Unsupervised clustering of B cell samples on the basis of protein profiles.

<p>IM-B Ramos samples clustered according to time after anti-IgM stimulation (1–120 h) and were distinctive from early pre-B (RS4;11, 380 and REH), pre-B (697, Nalm-1 and Nalm-6) and plasma cell U-266 samples. The degree of similarity in profiles is shown by colour (base-two logarithmical scale below the figure). Red and green indicate high and low levels of protein expression in relative to internal standard. The average protein abundance of the 175 identified proteins (labelled with Cy3 and Cy5 dyes) is calculated relative to internal standard (labelled with Cy2 dye). Cluster numbers (1–19) are shown to the right and refer to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-t001" target="_blank">Table 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-t002" target="_blank">Table 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-t003" target="_blank">Table 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s004" target="_blank">Table S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s006" target="_blank">Table S4</a>.</p

Biological process GO terms and their p-values for co-expressed proteins.

a)<p>SSP referring to those numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-g002" target="_blank">Figure 2</a> and short protein names referring to those proteins listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s004" target="_blank">Table S2</a>.</p>b)<p>P-value adjusted by the Benjamini multiple test adjustment.</p>c)<p>Cluster numbers referring to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-g004" target="_blank">Figure 4</a>.</p

Molecular function GO terms and their p-values for co-expressed proteins.

a)<p>SSP referring to those numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-g002" target="_blank">Figure 2</a> and short protein names referring to those proteins listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s004" target="_blank">Table S2</a>.</p>b)<p>P-value adjusted by the Benjamini multiple test adjustment.</p>c)<p>Cluster numbers referring to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-g004" target="_blank">Figure 4</a>.</p

Unsupervised clustering of proteins affected by anti-IgM stimulation.

<p>The heat map shows the comparison between anti-IgM stimulated and control Ramos samples during the time course 1–120 h. Protein abundance difference is calculated between stimulated and corresponding control samples for the 69 identified proteins, which passed the significance criteria (student’s t-test p≤0.05). Red and green colours indicate up- and down-regulation in response to anti-IgM stimulation (base-two logarithmical scale below the figure). Cluster numbers (1R-11R), short protein names and SSP are shown to the right and refer to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-t001" target="_blank">Table 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-t002" target="_blank">Table 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone-0077894-t003" target="_blank">Table 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s004" target="_blank">Table S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s006" target="_blank">Table S4</a>.</p

Reference 2D-DIGE map of Cy2-labelled internal standard.

<p>The 233 identified proteins are numbered in the gel and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077894#pone.0077894.s004" target="_blank">Table S2</a>.</p

PID-related proteins in B cell maturation pathway.

<p>Defects in proteins and genes important for maturation cause PIDs and block the maturation on certain stage. The PID-related proteins and stages where diseases affect the development are shown on top. The cell lines (RS4;11, 380, REH, 697, Nalm-1, Nalm-6, Ramos and U-266) are marked inside the corresponding cells representing their normal B cell counterparts.</p

Differences in proteome profiles between IM-B and early pre-B cell stages.

<p>Early pre-B 380 and IM-B Ramos (120 h time point) proteome samples were labelled with Cy5 and Cy3 dyes, respectively, and separated in a 2D-DIGE gel (nonlinear pH gradient 3–10) with internal standard sample labelled with Cy2 dye. Triple image overlay shows proteins highly expressed in Ramos (red) and 380 (green) samples in relative to internal standard sample (blue). Protein identities are indicated for those proteins with major changes in expression profile. IgM chains, ER and membrane bound vesicle proteins were highly expressed in Ramos cells, whereas cytoskeleton and immunity related proteins were expressed in 380 cells.</p

The full length human PEX3 protein sequence.

<p>A) The red letters indicate amino acids which are not expressed in the recombinant protein. The black letters indicate amino acids modelled in the X-ray structure (PDB ID: 3AJB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101-Sato1" target="_blank">[40]</a>. The blue letters indicate amino acids which are not modelled in the X-ray structure. B) X-ray structure of PEX3 with a PEX19 peptide bound (PDB ID: 3AJB). The secondary structure of PEX3 is colored as a) helix (orange), b) sheet (dark blue) and c) coil (pink). The PEX19 peptide is shown in turquoise.</p

Comparison of hydrogen exchange in PEX19 alone and in complex with PEX3 monitored by MS.

<p>(<b>A</b>) HXMS heat map of PEX19 monomer summarizing the deuterium uptake over time. (<b>B</b>). HXMS heat map of PEX19 in the heterodimer with PEX3 summarizing the deuterium uptake over time. (<b>A and B</b>). The sequence of the protein is shown above the heat map. Although the recombinant protein includes an N-terminal tag only the full sequence of human PEX19 is shown with the numbering starting at the initial PEX19 methionine. The heat maps were assembled from individual peptic peptides using MSTools <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101-Kavan1" target="_blank">[47]</a>. The extent of the peptide is demarcated by vertical lines in each block, running through all four time point bars, when there is a difference in uptake from the preceding or following peptide. All peptic peptides are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101.s001" target="_blank">Figure S1</a></b>. The scale bar at the bottom of each heat map illustrates the color coding for deuterium uptake as a percentage. The horizontal bars from the top to the bottom in each block of the heat map indicate incubation times of 0, 3, 7, 40 and 60 minutes, respectively. White bars represent the residues for which no data were available.</p

The heat map of PEX3 plotted on the X-ray structure of PEX3 with a PEX19 peptide bound (PDB ID: 3AJB) [40].

<p>Regions where the hydrogen/deuterium exchange decreased in the heterodimer compared to PEX3 alone are shown in red. Regions where the exchange remained the same are shown in blue. The left hand panel is rotated 180° along the Y axis compared to the right hand panel.</p