A.N.K. acknowledges support from NIH R33 CA225458 and R35 GM119455. We thank the Kettenbach and Gerber labs for their helpful discussion.

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M., Shenolikar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+promiscuity+to+precision:+protein+phosphatases+get+a+makeover.">From promiscuity to precision: protein phosphatases get a makeover.</a> Molecular Cell. 33 (5), 537-545 (2009).</li><li>Bollen, M., Peti, W., Ragusa, M. J., Beullens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extended+PP1+toolkit:+designed+to+create+specificity.">The extended PP1 toolkit: designed to create specificity.</a> Trends in Biochemical Sciences. 35 (8), 450-458 (2010).</li><li>Qian, J., Winkler, C., Bollen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4D-networking+by+mitotic+phosphatases.">4D-networking by mitotic phosphatases.</a> Current Opinion in Cell Biology. 25 (6), 697-703 (2013).</li><li>Heroes, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PP1+binding+code:+a+molecular-lego+strategy+that+governs+specificity.">The PP1 binding code: a molecular-lego strategy that governs specificity.</a> The FEBS Journal. 280 (2), 584-595 (2013).</li><li>Eichhorn, P. J., Creyghton, M. P., Bernards, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+regulatory+subunits+and+cancer.">Protein phosphatase 2A regulatory subunits and cancer.</a> Biochimica et Biophysica Acta. 1795 (1), 1-15 (2009).</li><li>Bantscheff, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+chemical+proteomics+reveals+mechanisms+of+action+of+clinical+ABL+kinase+inhibitors.">Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors.</a> Nature Biotechnology. 25 (9), 1035-1044 (2007).</li><li>Klaeger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+proteomics+reveals+ferrochelatase+as+a+common+off-target+of+kinase+inhibitors.">Chemical proteomics reveals ferrochelatase as a common off-target of kinase inhibitors.</a> ACS Chemical Biology. 11 (5), 1245-1254 (2016).</li><li>Duncan, J. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+chemical+proteomic+strategy+for+profiling+phosphoprotein+phosphatases+from+yeast+to+humans.">A quantitative chemical proteomic strategy for profiling phosphoprotein phosphatases from yeast to humans.</a> Molecular and Cellular Proteomics. 17 (12), 2448-2461 (2018).</li><li>Nasa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinase+and+phosphatase+profiling+reveal+that+CDK1+phosphorylates+PP2Ac+to+promote+mitotic+entry.">Quantitative kinase and phosphatase profiling reveal that CDK1 phosphorylates PP2Ac to promote mitotic entry.</a> Science Signaling. 13 (648), (2020).</li><li>Swingle, M., Ni, L., Honkanen, R. 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The authors have nothing to disclose and no conflicts of interest.

PIB-MS is a chemical proteomics approach used to quantitatively profile the PPPome from various sample sources in a single analysis. Much work has been done using kinase inhibitor beads to study the kinome and how it changes in cancer and other disease states10,11,12,13. Yet, the study of the PPPome lags behind. We anticipate that this approach is able to fill this gap and shed light on the reg...

Protein phosphorylation controls most cellular processes, including but not limited to the response to DNA damage, growth factor signaling, and the passage through mitosis1,2,3. In mammalian cells, the majority of proteins are phosphorylated at one or more serine, threonine, or tyrosine residues at some point in time, with phosphoserines and phosphothreonines comprising approximately 98% of all phosphorylation sites2,3. While kinases have been extensively studied in cellular signaling, the role of PPPs in the regulation of dynamic cellular processes is still emerging.
Phosphorylation dynamics are controlled by the dynamic interplay between kinases and phosphatases. In mammalian cells, there are more than 400 protein kinases that catalyze serine/threonine phosphorylation. Over 90% of these sites are dephosphorylated by phosphoprotein phosphatases (PPPs), a small family of enzymes that consists of PP1, PP2A, PP2B, PP4-7, PPT, and PPZ2,3. PP1 and PP2A are responsible for the majority of phosphoserine and phosphothreonine dephosphorylation within a cell2,3,4. The notable difference in number between kinases and phosphatases and the lack of specificity of PPP catalytic subunits in vitro led to the belief that kinases are the main determinant of phosphorylation2,3. However, multiple studies have shown phosphatases to establish substrate specificity through the formation of multimeric holoenzymes5,6,7,8,9. For example, PP1 is a heterodimer that consists of a catalytic subunit and, at a given time, one out of the more than 150 regulatory subunits6,7,8. Conversely, PP2A is a heterotrimer that is formed of a scaffolding (A), a regulatory (B), and a catalytic (C) subunit2,3,9. There are four distinct families of PP2A regulatory subunits (B55, B56, PR72, and striatin), each with multiple genes, splice variants, and localization patterns2,3,9. The multimeric nature of PPPs fills the gap in the number of kinases and PPP catalytic subunits. However, it creates analytical challenges for studying PPP signaling. To comprehensively analyze PPP signaling, it is critical to investigate the various holoenzymes within a cell or tissue. Great advances have been made in studying the human kinome through the use of kinase inhibitor beads, termed multiplex inhibitor beads or kinobeads, a chemical proteomic strategy where kinase inhibitors are immobilized on beads and mass spectrometry is used to identify enriched kinases and their interactors10,11,12,13.
We have established a similar approach to study PPP biology. This technique involves affinity capture of PPP catalytic subunits using beads with an immobilized, non-selective PPP inhibitor called microcystin-LR (MCLR) termed phosphatase inhibitor beads (PIBs)14,15. Unlike other methods that require the endogenous tagging or expression of exogenous PPP subunits that could alter protein activity or localization, PIB-MS allows for the enrichment of endogenous PPP catalytic subunits, their associated regulatory and scaffolding subunits, and interacting proteins (termed the PPPome) from cells and tissues at a given time point or under specific treatment conditions. MCLR inhibits PP1, PP2A, PP4-6, PPT, and PPZ at nanomolar concentrations, making PIBs highly effective at enriching for the PPPome16. This method can be scaled for use on any starting material from cells to clinical samples. Here, we describe in detail the use of PIBs and mass spectrometry (PIB-MS) to efficiently capture, identify, and quantify the endogenous PPPome and its modification states.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63805/63805fig1v2.jpg" /> 
Figure 1: Visual summary of the PIB-MS protocol. In a PIB-MS experiment, samples can be obtained in various forms, from cells to tumors. The sample is collected, lysed, and homogenized prior to PPP enrichment. To enrich for PPPs, the lysate is incubated with PIBs with or without a PPP-inhibitor, such as MCLR. The PIBs are then washed, and PPPs are eluted in denaturing conditions. The samples are prepared for mass spectrometry analysis by the removal of detergents through SP3 protein enrichment, tryptic digestion, and desalting. Samples can then be optionally TMT-labeled prior to mass spectrometry analysis. <a href="https://www.jove.com/files/ftp_upload/63805/63805fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
PIB-MS involves lysis and clarification of cells or tissues, incubation of the lysate with PIBs, elution, and analysis of the eluate via western blotting or mass spectrometry-based approaches (Figure 1). The addition of free MCLR can be used as a control to distinguish specific PIB binders from non-specific interactors. For most applications, a label-free approach can be used to directly identify proteins in eluates. In cases where greater precision in quantification or the identification of low-abundance species is needed, further processing with tandem mass-tag (TMT) labeling can be used to increase coverage and decrease input.

<table><tbody><tr><td>Acetonitrile (ACN)</td><td>Honeywell</td><td>AH015-4</td><td>CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood.</td></tr><tr><td>Anhydrous Acetonitrile</td><td>Sigma-Aldrich</td><td>271004-100ML</td><td>CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood.</td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>model no. 5424</td><td></td></tr><tr><td>Beta-glycerophosphoric acid, disodium salt pentahydrate</td><td>Acros Organics</td><td>410991000</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>model no. 5810 R 15 amp version</td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>Dounce tissue grinder</td><td>Fisherbrand Pellet Pestles</td><td>12-141-363</td><td></td></tr><tr><td>Empore solid phase extraction disk, C18</td><td>CDS Analytical</td><td>76333-132</td><td></td></tr><tr><td>Eppendorf tubes, 1.5 mL</td><td>Eppendorf</td><td>22363204</td><td>CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis.</td></tr><tr><td>Eppendorf tubes, 2 mL</td><td>Eppendorf</td><td>22363352</td><td>CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis.</td></tr><tr><td>Extraction plate manifold</td><td>Waters</td><td>WAT097944</td><td></td></tr><tr><td>Falcon tubes, 50 mL</td><td>VWR</td><td>21008</td><td></td></tr><tr><td>Generic blunt end needle and plunger</td><td></td><td></td><td></td></tr><tr><td>Generic magnetic separation rack</td><td></td><td></td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hydrogen chloride (HCl)</td><td>VWR Chemicals BDH</td><td>BDH3028</td><td>CAUTION: HCl is corrosive; wear gloves and work in a chemical fume hood.</td></tr><tr><td>Hydroxylamine solution 50% (wt/vol)</td><td>Sigma-Aldrich</td><td>467804</td><td></td></tr><tr><td>Incubator, 65 &amp;deg;C</td><td>VWR</td><td>model no. 1380FM</td><td></td></tr><tr><td>Koptec Pure Ethanol, 200 Proof</td><td>Decon Labs</td><td>V1001</td><td></td></tr><tr><td>Methanol for HPLC (MeOH)</td><td>Sigma-Aldrich</td><td>34860-4L-R</td><td>CAUTION: MeOH is flammable and toxic; wear gloves, and work in a chemical fume hood.</td></tr><tr><td>Microcystin LR (MCLR)</td><td>Cayman Chemical</td><td>10007188</td><td>CAUTION: MCLR is toxic; wear gloves when handling and avoid skin contact.</td></tr><tr><td>PBS, 1&amp;times; without calcium and magnesium, pH 7.4 &amp;plusmn; 0.1</td><td>Corning</td><td>&amp;nbsp;21-040-CV</td><td></td></tr><tr><td>pH test strips, such as MilliporeSigma MColorpHast pH test strips and indicator papers</td><td>Fisher Scientific</td><td>M1095310001</td><td></td></tr><tr><td>PIBs</td><td></td><td></td><td>For protocol for the generation of PIBs, see Moorhead et al., 2007.</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Pipette tips, 10 &amp;mu;L</td><td>Eppendorf</td><td>22491504</td><td>CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.</td></tr><tr><td>Pipette tips, 1000 &amp;mu;L</td><td>Eppendorf</td><td>22491555</td><td>CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.</td></tr><tr><td>Pipette tips, 200 &amp;mu;L</td><td>Eppendorf</td><td>22491539</td><td>CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.</td></tr><tr><td>plastic syringe, 10 mL</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Protease inhibitor cocktail III</td><td>Research Products International</td><td>P50700-1</td><td></td></tr><tr><td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer, Oribtrap Fusion, Orbitrap Fusion Lumos, or Orbitrap Eclipse Tribrid Mass Spectrometer&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Refrigerated benchtop centrifuge</td><td>Eppendorf</td><td>model no. 5424 R</td><td></td></tr><tr><td>Rotator (Labquake Shaker Rotisserie)</td><td>Thermo Scientific</td><td>13-687-12Q</td><td>8 rpm rotation</td></tr><tr><td>Sample collection plate, 96- well, 1 mL</td><td>Waters</td><td>WAT058957</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP1311-1</td><td></td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega</td><td>V511C</td><td></td></tr><tr><td>Sodium azide</td><td>EMD Chemicals</td><td>SX0299-1</td><td>CAUTION: Sodium azide is explosive and toxic; wear gloves, work in a chemical fume hood and avoid contact with metals.</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Chemical</td><td>S27110</td><td></td></tr><tr><td>Sonicator (Branson digital sonifier)</td><td></td><td>model no. SFX 250</td><td></td></tr><tr><td>SPE C18 desalting plate</td><td>Waters</td><td>186001828BA</td><td></td></tr><tr><td>SpeedBeads magnetic carboxylate modified particles (SP3 beads)</td><td>Cytiva</td><td>6.51521E+13</td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>model no. 5350</td><td></td></tr><tr><td>TMT10plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent, 3 &amp;times; 0.8 mg per tag</td><td>ThermoFisher</td><td>A37725</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Honeywell</td><td>T6508-25ML</td><td>CAUTION: TFA is corrosive and will irritate skin on contact. Wear gloves and eye protection, and work in a chemical fume hood.</td></tr><tr><td>Tris Base</td><td>Research Products International</td><td>T60040</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Vacuum centrifuge and vapor trap</td><td>Thermo Scientific</td><td>model nos. SpeedVac SPD120 and RVT5105</td><td></td></tr><tr><td>Vortexer (Vortex-Genie 2)</td><td>Scientific Industries</td><td></td><td></td></tr><tr><td>Water LC-MS</td><td>Honeywell</td><td>LC365-4</td><td></td></tr></tbody></table>

a mass spectrometry-based approach to identify phosphoprotein phosphatases and their interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63805/63805fig02.jpg" /> 
Figure 2: Identification of specific PIBs binders. (A) A variety of tissue types or cells can be analyzed via PIB-MS. HeLa cells in biological triplicate were either treated with DMSO or the PPP-inhibitor MCLR, incubated with PIBs, and analyzed via LC-MS/MS. (B) Volcano plot of PIB-MS anal...

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A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

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2.4K Views.  Geisel School of Medicine at Dartmouth. PIB-MS is een nieuwe techniek die wordt gebruikt om te verrijken voor endogene fosforproteïnefosfatasen en ze interageren eiwitten uit cellen en weefsels. Het gaat om de identificatie en kwantificering van deze eiwitten met behulp van op massaspectrometrie gebaseerde proteomics. Deze techniek vereist niet de exogene expressie van gelabelde versies van fosforproteïnefosfatasen die de eiwitactiviteit of lokalisatie kunnen veranderen.Ook maakt het geen gebruik van antilichamen die mogel...

Video: A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Cancer Research

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Biology

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

Developmental Biology

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

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