoing. To that end, depletion/enrichment strategies for protein analysis can be of great convenience. We have thus developed a method based on the use of EDTA-functionalized magnetic nanoparticles (NPs@EDTA), to fractionate urine samples before liquid chromatography-mass spectrometry analysis and compared the identified proteins with those obtained from ultrafiltrated/unfractionated (UF) urine samples. NPs@EDTA allowed larger urine volumes to be processed, resulting in a greater number of protein identifications (~2-fold) at a lower cost when compared to UF samples. Proteins of greater abundance (such as albumin and uromodulin) were, at least partially, depleted with NPs@EDTA while those of lower abundance were enriched. Bioinformatics analysis showed that approximately 27% of NPs@EDTA-enriched proteins were annotated as displaying enzymatic activity, most of these being hydrolytic enzymes (56%), particularly proteases/peptidases (48%). Also, post-translational modifications were prominently predicted across NPs@EDTA-enriched proteins (90%), particularly glycosylation (52%), phosphorylation (47%) and acetylation (30%). NPs@EDTA allowed the identification of 109 proteins in urine for the first time, showing high potential as a platform for urine's fractionation prior to proteomic analysis.

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans

“…Depleting albumins and immunoglobulins by affinity chromatography prior to MS analysis makes it possible to identify many LAPs in blood plasma (15). EDTA-functionalized nanoparticles effectively double the number of proteins detected in urine samples (16). Extraction protocols that favor recovery of LAPs over HAPs (beta-conglycinin and glycinin) from soybean seeds allow more LAPs to be identified (17).…”

An improved TurboID pipeline in C. elegans by biochemical depletion of endogenously biotinylated carboxylases

“…Depleting albumins and immunoglobulins by affinity chromatography prior to MS analysis makes it possible to identify many LAPs in blood plasma (15). EDTA-functionalized nanoparticles effectively double the number of proteins detected in urine samples (16). Extraction protocols that favor recovery of LAPs over HAPs (beta-conglycinin and glycinin) from soybean seeds allow more LAPs to be identified (17).…”