Intraendosomal membranes association with pycnosomes.

<p><i>D</i>. <i>discoideum</i> cells were treated for 2h with U18666A to induce the formation of intraluminal membranes in the endocytic compartments, then fixed and processed for electron microscopy. Pycnosomes (stars) often appeared continuous with internal membranes. Arrowheads point to regions where the continuity between pycnosomal material and endosomal membranes was most apparent. Bar: 500 nm.</p

Pycnosomes present in <i>D</i>. <i>discoideum</i> endocytic compartments are secreted in the extracellular medium.

<p><i>D</i>. <i>discoideum</i> cells grown in axenic medium were fixed and processed for electron microscopy. (A) Most endocytic compartments appeared empty or contained occasionally a few vesicles (arrowhead). (B) Dense bodies (pycnosomes) appeared as amorphous structures in the endosomal lumen (star). (C) Secreted pycnosomes were recovered from the extracellular medium by differential centrifugation. Bars: 500 nm.</p

The Sct family of proteins.

<p>The SctA protein was identified by mass spectrometry as the product of the DDB_G0278725 gene. The <i>D</i>. <i>discoideum</i> genome encodes three proteins exhibiting sequence homology to SctA: SctB, SctC and SctD. All Sct proteins harbor a signal peptide (not shown) and a conserved pair of cysteines residues (*). The regions of homology between all Sct proteins were aligned using the Multalin software and treated with Boxshade. The consensus sequence is indicated. The SctC protein contains a long extension rich in glycines and serines, just downstream of the predicted signal peptide (not shown on the figure).</p

SctA-positive material appears continuous with intraendosomal membranes.

<p><i>D</i>. <i>discoideum</i> cells were treated for 2h with U18666A, then fixed and processed for immuno-electron microscopy. (A) In sections labeled with the H161 anti-p80 antibody, the p80 protein was seen associated with the limiting endosomal membrane as well as with internal membranes, but was not detected in pycnosomes (stars). (B-C) SctA-positive structures with the dense morphology of pycnosomes were also detected, and often appeared continuous with intraluminal membranes (arrowhead). Bar: 500 nm.</p

SctA is massively associated with secreted pycnosomes.

<p>(A) The SctA and B proteins were expressed in bacteria as GST-fusion proteins, purified, and analyzed by Western blot with anti-GST and B4.2 antibodies. While an anti-GST antibody labeled both proteins, the B4.2 antibody only recognized GST-SctA and not SctB. (B) Supernatants (S6, S15, S100) and corresponding pellets (P15, P100) obtained by differential centrifugation of <i>D</i>. <i>discoideum</i> cell culture medium (600, 15’000, 100’000 x <i>g</i>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154875#pone.0154875.g002" target="_blank">Fig 2</a>) were analyzed by Western blot using the B4.2 antibody. Equal volumes were loaded for all samples. (C) <i>D</i>. <i>discoideum</i> cells were cultured for 4 days. Cells and a fraction enriched in secreted pycnosomes were recovered by successive centrifugation of cell suspension at 600 x <i>g</i> and 100’000 x <i>g</i> respectively. Serial two-fold dilutions from the two fractions were analyzed by Western blot using the B4.2 antibody, the H161 anti-p80 and an antibody against mitochondrial porin. The number of cells loaded on each lane is indicated above each band. The fraction of each protein associated to secreted pycnosomes is indicated on the right.</p

Electron microscopy reveals SctA-enriched endosomal pycnosomes.

<p><i>D</i>. <i>discoideum</i> cells grown in axenic medium were processed for immuno-electron microscopy. (A-B) Sections were labeled with the H161 anti-p80 antibody. The p80 protein was abundantly present in endosomal membranes, and only small amounts of p80 were found associated with pycnosomes (stars) in the lumen. (C-D) The B4.2 antibody revealed a high concentration of SctA in endosomal pycnosomes. In some pictures, pycnosomes appeared associated with some membranous elements (arrowheads). Bar: 500 nm.</p

SctA is a major constituent of secreted dense bodies.

<p>(A) <i>D</i>. <i>discoideum</i> cells were pelleted by centrifugation at 600 x g (pellet P6) and the supernatant (S6) was sequentially subjected to centrifugations at 15’000 and 100’000 x g. The corresponding supernatants (S15, S100) and pellets (P15, P100) resuspended in an equivalent volume were diluted in reducing sample buffer and separated on a 15% acrylamide gel. Equal volume of samples were loaded except for P6 that was diluted 1/10. The main proteins were revealed by silver staining. (B) The same amount of the 15 000 x g pellet was resuspended in reducing (+DTT) or non reducing (-DTT) sample buffer and analyzed by SDS-PAGE and Coomassie staining. Molecular weights (in kDa) are indicated, as well as the SctA protein (arrowheads).</p

Introducing AAA-MS, a Rapid and Sensitive Method for Amino Acid Analysis Using Isotope Dilution and High-Resolution Mass Spectrometry

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins

<i>DIGESTIF</i>: A Universal Quality Standard for the Control of Bottom-Up Proteomics Experiments

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called <i>DIGESTIF</i>, that can be added to any biological sample. The <i>DIGESTIF</i> standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC–MS system. Thus, <i>DIGESTIF</i> can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications