Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry I.Profiling an unfractionated tryptic digest

Abstract

The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

Effects of endurance exercise on the urinary proteome analyzed by 2‐D PAGE and Orbitrap MS

“…Each sample was prepared twice to enable the analysis with two different IPG strips (pH 3-6 and pH [5][6][7][8] to yield an optimal separation of proteins and cover a wide pH range. An equal amount of 400 mg of protein was used for the isoelectric focusing and MCSS was added to the sample to reach a total volume of 100 mL.…”

“…While glomerular dysfunction results in an increased excretion of medium and high molecular weight proteins, tubular changes are indicated by an increased appearance of low molecular weight proteins. The influence of exercise on the urinary proteome was investigated using different techniques for separation and identification of urinary proteins and a 2-D map of the urinary proteome in healthy volunteers was established with over 1500 proteins [6][7][8][9]. A recent study described the influence of different types of exercise on the 2-D PAGE pattern [10] and this study was expanded in the current work.…”

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

“…For healthy individuals, 30% of the urinary proteome has been estimated to originate from the plasma filtrate whereas the remaining 70% is believed to be derived from the kidneys and the urothelium (2). Until 2005, ϳ800 urinary proteins had been identified by various proteomic approaches (3)(4)(5)(6)(7). In 2006, a comprehensive proteomic study identified more than 1500 proteins from healthy human urine samples, simultaneously reflecting the complexity and the potential information concealed in the urinary proteome (8).…”

Concanavalin A-captured Glycoproteins in Healthy Human Urine

“…Several research teams have worked on profiling the healthy human urine proteome using electrophoresis and/or liquid chromatography followed by mass spectrometry identification (1)(2)(3)(4)(5).…”

“…Even for the other 83 proteins identified by single peptide with multiple hits, the reliability was still more than 97%. 94 proteins were identified in previous urine proteome studies (1)(2)(3)(4)(5). 43 proteins were also identified in serum Nglycoproteome (11)(12)(13).…”