Mascot analysis of fibrin clot-bound proteins.

<p>Protein spots shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041966#pone-0041966-g001" target="_blank">Fig. 1A</a> were analyzed by mass spectrometry. Proteins with an asterisk were analyzed with MALDI-ToF and the other protein spots were analyzed with nanoflow LC-MS/MS. Accession number of the NCBInr database, protein description, Mowse score, sequence coverage (%), calculated molecular weight (Mw), total identified peptides, unique identified peptides and the calculated pI are given.</p><p>HFREP-1; hepatocyte-derived fibrinogen related protein-1.</p

An overview of non-covalently fibrin clot-bound plasma proteins.

<p>Plasma clots were made by adding CaCl₂, thrombin and aprotinin to platelet-poor citrated normal plasma, unbound proteins were washed away and bound proteins were extracted. These proteins were separated with 2D gel electrophoresis and visualized by Sypro Ruby. A) The numbers and arrows indicate the protein spots that were excised from gel and analyzed with mass spectrometry. B) The trains of spots that resemble the same protein are indicated by white ellipses. They include: fibronectin (I), α₂-macroglobulin (II, III and VIII), plasminogen (IV), FXIII A chain (V), albumin (VI), α₁-antitrypsin (VII), apolipoprotein J (IX), apolipoprotein E, HFREP-1 (X) and apolipoprotein A-I (XI). C) A zoomed image of the 2D gel with a lower fluorescent signal. The isoforms of α₁-antitrypsin (A), apolipoprotein J (B) and apolipoprotein A-I (C) are indicated by white ellipses.</p

Western blot analysis with specific α₂-macroglobulin antibodies.

<p>Fibrin clot-bound plasma proteins were separated with 2D gel electrophoresis and analyzed with Western blot analysis using specific α₂-macroglobulin antibodies. The arrows indicate the three different α₂-macroglobulin trains that were also identified as α₂-macroglobulin with mass spectrometry (protein spots 2, 3 and 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041966#pone-0041966-g001" target="_blank">figure 1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041966#pone-0041966-t001" target="_blank">table 1</a>). The molecular mass of the protein marker is indicated in kDa.</p

Western blot analysis for apolipoproteins in purified fibrinogen.

<p>Fibrinogen was isolated from plasma with immunoaffinity chromatography and run on SDS-PAGE. Different apolipoproteins were detected with Western blot analysis using specific antibodies. Lane 1: protein marker, lane 2: apolipoprotein A-I (Mw = 28,900), lane 3: apolipoprotein J (Mw = 37,000), lane 4: apolipoprotein A-II (Mw = 8,700). Only the relevant section of the gel is shown.</p

Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in <i>Drosophila</i>

The ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in <i>Drosophila</i> S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, with the overall impact on the ubiquitinome being the most dramatic. The abundances of ∼10% of all proteins are increased, while the abundances of the far majority of over 14 000 detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Because this is to our knowledge the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the <i>Drosophila</i> community

Integrative Analysis of Genomics and Proteomics Data on Clinical Breast Cancer Tissue Specimens Extracted with Acid Guanidinium Thiocyanate–Phenol–Chloroform

Acid guanidinium thiocyanate, phenol, and chloroform extraction (AGPC) is a commonly used procedure to extract RNA from fresh frozen tissues and cell lines. In addition, DNA and proteins can be recovered, which makes AGPC an attractive source for integrative analysis on tissues of which little material is available, such as clinical specimens. Despite this potential, AGPC has only scarcely been used for proteomic analysis, mainly due to difficulties in extracting proteins. We have used a quantitative mass spectrometry method to show that proteins can readily be recovered from AGPC extracted tissues with high recovery and repeatability, which allows this method to be used for global proteomic profiling. Protein expression data for a selected number of clinically relevant markers, of which transcript and protein levels are known to be correlated, were in agreement with genomic and transcriptomic data obtained from the same AGPC lysate. Furthermore, global proteomic profiling successfully discriminated breast tumor tissues according to their clinical subtype. Lastly, a reference gene set of differentially expressed transcripts was strongly enriched in the differentially abundant proteins in our cohort. AGPC lysates are therefore well suited for comparative protein and integrative analyses