2‐D hydrophilic interaction liquid chromatography‐RP separation in urinary proteomics – Minimizing variability through improved downstream workflow compatibility

Optimization of every step in a bottom‐up urinary proteomics approach was studied with respect to maximize the protein recovery and making the downstream steps in the workflow fully compatible without compromising on the amount of information obtained. Sample enrichment and desalting using centrifugal filtration (5 kDa cut‐off) yielded protein recoveries up to 97% when 8 M urea was used. Although yielding lower recoveries (88%), addition of Tris‐HCl/NaCl was considered a better choice due to good down‐stream compatibility. The consecutive depletion of HSA, using an immunoaffinity column was successfully adapted for use in urine. Separation of the trypsin generated peptides in an off‐line 2‐D chromatographic system consisting of a hydrophilic interaction liquid chromatography column, followed by a RP chromatography column showed a high peak capacity and good repeatability in addition to a high degree of orthogonality. All operations were modified in order to keep sample handling between every step to a minimum, reducing the variability of each process. In order to test the suitability of the full method in an extensive proteomic experiment, a urine sample from a kidney‐transplanted patient was analyzed ( n =6). The total variability of the method was identified with RSD values ranging from 11 to 30%. Eventually, we identified a total of 1668 peptides and 438 proteins from a single urine sample despite the use of low‐resolution MS/MS equipment. The optimized and “streamlined” complex method has shown potential for use in future urinary proteomic studies.