Synthetic 2-DE image representing all protein spots present in plasma samples in the comparison between normal healthy controls and cirrhosis patients.

<p>Gels were run using pH 3–5.6 nonlinear immobilized pH gradient DryStrips with 9–16% (w/v) SDS-PAGE gradient gels and were stained using the fluorescent dye OGT 1238. The synthetic image shown was created using accurate spot matching as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603-Garcia1" target="_blank">[19]</a>. Differentially expressed features are indicated by arrows and the Swiss-Prot entry names are shown in parentheses. The names of selected proteins are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone-0039603-t002" target="_blank">Table 2</a> and a full list of all proteins shown on this image can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s008" target="_blank">Table S2</a>. N, feature present only in gels of plasma from normal healthy controls; C, feature present only in gels of plasma from cirrhosis patients; *, features present in gels of plasma from both normal healthy controls and cirrhosis patients but expressed to a higher extent in the group indicated. For complete gel figures, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s001" target="_blank">Figure S1</a>.</p

Western blot band densitometry.

<p>The five plots on the left show densitometry data for our five markers; from top to bottom: LTIP, complement C3d, apolipoprotein L1, apolipoprotein J and corticosteroid-binding globulin. The five plots on the right show densitometry data for all the markers that were blotted for in the ELF test (TIMP1 and PIIIP), FibroTest (apolipoprotein A1, alpha 2 macroglobulin and haptoglobin), Hepascore (alpha 2 macroglobulin) and FIBROSpect (TIMP-1, and alpha 2 macroglobulin). Each point represents the average band intensity for four patient samples. Error bars show +/− standard error.</p

Validation of the novel fibrosis markers by Western blotting indicates that they are promising compared to the markers in ELF test, FibroTest, Hepascore and FIBROSpect.

<p>The novel markers of fibrosis were validated alongside the markers for the ELF test, FibroTest, Hepascore and FIBROSpect using plasma samples from individuals in each of the seven Ishak stages of hepatic scarring as indicated at the top of the figure. (A) Western blots of our novel markers of fibrosis: LTIP, complement C3d, apolipoprotein L1 (ApoL1), apolipoprotein J (ApoJ), corticosteroid-binding globulin (CBG); (B) ELF test, FibroTest, Hepascore and FIBROSpect markers. Western blots of TIMP-1, PIIIP, apolipoprotein A1 (Apo A1), alpha 2 macroglobulin (a2M) and haptoglobin, ELISA data for hyaluronic acid (HA) and levels of bilirubin and gamma glutamyltranspeptidase. For hyaluronic acid, normal individuals are recognised to have hyaluronic acid below 120 ng/ml and cirrhotic patients above 250 ng/ml as indicated with the dashed lines. The two letter codes indicate if the marker is used in ELF test (EL), FibroTest (FT), Hepascore (HS) or FIBROSpect (FS).</p

Magnified regions of the gels showing changes for selected potential novel fibrosis biomarkers.

<p>The relative position of the identified protein is circled. (A) LTIP is present in normal plasma but decreased in plasma from cirrhotic patients; (B) Zinc-alpha-2-glycoprotein is present in normal plasma and decreased in plasma from cirrhotic patients; (C) Decreased feature of beta haptoglobin at pH 5.46–5.49. The top panel shows evenly spaced array of beta haptoglobin spots showing no significant difference between normal plasma and plasma from cirrhotic patients. The bottom panel shows zoomed image of the beta haptoglobin spot observed at approximately pH 5.46–5.49 which is present in normal plasma and decreased in plasma from cirrhotic patients; (D) Complement C3dg is absent in normal plasma but present in plasma from cirrhotic patients.</p

Uncleaved C3dg is elevated in hepatic cirrhosis.

<p>Using pH 3–5.6 gels, complement C3 was identified in a feature at approximately pH 4.9, MWt 38 kDa, only in gels for cirrhotic plasma. The full length sequence of complement C3 is shown with the alpha chain underlined, beta chain in italics, C3dg in bold and identified peptides highlighted in grey. Highlighted in black is the thioester site which is known to be cleaved by the fibrinolytic enzyme plasmin.</p

Details of the 20 plasma samples used for 2-DE and Western blotting.

<p>Sample name for 2-DE (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s001" target="_blank">Figure S1</a>) and lane number for Western blotting (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone-0039603-g004" target="_blank">Figure 4</a>) are shown.</p><p>na  =  not analysed.</p><p>Other clinical details for these samples and the other 30 plasma samples studied are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s007" target="_blank">Table S1</a>.</p