One-Step Sample Concentration, Purification, and Albumin Depletion Method for Urinary Proteomics

Abstract

Workflows in urinary proteomics studies are often complex and require many steps to enrich, purify, deplete and separate the complex mixture. Many of these methods are laborious, timeconsuming and have the potential for error. Although individual steps of these methods have been previously studied, their downstream compatibilities with fractionation technologies such as OffGel electrophoresis have not been investigated. We developed a One-Step sample preparation workflow that simultaneously i) concentrates proteins, ii) purifies by removing salts and other low molecular weight compounds and iii) depletes (albumin) from urine samples. This simple and robust workflow can be multiplexed and is compatible with a diverse range of downstream multidimensional separation technologies. Additionally, due to its high reproducibility and flexibility in processing samples with different volumes and concentrations, it has the potential to be used for standardization of urinary proteomics studies, as well as for studying other body fluids of similar complexity.

Urinary Proteomics Yield Pathological Insights for Ureteropelvic Junction Obstruction

“…All samples were negative for infection, blood, and protein. All samples were centrifuged at 4500 g for 20 min to remove cellular debris, aliquoted, and frozen at -80 C in 1.5 ml centrifuge tubes (Eppendorf) until sample preparation by our previously published protocol (10). An overview of the sample preparation is shown in Fig.…”

Untitled

“…The depletion of albumin in urine was performed according to a published one-step protocol [15], and the protein concentration was measured by the Bradford assay in triplicate. Approximately 250 µg of depleted urinary proteins from each donor were further processed according to the GlycoFilter platform to obtain released N-glycans and tryptic peptides [8], respectively.…”

“…Tryptic peptides from three different urine samples were focused into 24 fractions using a 3100 OFFGEL fractionator (Agilent, Santa Clara, CA) as described previously [15]. Briefly, the 24cm, pH 3-10 IPG DryStrips (GE healthcare) were rehydrated for 20min with the IPG buffer pH 3-10.…”

Applicability of 2D gel electrophoresis and liquid chromatography in proteomic analysis of urine using mass spectrometry MALDI-TOF

“…To make this stage of investigations more efficient, the column should be coupled with the spectrometer or a small-mass marker, which will appear as the first in the effluent should be used. It should eluate close to the retention time of the first of the proteins to wash out all interferents of the mass close to the lightest protein with its simultaneous "rescue" (Vaezzadeh et al 2010, Court et al 2011). An important factor affecting the quality of the sample prepared for 2D electrophoresis is its conductivity.…”