Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling

In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein–protein interactions, and changes in post-translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors.

Basic Protocol 1 : Quantifying protein phosphorylation via immunoblotting and near-infrared imaging

Alternate Protocol : Visualizing immunoblots using chemiluminescence

Basic Protocol 2 : Enriching target proteins and isolation of protein complexes by immunoprecipitation

Support Protocol : Covalent conjugation of antibodies to functionalized beads

Basic Protocol 3 : Quantifying proteins and post-translational modifications by targeted mass spectrometry

Tyrosine kinases are critical mediators of immune cell activation and regulation (Hwang, Byeon, Kim, & Park, 2020; Lowell, 2011). The transfer of the ATP γ phosphate to a protein tyrosine residue initiates signaling cascades that alter cell survival, proliferation, and effector functions. The steric and electrostatic effects of tyrosine phosphorylation can induce conformational changes in proteins that expose docking sites, block autoinhibitory interactions, or deprotect motifs for trafficking, degradation, or further post-translational modification. Phosphotyrosine-containing peptides also serve as direct SH2 and PTB binding sites, nucleating higher-order signaling complexes that tune signal strength and kinetics and may even alter the phase properties of signaling complexes (Case, Ditlev, & Rosen, 2019; Oh et al., 2012). The actions of tyrosine kinases initiate an array of immune cell functions, including pathogen detection and killing, phagocytosis, clonal expansion, and migration to sites of infection or damage.

Accordingly, dysregulation of tyrosine kinase signaling pathways is associated with many diseases, including autoimmune and inflammatory disease and cancer. Analysis of activated signaling pathways, therefore, is critical for understanding how immune cells participate in health and disease.

In this article we highlight genetic and chemical tools—including competitive inhibitors, designer kinase–inhibitor pairs, small interfering RNA (siRNA), and CRISPR/Cas9 gene editing—for dissecting tyrosine kinase signaling in immune cells. We present protocols for quantitative evaluation of signaling kinetics, amplitude, and binding interactions and for identifying sites of post-translational modification. Our protocols feature adherent bone marrow–derived macrophages (BMDMs), but we describe adaptations for use with lymphocytes and other cells in suspension. Basic Protocol 1 describes a method for quantitative immunoblotting. Basic Protocol 2 describes a method for (co-)immunoprecipitation of proteins from cell lysates, which can be used in conjunction with immunoblotting or quantitative, targeted mass spectrometry described in Basic Protocol 3. These cell perturbation and protein enrichment strategies can also be used as precursors to flow cytometry or proteomic methods (see Current Protocols articles: Breitkopf & Asara, 2012; Schulz, Danna, Krutzik, & Nolan, 2012).

The procedure for immunoblotting (Western blotting) was developed in the early 1980s. Subsequent advances in monoclonal antibody production, secondary antibody fluorophore conjugation, transfer methods, visualization strategies, and methods for quantification have made immunoblotting a workhorse method for quantifying biochemical changes in cells (Janes, 2015). In this protocol denatured cell lysates are resolved by size via reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE); other protein separation methods such native, nonreducing, and 2D methods may be substituted. Proteins are loaded onto polyvinylidene difluoride (PVDF) membranes via wet electrophoretic transfer, and cellular components are then quantified via antibody recognition of epitopes and subsequent coupling to a luminescent readout. This protocol contains instructions for quantification of total protein and phosphoprotein content with near-infrared imaging of fluorophore-conjugated secondary antibodies. Near-infrared imaging (LI-COR Odyssey or equivalent) has a broad dynamic range amenable to densitometry quantification in LI-COR Image Studio Lite or other software package (e.g., NIH ImageJ; see Internet Resources). The Alternate Protocol describes visualization of blots by chemiluminescence imaging.

