Phosphorylation Analysis of Primary Human T Lymphocytes Using Sequential IMAC and Titanium Oxide Enrichment

Carrascal, Montserrat; Ovelleiro, David; Casas, Vanessa; Gay, Marina; Abian, Joaquín

doi:10.1021/pr800500r

Cited by 71 publications

(71 citation statements)

References 58 publications

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“…We observed reproducibility in phosphopeptide identifications comparable to that obtained when analyzing technical replicates of K562 lysate, suggesting that our RP-SAX-RP platform is very robust with respect to variation across biological replicates. These results highlight the analytical capabilities of RP-SAX-RP, and are particularly encouraging in the context of two recent studies, one of which identified 281 phosphorylation sites from 2 mg of biological lysate derived from partially sorted, primary lymphocytes (110), and a second which reported a total of 6248 phosphopeptides across 46 LC-MS/MS analyses of whole mouse spleens (Ϸ2 mg total protein per analysis, or 92 mg total input) (111) in a model designed to study immune response to anthrax exposure. In both studies, the samples were composed of heterogeneous cell populations, each of which will contribute a distinct signaling response to the final ensemble of phosphopeptides measured.…”

Section: Discussionsupporting

confidence: 68%

Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis

Ficarro

Zhang

Carrasco-Alfonso

et al. 2011

Molecular & Cellular Proteomics

110

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Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at high pH in the first dimension of a twodimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimension in a three-dimensional reversed phase ( Reversible phosphorylation plays a central role in the regulation of normal cell physiology. The strong links between aberrant signaling and human disease, along with the potential for specific inhibition of disrupted kinase activity, continue to drive efforts aimed at systematic and largescale analysis of phosphorylation in cells and tissues. Shortly after introduction of immobilized metal affinity chromatography (IMAC) 1 as an enrichment tool prior to mass spectrometry (MS) analysis (1-3), several laboratories demonstrated the feasibility of phosphopeptide identification and quantitation en masse (4 -7). In the ensuing years, despite widespread proliferation of improved and innovative (8 -14) phosphoproteomics methods, the field struggled with low specificity and poor reproducibility within and across protocols and laboratories. These limitations effectively made dynamic range a secondary issue for the majority of studies. Over the past ca. 5 years, the performance of phosphopeptide enrichment protocols and related methods has stabilized; in fact several groups (15-23) have successfully coupled phosphopeptide enrichment with online or offline fractionation schemes to achieve, in some cases, over 10,000 phosphopeptide identifications. Although these strategies provide for larger phosphosite catalogs, closer inspection reveals that the analytical efficiency, as measured by the number of phosphopeptide identifications per microgram of biological lysate consumed, has remained surprisingly consistent at Ϸ1-10 phosphopeptides/g across a wide range of sample types (Table I). One explanation is that the physicochemical properties of phosphopeptides render them less amenable to fractionation by commonly used techniques. For example, although the combination of strong cation exchange (SCX) with reversed phase (RP) has been tremendously successful for 1 The abbreviations used are: IMAC, immobilized metal affinity chromatography; AML, Acute Myeloid Leukemia; CAD, collisionally activated dissociation; ESI, electrospray ionization; FDR, False Discovery Rate; FL, FLT3 ligand; FLT3, FMS-like tyrosine kinase 3; LC/MS, liquid chromatography/mass spectrometry; Lm-OVA, Recombinant Listeria monocytogenes expressing chicken ovalbumin; ITD, internal tandem duplication; MS/MS, mass spectrometry/mass spectrometry or tandem mass spectrometry; NTA, nitrilotetra...

