Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3epsilon, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) zeta was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRzeta, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRzeta in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRzeta when both were coexpressed in 293T cells, establishing TCRzeta as a direct substrate of PTPN22.

Two liquid crystal compounds, cholesterol-10-undeceneoate and 4-cyano-4‘-pentoxybiphenyl, are attached to the inner wall of a fused-silica capillary that has been etched by ammonium hydrogen fluoride. The bonding process involves formation of a hydride layer on the etched surface via a silanization reaction with triethoxysilane followed by attachment of the liquid crystal using a hydrosilation reaction. The etched surface is characterized by photoelectron spectroscopy (ESCA) and the inner wall after being chemically modified with the liquid crystal is analyzed by diffuse reflectance infrared Fourier transform spectroscopy. The surface properties of these materials are further probed by measuring electroosmotic flow as a function of pH. The performance of the etched chemically modified capillaries as a separation medium is evaluated by electrochromatographic experiments using mixtures of proteins, pyrimidine/purine bases and a nucleoside, benzodiazepines, the synthetic and metabolic compounds of serotonin, and other small basic molecules. In all cases, peak symmetry is good and efficiency is generally considerably higher than in packed capillary CEC. In some instances, there are significant differences in selectivity between the two capillaries indicating that the type of liquid crystal (cholesteric or nematic) is an important factor in the separation mechanism. Finally, during the evaluation period for the capillaries used in this study there was no significant loss in chromatographic performance, indicating that the long-term stability of these materials is good.

Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), gives rise to devastating crop losses in rice. Disease resistant rice cultivars are the most economical way to combat the disease. The TP309 cultivar is susceptible to infection by Xoo strain PXO99. A transgenic variety, TP309_Xa21, expresses the pattern recognition receptor Xa21, and is resistant. PXO994rax-ST, a strain lacking the raxST gene, is able to overcome Xa21-mediated immunity. We used a single extraction solvent to demonstrate comprehensive metabolomics and transcriptomics profiling under sample limited conditions, and analyze the molecular responses of two rice lines challenged with either PXO99 or PXO994raxST. LC-TOF raw data file filtering resulted in better within group reproducibility of replicate samples for statistical analyses. Accurate mass match compound identification with molecular formula generation (MFG) ranking of 355 masses was achieved with the METLIN database. GC-TOF analysis yielded an additional 441 compounds after BinBase database processing, of which 154 were structurally identified by retention index/MS library matching. Multivariate statistics revealed that the susceptible and resistant genotypes possess distinct profiles. Although few mRNA and metabolite differences were detected in PXO99 challenged TP309 compared to mock, many differential changes occurred in the Xa21-mediated response to PXO99 and PXO994raxST. Acetophenone, xanthophylls, fatty acids, alkaloids, glutathione, carbohydrate and lipid biosynthetic pathways were affected. Significant transcriptional induction of several pathogenesis related genes in Xa21 challenged strains, as well as differential changes to GAD, PAL, ICL1 and Glutathione-S-transferase transcripts indicated limited correlation with metabolite changes under single time point global profiling conditions.

Cellular response to the external environment is often controlled by one or more protein kinases. We report a methodology for simultaneously monitoring multiple kinase activities across multiple signal-transduction pathways using ProteinChip Array technology. Based on the addition of specific peptide reporters, kinase activity is detected by the presence of a mass shift of 80 Da (or multiple thereof) corresponding to the addition of one or more phosphate groups. These phosphorylated peptide substrates are then enriched using an immobilized metal affinity capture (IMAC)-Ga array and detected directly by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). SELDI-TOF MS is sensitive, tagless (nonradioactive, nonfluorescent), can be easily multiplexed for the analysis of several different kinases in a single reaction mixture (limited only by the specificity of the kinase for its substrate peptides), and is directly scalable through the use of robotic sample processing. By multiplexing kinase assays, one can dramatically increase the amount of information obtained from rare or volume-limited samples. More important, results reflect closely the complex interrelationships between kinases and show high correlation with in vivo assays.

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