Optimisation of urine sample preparation for shotgun proteomics

Urine 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.