Evaluation of Urinary Protein Precipitation Protocols for the Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network

Abstract

Standardization of sample collection, shipping, and storage has been a major focus of biorepositories servicing large, multi-institute studies. The standardization of total protein concentration measurements may also provide an important metric for characterizing biospecimens. The measurement of total protein concentration in urine is challenging because of widely variable sample dilutions obtained in the clinic and the lack of a reference matrix for use with a standard curve and blank subtraction. Urinary proteins are therefore typically precipitated and reconstituted in a reference solution before quantitation. We have tested three different methods for protein precipitation and evaluated them using variability in total protein concentration measurement as a metric. The methods were tested on four urine samples ranging from very concentrated to very dilute. A method using a commercially available kit provided the most reproducible results, with average coefficients of variation <10%. Addition of a freeze/thaw did not lead to significant protein loss or additional variability. Samples were titrated and the measurements obtained appeared to be linearly correlated with sample starting volume. This method was applied to analysis of 77 urine biorepository samples and provided reproducible results when the same sample was assayed on different microwell plates.

Urinary Proteomic/Peptidomic Biosignature of Breast Cancer Patients Using 1D SDS-PAGE Combined with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry

“…The primary objectives of preprocessing are the selection of the m/z along with properly accurate intensity, and the designation of peaks with normalized intensities below a threshold as noise with consideration of the m/z values of interest [37]. A model of the spectra achieved from the BC patients after pre-treatment and peak alignment techniques following the Mass-UP options is exhibited in Figure 5.…”

Urine-HILIC: Automated Sample Preparation for Bottom-Up Urinary Proteome Profiling in Clinical Proteomics

“…The reported methods are based on precipitation [ 11 , 12 , 13 ], concentration [ 14 , 15 ], and on-membrane protein capture [ 16 , 17 , 18 ], thereby removing interfering compounds found in normal urine such as salts and other metabolites. The most common methods include acetone precipitation, trichloroacetic acid precipitation, ultracentrifugation, filter-aided sample preparation (FASP), and various combinations thereof [ 11 , 19 , 20 , 21 ]. After precipitation, protein resolubilization is often performed using urea-based buffers instead of more efficient detergent-based buffers, as detergent removal has historically been difficult to achieve [ 22 ].…”

Urine-HILIC: Automated sample preparation for bottom-up urinary proteome profiling in clinical proteomics

“…To date, a few groups have developed methods for urinary proteome profiling that are robust; however, reproducibility between laboratories remains a challenge. The reported methods are based on precipitation, concentration, and on-membrane protein capture [3][4][5][6], thus removing interfering compounds found in normal urine such as salts and other metabolites. The most common methods include acetone precipitation, acetone and trichloroacetic acid precipitation, ultracentrifugation, filter-aided sample preparation and various combinations thereof [3,4,7,8].…”

“…The reported methods are based on precipitation, concentration, and on-membrane protein capture [3][4][5][6], thus removing interfering compounds found in normal urine such as salts and other metabolites. The most common methods include acetone precipitation, acetone and trichloroacetic acid precipitation, ultracentrifugation, filter-aided sample preparation and various combinations thereof [3,4,7,8]. After precipitation, protein resolubilisation is often performed using urea-based buffers instead of more efficient detergent-based buffers, as detergent removal is difficult to achieve.…”