Establishment of a near‐standard two‐dimensional human urine proteomic map

Abstract

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.

Proteomic Analysis of Peritoneal Dialysate Fluid in Patients with Different Types of Peritoneal Membranes

“…The PDE proteome profile is similar but not identical to the urinary proteome profile previously reported in several studies, [8][9][10] indicating that CPD can partially work as an artificial kidney, but cannot completely replace the native kidney, a sophisticated organ for maintaining the normal physiology or homeostasis. Quantitative intensity analysis and ANOVA with Tukey's posthoc multiple comparisons revealed five protein spots whose intensity levels significantly differed among groups (see Table 2).…”

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

“…25 It is interesting to note that no more than 30 mL of urine was needed in our experiments while hundreds of milliliters of urine is typically used for gel-based analysis. [16][17][18][19]23 The pooling of samples from different individuals is typically observed in the published gel analysis of urines to increase the amount of proteins. 17,19 By pooling samples, one is likely to lose the individual information of the intrinsic components present in urine from a single patient at a given time.…”

“…[16][17][18][19]23 The pooling of samples from different individuals is typically observed in the published gel analysis of urines to increase the amount of proteins. 17,19 By pooling samples, one is likely to lose the individual information of the intrinsic components present in urine from a single patient at a given time. Thus, a reliable and accurate profiling technique employing a small amount of urine is essential for detecting urinary proteomes and for marker identification.…”

“…16 A near-standard 2-D map 17 and high-resolution 2-D gels 18 of urological tissues have contributed to the construction of a 2-D database. 11,19 The database also contains a listing of 339 proteins detectable in urine, 124 of which have been identified.…”

Optimizing a Proteomics Platform for Urine Biomarker Discovery

“…Impact of Abundant Protein Depletion-Depletion of abundant urine proteins such as albumin increases the number of identified protein spots on a two-dimensional gel (1,7,16). However, the effect of such depletion on the urine proteome, as perceived by iTRAQ, has not been addressed.…”