We describe a method for stimulating adherent BMDMs with depleted zymosan, a β-glucan preparation that binds the hemi-ITAM-containing receptor Dectin-1 (Underhill, 2003). This representative cell-activating stimulus can be coupled with pharmacological, transcriptional, or genetic disruption of tyrosine kinase function to test the contribution of these kinases to cell signaling. Alternative receptor ligation or inhibition of analog-sensitive Csk (Csk^AS) by the small molecule 3-IB-PP1 can be used as an alternative to Dectin-1 clustering. In the latter approach, 3-IB-PP1 inhibits a sensitized form of Csk, the tyrosine kinase that negatively regulates the Src family tyrosine kinases (SFKs). When Csk^AS is inhibited, SFKs become activated and initiate signaling through many pathways (see Background Information; Brian et al., 2019; Freedman et al., 2015; Schoenborn, Tan, Zhang, Shokat, & Weiss, 2011; Tan et al., 2014). Dectin-1 ligation is a useful positive control for myeloid cell activation via tyrosine kinase–dependent signaling (Freedman et al., 2015; Goodridge et al., 2011), but the choice of controls for a given experiment should reflect the cell and pathway of interest. Where appropriate, we include adaptations applicable to lymphocytes and other cells in suspension.

Materials

• Cells of interest (e.g., BMDMs)

• Cell culture medium (e.g., Dulbecco's modified Eagle medium [DMEM-10]; see recipe)

• Phosphate-buffered saline (PBS), without calcium or magnesium (e.g., Corning, MT21031CV)

• Cell dissociation buffer (e.g., Gibco, 13151014)

• Polarization agent

• Depleted zymosan (e.g., Sigma, Z4250; for preparation see Underhill, 2003)

• Kinase inhibitor (e.g., PP2; Thermo, PHZ1223)

• 3-IB-PP1 (e.g., Millipore, 529598)

• SDS sample buffer (see recipe)

• 1 M dithiothreitol (DTT; e.g., Fisher Scientific, BP172-5)

• 1× running buffer (see recipe)

• 1× transfer buffer (see recipe)

• Tris-acetate protein gel (e.g., Fisher Scientific, EA03585BOX)

• Molecular weight marker (e.g., Bio-Rad, 161-0394)

• Methanol (e.g., Honeywell, AH230-4)

• Total protein stain (e.g., LI-COR, 926-11021)

• Total protein wash (see recipe)

• Total protein removal solution (see recipe)

• 1× tris-buffered saline (TBS; see recipe)

• Blocking buffer (see recipe or purchase from commercial source; e.g., LI-COR, 927-50003)

• Primary antibody

• Primary diluent (see recipe)

• 1× TBS containing Tween-20 (TBST; see recipe)

• Secondary antibody appropriate for primary antibody (e.g., LI-COR)

• Secondary diluent (see recipe)

• Cell culture incubator

• 150-mm² non-tissue culture-treated plate

• Midspeed centrifuge (e.g., Sorvall Legend XTR)

• Hemocytometer

• 6-well non-tissue culture-treated plate (e.g., Corning, 351146)

• Cell scraper (e.g., Corning, 353085)

• 1.5 ml snap-lock microcentrifuge tubes (e.g., Eppendorf, 022363611)

• Sonicator (e.g., Diagenode Bioruptor Pico or other small-capacity bath or probe sonicator)

• Thermomixer (e.g., Eppendorf, 2231000033) or heat block

• Refrigerated microcentrifuge (e.g., Eppendorf, 5415R)

• Electrophoresis and wet transfer running unit (e.g., Invitrogen, EI0002)

• Power source (e.g., Invitrogen, PS0300)

• Sponges (e.g., Invitrogen, EI9052)

• Immobilon-FL PVDF membrane (e.g, Millipore, IPFL00010)

• Filter paper (e.g., GE Healthcare, 30306132)

• Nontranslucent incubation box (e.g., LI-COR, 929-96310)

• Orbital rocker

• Near-infrared imaging system (e.g., LI-COR Odyssey)

• Computer running LI-COR Image Study Lite or similar software and spreadsheet analysis software (e.g., Microsoft Excel)

Stimulation and cell lysis

1.Grow BMDMs according to published protocols (Freedman et al., 2015; Zhu, Brdicka, Katsumoto, Lin, & Weiss, 2008). Seed BMDMs on 150-mm² plates.

