Protein Quantification Using a Cleavable Reporter Peptide

Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC–MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described

Biochemical and Physical Characterisation of Urinary Nanovesicles following CHAPS Treatment

<div><p>Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, <em>e.g.</em> by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.</p> </div

Protein identification in DTT and CHAPS supernatant.

<p>Partial list of proteins not previously reported in urinary exosomes and in 200,000 g supernatants.</p>a<p>Unique peptides on the total number of peptides.</p

TEM analysis.

<p>Transmission electron micrographs of <b>P18</b> (Panel A) and <b>P200</b> (Panel B) at 10,000× and 5,000× magnifications, respectively. High-magnification (50,000×) of CHAPS- (Panels C-F) and DTT-treated (Panels G-I) vesicle preparations are represented.</p

Protein identification comparisons.

<p>Venn diagram showing the distribution of the number of identified proteins presents in SN 200,000 g after CHAPS and DTT treatments. Protein identifications from the current study were compared to two other studies which were carried out using high-resolution mass spectrometers in gels on 200,000 g pellets after DTT treatment (Gonzales et al. 2008) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Gonzales1" target="_blank">[5]</a> and 200,000 g supernatants (Kentsis et al. 2009) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Kentsis1" target="_blank">[23]</a>.</p

Western blotting analysis.

<p>Rabbit anti-CD63, Rabbit anti-TSG101, rabbit anti-MGF-E8/lactadherin and rabbit anti-nephrin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Hara1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Khatua1" target="_blank">[50]</a>. Ten µg of protein of fractions obtained in Method 1 were loaded on the gels.</p

SDS-PAGE.

<p><b>Panel A:</b> Gel Acrylamide T 12% constant. Fifteen µg of protein per lane of crude preparation <b>Panel B:</b> Gel Acrylamide T 8% constant. Ten µg of protein per fraction obtained in Method 1. <b>Panel C:</b> Gel Acrylamide T 12% constant. Ten µg of protein per fraction obtained in Method 2.</p

Proteases activity.

<p>Membrane-bound DPP IV (Panel A) and NEP (Panel C) peptidase activity profiles recorded in absence and presence of 10 mM DTT. Samples were dialysed at a MWCO of 300 kDa. DTT pellet 200,000 g (sample 1), DTT SN 200,000 g (sample 3), CHAPS pellet 200,000 g (sample 2) and CHAPS SN 200,000 g (sample 4) are represented. Columns compare DTT vs CHAPS after dialysis with a membrane of MWCO 300 kDa and in the presence of 5 mM DTT. Values represent mean ± SD of units of peptidase (UP) per milligram of protein per minute. Panel B represents the Coomassie gel and DDP immunodetection of the same samples. Ten µg of protein per fraction obtained in Method 1 were loaded on the gels after 300 kDa MWCO dialysis.</p