Protein kinases and their substrates comprise extensive signaling networks that regulate many diverse cellular functions. However, methods and techniques to systematically identify kinases directly responsible for specific phosphorylation events have remained elusive. Here we describe a novel proteomic strategy termed fluorescence complementation mass spectrometry (FCMS) to identify kinase–substrate pairs in high throughput. The FCMS strategy employs a specific substrate and a kinase library, both of which are fused with fluorescence complemented protein fragments. Transient and weak kinase–substrate interactions in living cells are stabilized by the association of fluorescence protein fragments. These kinase–substrate pairs are then isolated with high specificity and are identified and quantified by LC–MS. FCMS was applied to the identification of both known and novel kinases of the transcription factor, cAMP response element-binding protein (CREB). Novel CREB kinases were validated by in vitro kinase assays, and the phosphorylation sites were unambiguously located. These results uncovered possible new roles for CREB in multiple important signaling pathways and demonstrated the great potential of this new proteomic strategy.

Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

Protein–ligand interaction plays a critical role in regulating the biochemical functions of proteins. Discovering protein targets for ligands is vital to new drug development. Here, we present a strategy that combines experimental and computational approaches to identify ligand-binding proteins in a proteomic scale. For the experimental part, we coupled pulse proteolysis with filter-assisted sample preparation (FASP) and quantitative mass spectrometry. Under denaturing conditions, ligand binding affected protein stability, which resulted in altered protein abundance after pulse proteolysis. For the computational part, we used the software Patch-Surfer2.0. We applied the integrated approach to identify nicotinamide adenine dinucleotide (NAD)-binding proteins in the Escherichia coli proteome, which has over 4200 proteins. Pulse proteolysis and Patch-Surfer2.0 identified 78 and 36 potential NAD-binding proteins, respectively, including 12 proteins that were consistently detected by the two approaches. Interestingly, the 12 proteins included 8 that are not previously known as NAD binders. Further validation of these eight proteins showed that their binding affinities to NAD computed by AutoDock Vina are higher than their cognate ligands and also that their protein ratios in the pulse proteolysis are consistent with known NAD-binding proteins. These results strongly suggest that these eight proteins are indeed newly identified NAD binders.Keywords: binding pocket comparison; binding pocket screening; Patch-Surfer2.0; protein−ligand interaction; pulse proteolysis; quantitative mass spectrometry; structural bioinformatics; tandem mass tags labeling;

Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

Our understanding of the complex cell entry pathways would greatly benefit from a comprehensive characterization of key proteins involved in this dynamic process. Here we devise a novel proteomic strategy named TITAN (Tracing Internalization and TrAfficking of Nanomaterials) to reveal real-time protein–dendrimer interactions using a systems biology approach. Dendrimers functionalized with photoreactive cross-linkers were internalized by HeLa cells and irradiated at set time intervals, then isolated and subjected to quantitative proteomics. In total, 809 interacting proteins cross-linked with dendrimers were determined by TITAN in a detailed temporal manner during dendrimer internalization, traceable to at least two major endocytic mechanisms, clathrin-mediated and caveolar/raft-mediated endocytosis. The direct involvement of the two pathways was further established by the inhibitory effect of dynasore on dendrimer uptake and changes in temporal profiles of key proteins.

Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration.

Characterization of ligand–protein binding is of crucial importance in drug discovery. Classical competition binding assays measure the binding of a labeled ligand in the presence of various concentrations of unlabeled ligand and typically use single purified proteins. Here, we introduce a high-throughput approach to study ligand–protein interactions by coupling competition binding assays with mass spectrometry-based quantitative proteomics. With the use of a phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide as a model, we characterized pITAM-interacting partners in human lymphocytes. The shapes of competition binding curves of various interacting partners constructed in a single set of quantitative proteomics experiments reflect relative affinities for the pITAM peptide. This strategy can provide an efficient approach to distinguish specific interacting partners, including two signaling kinases possessing tandem SH2 domains, SYK and ZAP-70, as well as other SH2 domain-containing proteins such as CSK and PI3K, from contaminants and to measure relative binding affinities of multiple proteins in a single experiment.

Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events.

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.

Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high-throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO, that is multifunctionalized with titanium(IV) ions for specific binding to phosphoproteins and with biotin groups that allow for enzyme-linked spectrometric detection. The sensitivity, specificity, and quantitative nature of pIMAGO for phosphorylation assays were examined with standard phosphoproteins and with purified phosphoproteins from whole cell extracts. As low as 100 pg of phosphoprotein can be measured quantitatively with the pIMAGO chemiluminescence assay. The pIMAGO assay was applied to an in vitro kinase assay, kinase inhibitor screening, and measurement of endogenous phosphorylation events. The technique provides a universal, quantitative method for global phosphorylation analysis with high sensitivity and specificity.

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein–protein interactions using anti-GFP single-chain antibody VHH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain VHH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.

Bi-functionalized dendrimers leads to highly efficient quantitative proteomics and the determination of protease activities in snake venoms.

Current methods for phosphoproteome analysis have several limitations. First, most methods for phosphopeptide enrichment lack the specificity to truly purify phosphopeptides. Second, fragmentation spectra of phosphopeptides, in particular those of phosphoserine and phosphothreonine containing peptides, are often dominated by the loss of the phosphate group(s) and therefore lack the information required to identify the peptide sequence and the site of phosphorylation, and third, sequence database search engines and statistical models for data validation are not optimized for the specific fragmentation properties of phosphorylated peptides. Consequently, phosphoproteomic data are characterized by large and unknown rates of false positive and false negative phosphorylation sites. Here we present an integrated chemical, mass spectrometric and computational strategy to improve the efficiency, specificity and confidence in the identification of phosphopeptides and their site(s) of phosphorylation. Phosphopeptides were isolated with high specificity through a simple derivatization procedure based on phosphoramidate chemistry. Identification of phosphopeptides, their site(s) of phosphorylation and the corresponding phosphoproteins was achieved by the optimization of the mass spectrometric data acquisition procedure, the computational tools for database searching and the data post processing. The strategy was applied to the mapping of phosphorylation sites of a purified transcription factor, dFOXO and for the global analysis of protein phosphorylation of Drosophila melanogaster Kc167 cells.

Bi-functionalized dendrimers leads to highly efficient quantitative proteomics and the determination of protease activities in snake venoms.