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Section: Discussionsupporting

confidence: 68%

Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis

Ficarro

Zhang

Carrasco-Alfonso

et al. 2011

Molecular & Cellular Proteomics

110

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“…Over 40 000 MS/MS spectra were acquired from two samples and the data were searched against a protein target/decoy human database. 21 The target/decoy strategy is based on the principle that incorrect matches have an equal probability of being derived from either the target or the decoy database. 22,23 In contrast, spectra with relevant sequence information will match their corresponding sequence in the target database but not in the decoy database.…”

Section: Resultsmentioning

confidence: 99%

“…Correct peptide sequence identifications were evaluated using both the Xcorr score from SEQUEST and the D value, which was calculated as described. 21 Correct identifications were those with scores within the area defining a false discovery rate of o1%.…”

Section: Methodsmentioning

confidence: 99%

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

et al. 2011

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The objective of this study was to characterize the peptide-binding motif of the major histocompatibility complex (MHC) class II HLA-DR8 molecule included in the type 1 diabetes-associated haplotype DRB1*0801-DQA1*0401/DQB1*0402 (DR8-DQ4), and compare it with that of other diabetes-associated MHC class II alleles; DR8-bound peptides were eluted from an HLA-DR homozygous lymphoblastoid cell line. The repertoire was characterized by peptide sequencing using a LTQ ion trap mass spectrometer coupled to a multidimensional liquid chromatography system. After validation of the spectra identification, the definition of the HLA-DR8 peptide-binding motif was achieved from the analysis of 486 natural ligands, based on serial alignments of all possible HLA-DR-binding cores. The DR8 motif showed a strong similarity with the peptide-binding motifs of other MHC class II diabetes-associated alleles, HLA-DQ8 and H-2 I-A g7 . Similar to HLA-DQ8 and H-2 I-A g7 , HLA-DR8 preferentially binds peptides with an acidic residue at position P9 of the binding core, indicating that DR8 is the susceptibility component of the DR8-DQ4 haplotype. Indeed, some DR8 peptides were identical to peptides previously identified as DQ8-or I-A g7 ligands, and several diabetes-specific peptides associated with DQ8 or I-A g7 could theoretically bind to HLA-DR8. These data further strengthen the association of HLA-DR8 with type I diabetes.

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“…Although Jurkat cells are a very commonly accepted model to study TCR signaling (10,11,14), we have chosen here primary cells, because Jurkat cells lack specific signaling proteins as phosphatase and tensin homolog (8,15), which could result in important differences in the phosphorylation profiles after TCR stimulation. Because of the usage of primary cells, isobaric mass tags (iTRAQ) (16,17) were employed to measure differences in the phosphorylation profile after TCR stimulation.…”

mentioning

confidence: 99%

Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells

Ruperez

Gago-Martı́nez

Burlingame

et al. 2012

Molecular & Cellular Proteomics

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Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. T lymphocytes are able to recognize specific antigenic peptides presented by molecules of the major histocompatibility complex on the surface of other cell types. This interaction is mediated by a dimeric specialized molecule called T cell receptor (TCR), 1 which is part of a larger membrane complex in association with CD3 ␥, ␦, , and chains. The binding between TCR and the major histocompatibility complex-antigen is of relatively low affinity, and it is stabilized by the association with co-receptors (CD4 or CD8). All of these molecules in turn recruit, via their intracellular domains, different polypeptides to carry out signal transduction. In addition to antigen recognition, coactivation by CD28 is required to trigger full activation of the T cell, which expresses then different cell surface molecules and releases soluble mediators (cytokines) that promote changes in the activity of different target cell types (1).During the TCR-major histocompatibility complex-antigen recognition, T cells undergo considerable membrane and cytoskeletal rearrangements that lead to the formation of the immunological synapse (IS). During this maturation, precise molecular reorganizations occur at the interface between T cells and an antigen presenting cell. Cell motility, polarization, and receptor relocalization events are dependent on the lymphocyte cytoskeleton and are necessary for the maturation of the IS. TCR, co-receptors, intracellular signaling molecules, and adhesion receptors polarize to the IS and form small aggregates known as microclusters (2, 3), processes all dependent on functional microtubule and actin cytoskeleton. This results in the stabilization and functional maturation of the signaling complexes.Protein phosphorylation...

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Phosphorylation Analysis of Primary Human T Lymphocytes Using Sequential IMAC and Titanium Oxide Enrichment

Cited by 71 publications

References 58 publications

Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis

Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells

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