Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach

Abstract

In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time.Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E 2 (PGE 2 ) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i. The identification and quantification of protein phosphorylation under system perturbations is an integral part of systems biology (1, 2). The combination of phosphopeptide enrichment (3-6), stable isotope labeling, and high-resolution mass spectrometry (MS) methods (7-9) has become the method of choice for the identification of novel phosphorylation sites and for the quantitation of temporal dynamics within signaling networks (10, 11), allowing the behavior of thousands of phosphorylation sites to be studied in a single experiment (10, 12, 13). Nowadays, one of the most commonly adopted high-throughput phosphoproteomics strategies utilizes two consecutive separation steps: (i) an initial fractionation to reduce the sample complexity, and (ii) a phosphopeptide-specific affinity purification. Such techniques include strong cation exchange fractionation under acidic conditions (3), followed by a chelation-based method with the use of metal ions (i.e. immobilized metal ion affinity chromatography (4), metal oxide affinity chromatography (10, 14), or Ti 4ϩ immobilized metal ion affinity chromatography (6)). Alternatives to strong cation exchange for the first sample fractionation step have also been reported, including the use of electrostatic repulsion liquid chromatography (15, 16), which is well suited for the identification of multiply phosphorylated peptides, or hydrophilic interaction chromatography (17). Although the number of detected phosphorylated peptides is nowadays impressive, these kinds of methodologies are still inclined to identify/quantify the more abundant phosphoproteins present in a sample. For example, phosphotyrosine peptides are underrepresented because of their relatively lower abundance.In order to analyze key signaling events that may occur on less abundant phosphoproteins, more targeted approaches, focused on a specific pathway or a specific post-translational modification, are thus still essential. Studies examining posttranslational modifications are ...

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis

“…As shown in Figure 3, the overlap between a given antibody enrichment and the IMAC enrichment ranged from roughly 16% with the all Ser/Thr antibody mix, to a low of only 3.6% with phosphotyrosine pY-1000. No fractionation of samples was performed prior to LC-MS/MS analysis in this study, and it is possible fractionation could increase the overlap between antibody and IMAC enrichment, however, the complementarity observed here between antibody enrichment and IMAC is consistent with other studies that fractionated samples prior to enrichment to compare the two methods [53,57,58,70,71]. These data demonstrate that to maximize the amount of information gleaned from each sample, both IMAC and antibody enrichment should be performed.…”

“…These motif antibodies have been incorporated into proteomic methods that have also proven useful in the study of disease signaling. Antibody-based methods have been developed including enrichment of tyrosine-phosphorylated peptides [39,43,44,50,51,52], peptides that share a common consensus kinase substrate motif [41,45,53,54], and peptides that are modified by PTMs other than phosphorylation, for which no metal affinity enrichment exists [37,38,40,42,46]. More recently, immunoaffinity reagents have been generated that target key regulators of known signaling pathways, or critical protein classes such as kinases, allowing multiplexed detection and quantitation of hundreds to thousands of regulatory sites in a single experiment [55,56].…”

“…Previous work has suggested that the two methods provide complementary coverage of phosphorylation sites [53,57,58]. This would be expected, as metal affinity methods will preferentially identify phosphopeptides present at higher levels (abundance-driven), while antibody-based methods will identify peptides that share the sequence characteristics targeted by the antibody itself (affinity-driven).…”

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells

“…The combined approach of using selective inhibitors with phosphoproteomic analysis builds on previous classical and phosphoproteomic studies (17) in several key aspects. First, the majority of previous phosphoproteomic studies used high concentrations of agents such as cAMP analogs or receptor agonists to increase cAMP globally.…”

“…In a few cases increased cAMP may even potentiate the T-cell activation signal (15), particularly at early stages of activation. Recent MS-based proteomic studies have been useful in characterizing changes in the phosphoproteome of T cells under various stimuli such as T-cell receptor stimulation (16), prostaglandin signaling (17), and oxidative stress (18), so much of the total Jurkat phosphoproteome is known. Until now, however, no information on the regulation of phosphopeptides by PDEs has been available in these cells.…”