Abstract
AbstractUrine reflects the renal function and urinary and kidney systems, but it may also reflect the presence of cancer in other parts of the body. Urine also has potential for providing prognostic information during therapeutic treatments thanks to non-invasive monitoring. A quick and reproducible protein purification procedure is essential to allow data comparison between proteomic studies in urine biomarker discovery. The article describes a simple, reproducible and cheap sample preparation procedure with a maximum protein yield (400 µg) obtained from only 10 mL of urine utilising cut-off filter desalting and digestion. The reported procedure removes yellowish background coloration residues and thus prevents the errors in spectrophotometric protein concentration determination. Different extraction solvents used in the presented procedure point to the possibility of partial elimination of abundant proteins (albumin and keratin family), as well as to the improvement of the sequence coverage of proteins identified, which helps to reveal changes in the urinary proteome. With this workflow, proteins can be easily obtained on standard laboratory equipment within 3 h. Data are available via ProteomeXchange with identifier PXD019738.
Highlights
The aim of preventive diagnostics in medicine is to find suitable biomarkers in accessible body fluids
Centrifugal Filter Units Centriprep® 3 K, 10 K, Amicon® Ultra-0.5 Centrifugal Filter Units, 3K, acetonitrile (ACN), ethanol and calcium chloride anhydrous (CaCl2) were obtained from Merck KGaA (Germany). 2-Dithiothreitol (DTT), tris(hydroxymethyl)aminomethane (TRIS) and urea were obtained from Bio-Rad GmbH (Germany); formic acid was obtained from Fisher Scientific (UK); iodoacetamide (IAA), sodium chloride (NaCl), ammonium bicarbonate (AMB) and acetone were from AppliChem GmbH (Germany)
The aim of the study was to optimize the method of protein extraction from urine samples
Summary
The aim of preventive diagnostics in medicine is to find suitable biomarkers in accessible body fluids. The human urine is a very complex matrix comprising 95% water and a mixture of water-soluble components such as urea, NaCl, KCl, various amounts of organic acids such as oxalic and citric acid, phosphates, poorly water-soluble uric acid and creatinine It contains soluble and insoluble proteins, peptides, extracellular vesicles, nucleic acids, cells and cell debris. The yellowish colour of the urine is mainly caused by urochrome, the haemoglobin degradation product, and by urobilin, an orange-brown pigment, and uroerythrin, which has a pink colour [6] All these colour components complicate proteomic determination, usually by clogging the HPLC nano-columns, and they influence protein concentration measurement by the Bradford method. Having a molar mass of less than 600 Da, they are expected to be
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