Protein tyrosine-O-sulfation analysis by exhaustive product ion scanning with minimum collision offset in a NanoESI Q-TOF tandem mass spectrometer.

Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, approximately 1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b2/y(max-2) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions.Journal Articleinfo:eu-repo/semantics/publishe

Total phosphorylation in the absence of LAT.

<p>(<b>A</b>) Phosphoproteomics workflow used in this study starts with labeling Jurkat cell lines in different SILAC media in two time series (TS1 and TS2) as indicated, each composed of three sets of differentially labeled cells. In each TS, cell are activated for the indicated time points and the total protein lysates are equally mixed prior to its digestion by trypsin. The resulted peptides are submitted to strong cation exchange chromatography (SCX) and titanium oxide (TiO2) affinity enrichment prior to liquid chromatography and mass spectrometry (LC-MS/MS) analysis. The data is then analyzed (<b>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077423#s2" target="_blank">Methods</a></b>) and the two time series are normalized using the common time point (0.5 min) so as to obtain a curve representing fold change versus activation time points. (<b>B</b>) Pie chart showing the distribution of the total 11,454 unique phosphorylation sites as phospho (p)-Serine (pS), p-Threonine (pT) and p-Tyrosine (pY). (<b>C</b>) Bar chart presenting the quantified (Qt) and significant change (SChg.) phosphorylated residues as the percent of the total detected sites in CL20 Jurkat cell lines (LAT+/+) or JCaM2.5 (LAT−/−). (<b>D, E</b>) Scatter plot of change in phosphorylation between consecutive time points (Red: 0 to 0.5 min, Green: 0.5 to 5 min, Blue: 0.5 to 10 min, Black: 10 to 20 min) versus the intensity of phosphopeptides - identified in both intact and perturbed cell lines (common peptides) - over each time interval. The grey zone delimits the 95% confidence interval (at ±0.42). (<b>F</b>) Bar chart quantifies the common phosphopeptides classified as significant in (<b>E</b>) as the percent of the total common peptides over the indicated time intervals.</p

Topology of TCR signaling networks and effects of LAT deficiency.

<p>(<b>A</b> and <b>B</b>) Phosphorylation-specific networks based on integration of our phosphoproteomic data with protein-protein interactions in the STRING database. Note that both networks show constellation of hubs characterized by interacting proteins function. Zooms into the signaling hub of LAT efficient (LAT+/+) or deficient (LAT−/−) cell lines shows first neighbors (pistachio green or orange circles) of CD3ζ, LCK and ZAP-70 (red or blue circles). (<b>C</b>) The number of edges and (<b>D</b>) degree distribution for experimental and random networks. Orange and pistachio-green triangles correspond to degree distribution in networks based on data from two replicas in Jurkat CL20 cell line. Blue triangles and purple crosses correspond to networks based on the data in LAT-deficient cell line and randomly selected proteins, respectively.</p

T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component*

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive “signaling waves” revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function

A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76

Di Bartolo V, Montagne B, Salek M, et al. A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76. J Exp Med. 2007;204(3):681-691.The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation

LAT regulates phosphorylation of ZAP70 and CD3Z.

<p>(<b>A</b>) The comparison of CD3ζ and ZAP70 phosphopeptides kinetics in CL20 and JCaM2.5 cell lines. (<b>B</b>) SILAC labeling strategy for direct comparison of peptide-specific phosphorylation in JCaM.2.5 and JCam2.5-LAT cell lines. Cell lines grown in SILAC media (Arginine: R and Lysine: K) were activated (or not) for the indicated time points. Total protein extract were equally mixed and subjected to phosphoproteomics analysis (for details see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077423#s2" target="_blank">Methods</a> and Fig. 1S</b>). Anti-pY detection of the cell lysates was used to control the activation. Anti-ZAP70 antibodies were used to control the loading. The arrow at 18 KDa shows a band that probably corresponds to CD3ζ. L, M and H stand for Light, Medium and Heavy amino acids combinations. (<b>C</b>) S-shape graphic showing log2-transformed ratios for tyrosine phosphorylated peptides signals in different experimental conditions: M/L (Blue diamonds; peptide signal from 0.5 min activated JCam2.5/resting JCam2.5LAT), H/L (light-red squares; JCaM2.5LAT activated/JCaM2.5 resting), H/M (Pistachio-green triangles; JCaM2.5LAT activated/JCaM2.5 activated). (<b>D</b>) Similar to <b>C</b> except that only activated cell lines were confronted, as indicated. The graphic shows log2-transformed H/L ratio (black diamonds; JCaM2.5LAT activated/JCaM2.5 activated). (<b>E</b>) LAT-dependent phosphorylation of ZAP70 was tested as indicated. (<b>F</b>) Quantitation of the immunoblots in (<b>E</b>). This file contains: additional information on the analysis of global dynamics of TCR-induced phosphorylation; methods to evaluate experimental error and define activation threshold; supporting references.</p