Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

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The ventral striatum (VS) is considered a key region that flexibly updates recent changes in reward values for habit learning. However, this update process may not serve to maintain learned habitual behaviors, which are insensitive to value changes. Here, using fMRI in humans and single-unit electrophysiology in macaque monkeys we report another role of the primate VS: that the value memory subserving habitual seeking is stably maintained in the VS. Days after object-value associative learning, human and monkey VS continue to show increased responses to previously rewarded objects, even when no immediate reward outcomes are expected. The similarity of neural response patterns to each rewarded object increases after learning among participants who display habitual seeking. Our data show that long-term memory of high-valued objects is retained as a single representation in the VS and may be utilized to evaluate visual stimuli automatically to guide habitual behavior.

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naïve and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILACbased quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis. Molecular & Cellular Proteomics 5:1131-1145, 2006.Comprehensive understanding of the biology of the nucleus will require complete identification of the proteome of this important organelle. The nucleus of the cell is where a number of key enzymatic processes occur, and most of these processes are critical for cellular homeostasis: DNA synthesis, DNA replication and repair, transcription, higher order chromatin organization, and gene and chromosomal silencing (1-8). A comprehensive list of proteins that reside in the nucleus, as well as the proteins that shuttle between multiple subcellular compartments and nucleus, has not been completed to date (9 -11). In addition, different classes of proteins are sequestered in the nucleus based on their affinity to DNA, nuclear matrix, nuclear membrane, nucleolus, specialized nuclear bodies such as speckles and PIKA (polymorphic interphase karyosomal association), or Cajal bodies (coiled bodies) (12-15). Thus, large scale identification of nuclear proteins from human cells and characterization of their associated physicochemical properties will likely provide insights into the biology of the nucleus.We have been interested in identifying proteins that control apoptosis in the nucleus. Apoptosis or programmed cell death is a process essential for the development and maintenance of cellular homeostasis of higher eukaryotes (16). One of the hallmarks of apoptosis is rapid chromatin condensation and DNA fragmentation (17). The exact mechanisms that control rapid DNA fragmentation and chromatin condensation in the nucleus are not fully understood, although some of the proteins that are crucial for these events have been identified (18 -25). For example, the caspase-activated DNase (CAD/DFF40) (18, 19), lamin protease (Caspase 6...

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Mass spectrometry-based phosphoproteomics holds promise for advancing drug treatment and disease diagnosis; however, its clinical translation has thus far been limited. This is in part due to an unstandardized and segmented sample preparation process that involves cell lysis, protein digestion, peptide desalting, and phosphopeptide enrichment. Automating this entire sample preparation process will be key in facilitating standardization and clinical translation of phosphoproteomics. While peptide desalting and phosphopeptide enrichment steps have been individually automated, integrating these two extractions and, further, the entire process requires more advanced robotic platforms as well as automation-friendly extraction tools. Here we describe a fully automated peptide desalting and phosphopeptide enrichment method using IMCStips on a Hamilton STAR. Using our established automated method, we identified more than 10,000 phosphopeptides from 200 µg of HCT116 cell lysate without fractionation with >85% phosphopeptide specificities. Compared with titania-based Spin Tip products, the automated IMCStips-based method gave 50% higher phosphopeptide identifications. The method reproducibility was further assessed using multiple reaction monitoring (MRM) to show >50% phosphopeptide recoveries after the automated phosphopeptide extraction with coefficients of variation (CVs) of <20% over a 3-week period. The established automated method is a step toward standardization of the sample preparation of phosphopeptide samples and could be further expanded upon to create a fully automated “cells to phosphopeptides” method.

Background Choline deficiency has numerous negative health consequences; although the preponderance of the US population consumes less than the recommended Adequate Intake (AI), clinical assessment of choline status is difficult. Further, several pathways involved in primary metabolism of choline are estrogen-sensitive and the AI for premenopausal women is lower than that for men. Objectives We sought to determine whether in vivo magnetic resonance spectroscopy (MRS) of liver and/or isotope-dilution MS of plasma could identify biomarkers reflective of choline intake (preregistered primary outcomes 1 and 2, secondary outcome 1). Determination of whether biomarker concentrations showed sex dependence was a post hoc outcome. This substudy is a component of a larger project to identify a clinically useful biomarker panel for assessment of choline status. Methods In a double-blind, randomized, crossover trial, people consumed 3 diets, representative of ∼100%, ∼50%, and ∼25% of the choline AI, for 2-wk periods. We measured the concentrations of choline and several metabolites using 1H single-voxel MRS of liver in vivo and using 2H-labeled isotope dilution MS of several choline metabolites in extracted plasma. Results Plasma concentrations of 2H9-choline, unlabeled betaine, and 2H9-betaine, and the isotopic enrichment ratio (IER) of betaine showed highly significant between-diet effects (q &lt; 0.0001), with unlabeled betaine concentration decreasing 32% from highest to lowest choline intake. Phosphatidylcholine IER was marginally significant (q = 0.03). Unlabeled phosphatidylcholine plasma concentrations did not show between-diet effects (q = 0.34). 2H9 (trimethyl)-phosphatidylcholine plasma concentrations (q = 0.07) and MRS-measured total soluble choline species liver concentrations (q = 0.07) showed evidence of between-diet effects but this was not statistically significant. Conclusions Although MRS is a more direct measure of choline status, variable spectral quality limited interpretation. MS analysis of plasma showed clear correlation of plasma betaine concentration, but not plasma phosphatidylcholine concentration, with dietary choline intake. Plasma betaine concentrations also correlate with sex status (premenopausal women, postmenopausal women, men). This trial was registered at clinicaltrials.gov as NCT03726671.