Viveka Mayya

Proteomics Analysis of Human Coronary Atherosclerotic Plaque A Feasibility Study of Direct Tissue Proteomics by Liquid Chromatography and Tandem Mass Spectrometry

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.

Transcriptional insights into the CD8+ T cell response to infection and memory T cell formation

After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity.

Absolute Quantification of Multisite Phosphorylation by Selective Reaction Monitoring Mass Spectrometry Determination of Inhibitory Phosphorylation Status of Cyclin-Dependent Kinases

Multisite phosphorylation is an important mechanism for achieving intricate regulation of protein function. Here we extended the absolute quantification of abundance (AQUA) methodology and validated its applicability to quantitatively study multisite phosphorylation. As a test case, we chose the conserved inhibitory site of the cyclin-dependent kinases (CDKs), Cdk1, Cdk2, and Cdk3, which are important regulators of cell cycle transitions and apoptosis. Inhibitory phosphorylation at Thr14 and Tyr15 of the CDKs is modulated by complex regulatory mechanisms involving multiple kinases and phosphatases. Yet the resulting quantitative dynamics among the four possible phosphorylated and non-phosphorylated versions of CDKs (T14p-Y15p, T14p-Y15, T14-Y15p, and T14-Y15) has not been investigated to date. Hence we used the heavy isotope-labeled tryptic peptides spanning the inhibitory site as internal standards and quantified all four versions by LC-selected reaction monitoring. Quantification of the phosphorylation status of the inhibitory site in the cell extracts provided novel quantitative insights. 1) The transition to mitotic phase was dominated by the conversion of “T14p-Y15p” to the “T14-Y15” form, whereas the two monophosphorylated forms were considerably lower in abundance. 2) The amount of all four forms decreased during the progression of apoptosis but with differing kinetics. Analysis of immunoprecipitated Cdk1 and Cdk2 revealed that the inhibitory site phosphorylation state of both kinases at different stages of the cell cycle followed the same trend. Quantitative immunoblotting using antibodies to Cdk1 and Cdk2 and to the T14-Y15p form suggested that quantification by AQUA was reliable and accurate. These results highlight the utility of internal standard peptides to achieve accurate quantification of multisite phosphorylation status.

A Systematic Characterization of Mitochondrial Proteome from Human T Leukemia Cells

Global understanding of tissue-specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one-dimensional gel electrophoresis in combination with the nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS). The use of only 40 μg of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular mass validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60 μg of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing subcellular proteomes form multiple cells and tissues.

Systematic Characterization of Nuclear Proteome during Apoptosis A Quantitative Proteomic Study by Differential Extraction and Stable Isotope Labeling

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 naive 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 SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.

Global Survey of Human T Leukemic Cells by Integrating Proteomics and Transcriptomics Profiling

A global protein survey is needed to gain systems-level insights into mammalian cell signaling and information flow. Human Jurkat T leukemic cells are one of the most important model systems for T cell signaling study, but no comprehensive proteomics survey has been carried out in this cell type. In the present study we combined subcellular fractionation, multiple protein enrichment methods, and replicate tandem mass spectrometry analyses to determine the protein expression pattern in a single Jurkat cell type. The proteome dataset was evaluated by comparison with the genome-wide mRNA expression pattern in the same cell type. A total of 5381 proteins were identified by mass spectrometry with high confidence. Rigorous comparison of RNA and protein expression afforded removal of the false positive identifications and redundant entries but rescued the proteins identified by a single high scoring peptide, resulting in the final identification of 6471 unique gene products among which 98% of the corresponding transcripts were detected with high probability. Using hierarchical clustering of the protein expression patterns in five subcellular fractions (cytosol, light membrane, heavy membrane, mitochondria, and nuclei), the primary subcellular localization of 2241 proteins was assigned with high confidence including 792 previously uncharacterized proteins. This proteome landscape can serve as a useful platform for systems-level understanding of organelle composition and cellular functions in human T cells.

Phosphoproteomics by Mass Spectrometry: insights, implications, applications, and limitations

Phosphorylation of proteins is a predominant, reversible post-translational modification. It is central to a wide variety of physiological responses and signaling mechanisms. Recent advances have allowed the global scope of phosphorylation to be addressed by mass spectrometry using phosphoproteomic approaches. In this perspective, we discuss four aspects of phosphoproteomics: the insights and implications from recently published phosphoproteomic studies and the applications and limitations of current phosphoproteomic strategies. Since approximately 50,000 known phosphorylation sites do not yet have any ascribed function, we present our perspectives on a major function of protein phosphorylation that may be of predictive value in hypothesis-based investigations. Finally, we discuss strategies to measure the stoichiometry of phosphorylation in a proteome-wide manner that is not provided by current phosphoproteomic approaches.

What Scales the T Cell Response

T cells are known to scale their clonal expansion and effector cytokine response according to the dose and strength of antigenic signal so as to balance their role of affecting protection with the intertwined and immunologically driven tissue damage. How T cells achieve this is now beginning to be understood. We underscore temporal integration of digital T cell receptor (TCR) signaling as the basis for achieving scaled response by means of accumulating crucial mediators over time. We also discuss the role of temporally integrated crosstalk between TCR and IL2 signaling in mediating a scaled, coherent, collective response by T cells. Finally, we highlight numerous known and putative regulatory interactions in the transcriptional program that are expected to quantitatively scale the T cell response, and also offer new mechanisms to hitherto unexplained observations.

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

Elucidating the rules for receptor triggering in cell–cell and cell–matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.Using a nanofabricated array platform to precisely control ligand organization at the single-molecule level, the authors illuminate the role of geometry in T cell receptor signalling.

Durable Interactions of T Cells with T Cell Receptor Stimuli in the Absence of a Stable Immunological Synapse

Summary T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses.