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Analytical Chemistry Jun 2017The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion...
The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript ( Anal. Chem. 2017 , DOI: 10.1021/acs.analchem.7b00213 ), we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90 min analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photoactivation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, an ∼23.5 kDa phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.
Topics: Animals; Brain; Chromatography, Liquid; Electron Transport; Ions; Mice; Phosphopeptides; Phosphoproteins; Phosphorylation; Proteomics; Tandem Mass Spectrometry
PubMed: 28383256
DOI: 10.1021/acs.analchem.7b00212 -
Journal of the American Chemical Society Feb 2015Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which...
Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome.
Topics: Ligands; Magnetite Nanoparticles; Phosphoproteins
PubMed: 25655481
DOI: 10.1021/ja511833y -
Trends in Endocrinology and Metabolism:... Nov 2011How does signalling via PI3K-PKB (AKT)-mTORC1-p70S6K and ERK-p90RSK mediate wide-ranging physiological responses to insulin? Quantitative proteomics and biochemical... (Review)
Review
How does signalling via PI3K-PKB (AKT)-mTORC1-p70S6K and ERK-p90RSK mediate wide-ranging physiological responses to insulin? Quantitative proteomics and biochemical experiments are revealing that these signalling pathways induce the phosphorylation of large and overlapping sets of proteins, which are then captured by phosphoprotein-binding proteins named 14-3-3s. The 14-3-3s are dimers that dock onto dual-phosphorylated sites in a configuration with special signalling and mechanical properties. They interact with the Rab GTPase-activating proteins AS160 and TBC1D1 to regulate glucose uptake into target tissues in response to insulin and energy stress. Dynamic patterns in the 14-3-3-binding phosphoproteome are providing new insights into how insulin triggers coherent shifts in metabolism that are integrated with other cellular response systems.
Topics: 14-3-3 Proteins; Animals; Humans; Insulin; Phosphoproteins; Phosphorylation; Proteome
PubMed: 21871813
DOI: 10.1016/j.tem.2011.07.005 -
Analytical Sciences : the International... Dec 2018A novel fluorescent staining protocol to detect phosphoproteins in sodium dodecyl sulfate-polyacrylamide gels using a fluorescence sensor,...
A novel fluorescent staining protocol to detect phosphoproteins in sodium dodecyl sulfate-polyacrylamide gels using a fluorescence sensor, 1-(2-hydroxy-1-naphthylazo)-2-naphthol-4-sulfonic acid sodium salt (Calcon), was developed. This method yields results within 135 min, with the sensitivities of 15 ng of α-casein and β-casein, and 62.5 ng of κ-casein, respectively. Since non-phosphoproteins have shown negative signals that are distinctly different from positive signals of phosphoproteins, this detection method allows one to monitor phosphoproteins with high specificity. Furthermore, a total protein profile can be achieved before a destaining step using a scanner with rapid and low-cost without further total protein staining.
Topics: Azo Compounds; Caseins; Electrophoresis, Polyacrylamide Gel; Fluorescence; Fluorescent Dyes; Phosphoproteins; Sensitivity and Specificity; Serum Albumin, Bovine; Staining and Labeling
PubMed: 30224566
DOI: 10.2116/analsci.18P298 -
Cell Communication & Adhesion 2006Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible... (Review)
Review
Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and "top-down" proteomics to phosphoprotein analysis.
Topics: Mass Spectrometry; Phosphoproteins; Phosphorylation
PubMed: 17162667
DOI: 10.1080/15419060601077917 -
Journal of Virology Sep 1986Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of...
Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.
Topics: Cytomegalovirus; Electrophoresis, Polyacrylamide Gel; Humans; Hydrogen-Ion Concentration; Magnesium; Phosphates; Phosphoproteins; Phosphorylation; Protein Kinases; Viral Proteins; Virion
PubMed: 3016333
DOI: 10.1128/JVI.59.3.714-727.1986 -
International Journal of Molecular... Feb 2021phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They... (Review)
Review
phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They have highly diverged, but their overall architecture is conserved. They are multidomain proteins, which all possess an oligomerization domain that separates - and -terminal domains. Large intrinsically disordered regions constitute their hallmark. Here, we exemplify their structural features and interaction potential, based on the P proteins. These P proteins are rather small, and their oligomerization domain is the only part with a defined 3D structure, owing to a quaternary arrangement. All other parts are either flexible or form short-lived secondary structure elements that transiently associate with the rest of the protein. P proteins interact with several viral and cellular proteins that are essential for viral transcription and replication. The combination of intrinsic disorder and tetrameric organization enables them to structurally adapt to different partners and to act as adaptor-like platforms to bring the latter close in space. Transient structures are stabilized in complex with protein partners. This class of proteins gives an insight into the structural versatility of non-globular intrinsically disordered protein domains.
Topics: Amino Acid Sequence; Animals; Binding Sites; Gene Expression Regulation, Viral; Humans; Intrinsically Disordered Proteins; Models, Molecular; Mononegavirales; Phosphoproteins; Pneumovirus; Protein Binding; Protein Conformation; Protein Folding; Protein Interaction Domains and Motifs; Respiratory Syncytial Virus, Human; Structure-Activity Relationship; Viral Proteins
PubMed: 33546457
DOI: 10.3390/ijms22041537 -
Genes & Development Aug 2000
Review
Topics: Adaptor Proteins, Signal Transducing; Adenomatous Polyposis Coli Protein; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cytoskeletal Proteins; Dishevelled Proteins; Glycogen Synthase Kinase 3; Humans; Neoplasms; Phosphoprotein Phosphatases; Phosphoproteins; Proto-Oncogene Proteins; Signal Transduction; Trans-Activators; Transcription Factors; Wnt Proteins; Zebrafish Proteins; beta Catenin
PubMed: 10921899
DOI: No ID Found -
Cell Structure and Function Oct 1999Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular... (Review)
Review
Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.
Topics: Animals; Binding Sites; Carrier Proteins; Cell Differentiation; Cell Division; Humans; Membrane Proteins; Microtubule Proteins; Microtubules; Models, Biological; Nerve Growth Factors; Neurons; Phosphoproteins; Phosphorylation; Protein Binding; Stathmin; Tubulin
PubMed: 15216892
DOI: 10.1247/csf.24.345 -
Current Protocols Sep 2022Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that...
Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).
Topics: Adenosine Triphosphate; Catalysis; Immunoprecipitation; Phosphoproteins; Phosphorylation
PubMed: 36135312
DOI: 10.1002/cpz1.539