SUMMARY Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H2O2. Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Patients with Tourette Syndrome often state that their sensitivity to sensations is equally or more disruptive than are motor tics. However, their sensory sensitivity is not addressed by standard clinical assessments nor is it a focus of research. This lapse likely results from our limited awareness and understanding of the symptom. In this study we 1) defined the patients’ experience of sensitivity to external stimuli in detail, and 2) tested two hypotheses regarding its origin. First, we administered a lengthy questionnaire and in-depth interviews to adult Tourette patients (n=19) and age-matched healthy volunteers (n=19). Eighty percent of patients described a heightened sensitivity to external stimuli, with examples among all 5 sensory modalities. Bothersome stimuli were characterized as faint, repetitive or constant, and non-salient, whereas intense stimuli were well-tolerated. We then determined whether the sensitivity could be due to an increased ability to detect faint stimuli. After measuring the threshold of detection for olfactory and tactile stimuli among the patients and healthy volunteers, we found no significant difference for either sensory modality. These results indicate that patients’ perceived sensitivity derives from altered central processing rather than from enhanced peripheral detection. Lastly, we assessed one aspect of processing: the perception of intensity. When subjects rated the intensity of near-threshold tactile and olfactory stimuli, there was a surprising difference: Tourette patients more frequently used the lowest range of the scale compared with healthy volunteers. Future research is necessary to define the anatomical and physiological basis of the patients’ experience of heightened sensitivity.

The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers.