Experimental workflow and chemical structure and properties of the crosslinking agents employed.

<p><b>A,</b> schematic representation of the experimental workflow used for growing cells, preparing microsomes, carbonate wash of membranes, chemical crosslinking of proteins and preparation of peptides for MS analysis. <b>B,</b> chemical structure and reactivity of the crosslinkers employed BS3 (bis[sulfosuccinimidyl] suberate) and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.</p

Mapping the Trypsin-resistant Proteome by Targeted Multi-enzyme Digestion of Large Tryptic Peptides

<p>Poster for HUPO 2009.</p> <p> </p

Molecular mass and scores of XLs found by pLink for Pma1p and BSA.

<p>Distributions of scores and calculated molecular masses of XLs generated by pLink by scanning through all mgf files from BS3 experiments (<b>A-D</b>) or EDC experiments (<b>E-H</b>) for Pma1p and BSA, a protein that is not present in the sample, are plotted as calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s007" target="_blank">S7 Table</a>. The cut off lines used to filter all pLink XLs (stippled red lines) indicate a minimal molecular mass of XL of 1’500 Da and scores of ≤ 7.5 x 10⁻⁴ for BS3-XLs, or ≤ 4 x 10⁻⁴ for EDC-XLs.</p

Frequency of XLs found by pLink for a given protein is correlated with its copy number.

<p>The number of XLs for 18 arbitrary chosen MSPs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s004" target="_blank">S4 Table</a> is plotted as a function of the copy number of the respective MSP (<b>A</b>) or its amino acid length (<b>B</b>). In panel <b>C</b>, the number of BS3- and EDC-generated XLs for these 18 proteins are plotted separately, as a function of copy number. In panel <b>D</b>, the distance in the primary sequence between crosslinked amino acids in 160 XLs found in 27 proteins of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s014" target="_blank">S14 Table</a> are plotted.</p

BS3 and EDC crosslinks mapped onto the structural models of Pma1p, Por1p and Sec61p.

<p>Structures of Pma1p (<b>A, B</b>), Por1p (<b>C-F</b>), and Sec61p (<b>G, H</b>) were homology modeled by HHPRED [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref015" target="_blank">15</a>] using plasma membrane H⁺-ATPase from <i>Neurospora crassa</i> (1mhs_A) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref016" target="_blank">16</a>], the voltage-dependent anion channel VDAC1 from <i>Mus musculus</i> (4c69_X) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref017" target="_blank">17</a>], and Sec61 from <i>Canis lupus/Bos Taurus</i> (3jc2_1) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref018" target="_blank">18</a>] as template. Structural models were visualized by PyMOL and the position of crosslinks connecting the Cα atoms of amino acids were added manually based on the experimental data from pLink.</p

BS3 crosslinks mapped onto the topological model of Fks1p, the catalytic subunit of 1,3-beta-D-glucan synthase.

<p><b>A,</b> Arch model of Fks1p indicating the positions of BS3 (red) XLs visualized using xVis. <b>B,</b> topology model of Fks1p proposed using the crosslinking data to validate a topology prediction visualized by Protter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref019" target="_blank">19</a>].</p

Improved phosphoproteome analysis by multi-enzymatic digestions targeting the Trypsin-Resistant Proteome (TReP)

<p>Poster for conference ASMS 2010.</p

Labeling, measurable parameters and data obtained from pcSILAC experiments.

<p><b>A</b>) Protein labeling scheme for pcSILAC experiments. Two cell cultures were fully labelled only on Arg residues before cell treatments. For example, cells to be treated later with HSP90 inhibitor were fully labelled with ¹³C₆¹⁵N₄-L-arginine (R10/K0) (“heavy” cells) while cells to be used as control were fully labelled with ¹³C₆-L-arginine (R6/KO) (“medium” cells). At the start of the experiment, “heavy” cells were transferred into a medium containing light R (R0) and “heavy” K (¹³C₆¹⁵N₂-L-Lys, referred to as R0/K8 medium), while “medium” cells were transferred to a medium containing light R (R0) and “medium” K (²H₄-L-lysine, referred to as R0/K4 medium). GA or DMSO as a control were added either simultaneously or later, depending on the experiment. Cells were harvested and lysed at various time points to produce extracts that were mixed before analysis. <b>B</b>) Conceptual view of the levels of a hypothetical protein in a mixture of two (control and treated) pcSILAC-labeled samples. Pre-existing protein is fully labeled R6/K0 and R10/K0 (light and dark brown), respectively for the control and treated sample. Newly synthesized protein is labeled R0/K4 (control, pink) and R0/K8 (treated, blue). The SILAC (H/M) ratios for R- and K-containing peptides therefore measure the ratios of preexisting and newly synthesized proteins at time t. <b>C)</b> examples of pcSILAC spectra for peptides from the tyrosine kinase LCK (left, peptide IPYPGMTNPEVIQNLER at t = 6h, z = 2) and the chaperone DNAJB1 ( = Hsp40) (right, peptide EGDQTSNNIPADIVFVLK at t = 20h, z = 2).</p

Validation of new Hsp90 clients.

<p><b>A</b>) Network analysis of a selected set of potentially new Hsp90 client proteins. stSILAC data and the Hsp90 interaction network Hsp90Int <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080425#pone.0080425-Echeverra1" target="_blank">[21]</a> were combined to identify interesting candidates (OGT, ITK and BRAT1) with no reported interactions with Hsp90 at the time of the analysis. Edges connecting candidate proteins with known Hsp90 interacting proteins are highlighted in red. <b>B</b>). Co-immunoprecipitation (co-IP) experiment demonstrating interactions between BRAT1, OGT and ITK with Hsp90β in Jurkat cells. Equal concentrations of specific antibodies against BRAT1 (rabbit), OGT (rabbit), ITK (mouse), Hsp90β (mouse), and the corresponding non-immune (NI) control antibodies from rabbit and mouse were used in co-IP experiments, and then analysed by immunoblotting (WB). <b>C</b>) GA-induced degradation of BRAT1, ITK and OGT in Jurkat cells. Lysates from cells treated with GA or with the equivalent volume of the solvent DMSO (control) for 6 and 20 h were analysed by WB for these three mentioned proteins and also for Hsp70 and CDK6 as positive controls of GA action.</p

Results from calculations of decay constants, synthesis rates and evolution of total protein levels.

<p>Indexes of variables are A (as in k_A, V_A) for control (+DMSO) and B (as in k_B, V_B) for treated (+GA) cells. <b>A</b>) Scatter plot of the values of the degradation constants for the control and treated sample (experiment 2, 911 proteins). The position of reference proteins is indicated. The dashed line indicates a 1:1 relationship <b>B</b>) Scatter plot of V_A and V_B (same dataset as A). Other heat shock proteins are shown in pink. The dashed line indicates a 1:1 relationship <b>C</b>) Kernel density estimate of log₂ of ratios of synthesis rates (V_B/V_A) and degradation constants (k_B,d/k_A,d) after correction for cell growth. <b>D</b>) Comparison of distributions of log₂ of ratios <i>S</i> of net protein levels (treated/control) calculated from pcSILAC data at t = 6, 12, 20h vs. ratios at steady-state (t = infinite) calculated from the model. <i>S</i> values were corrected for mixing inequalities.</p