2.On day 6 or 7, detach adherent BMDMs: Aspirate medium and wash once with PBS. Dispense 8 ml cell dissociation buffer. Return cells to incubator for ≤15 min, tapping to see if cells separate from the plastic. Remove cells by repeatedly pipetting cell dissociation buffer over the plate surface and rinsing once with fresh PBS. Centrifuge cells 5 min at 400 × g , 4°C. Resuspend cells in DMEM-10 and count.

3.Transfer cells to 6-well plate (10⁶ cells per 2 ml DMEM-10 with or without polarization agents).

4.Rest BMDMs overnight at 37°C in a 10% CO₂ incubator.

5.Prewarm a midspeed centrifuge to 37°C by spinning at 6000 × g.

6.Prepare 0.5 ml depleted zymosan in DMEM-10 with or without kinase inhibitor (e.g., 20 µM PP2, a SFK inhibitor) for each stimulation and time point.

7.Gently remove 1.5 ml DMEM-10 supernatant from each well, and return plates to incubators for at least 10 min to re-equilibrate the temperature.

8.Gently apply 0.5 ml sonicated and washed depleted zymosan or 3-IB-PP1 with or without kinase inhibitor (or alternative stimulation/perturbation). Quickly but gently place plates in the prewarmed centrifuge, and pulse spin 30 s at 5000 × g to synchronize deposition of depleted zymosan particles onto cells.

9.Stop signaling at the desired time point by placing the plate on ice. Quickly aspirate supernatant.

10.Lyse cells by adding 200 to 400 µl SDS sample buffer and DTT to 50 mM. Scrape cells off plate, and incubate at 37°C for 5 min. Pipette cell lysates into labeled 1.5-ml snap-lock tubes.

11.Lyse cells and shear DNA by sonication with chilling (e.g., three times for 1 min at 50% duty cycle with a chilled Diagenode Bioruptor Pico).

12.Incubate samples 15 min at ≥99°C. Microcentrifuge samples 30 s at 10,000 × g , room temperature.

Gel electrophoresis and wet transfer

13.Prepare running buffer and transfer buffer.

14.Prepare gels by removing the comb from the gel and rinsing each lane with running buffer to remove gel fragments.

15.Load ∼2.5 × 10⁴ cell equivalents into each lane, taking care not to puncture the gel. For best results load the same volume in each well. Load the left-most lane with molecular weight marker. Include positive and negative controls on each gel to facilitate quantification across blots. Load unused wells with SDS sample buffer.

16.Fill electrophoresis module with running buffer, and apply constant voltage (150 V) until the dye front has migrated out of the gel or the desired separation has occurred (∼80 min).

17.While the gel is running, prepare transfer apparatus. Cut Immobilon-FL PVDF membrane to size, and rinse membrane in methanol to activate. Rinse three times with distilled water. Place membrane in transfer buffer.

18.Assemble transfer apparatus according to the manufacturer's instructions. Place one corner of the membrane on top of the gel. Slowly place the opposite corner of the membrane onto gel, and lower the rest of the membrane onto the gel, taking care to avoid trapping bubbles. Orient transfer with the membrane on the positive (anode) side and gel on the negative (cathode) side.

19.Fill inner and outer chambers of the apparatus with cold transfer buffer. Place on ice or in a cold room.

20.Transfer 1.75 hr at low voltage (30 V) on ice.

21.Remove membrane from the apparatus, and dry in between two sheets of clean filter paper to fix proteins onto the membrane.

Total protein staining

22.Place membrane in an incubation box, and activate by soaking 1 min in methanol. Discard methanol and rinse three times for 30 s each with water.

23.Add 5 ml total protein stain. Rock 5 min in the dark at room temperature.

24.Discard total protein stain, and wash two times for 30 s each with total protein wash.

25.Rinse membrane three times with water, and image gel with a near-infrared imaging system.

26.Rinse membrane briefly in water. Replace solution with total protein removal solution. Rock 5 min in the dark at room temperature.

Immunoblotting

27.Discard solution and place membrane in methanol.

28.If cutting membrane into segments of different molecular weights, place membrane on clean filter paper, and cut with clean scissors or razor blade. Return to methanol.

29.Discard methanol and rinse three times for 30 s each with water.

30.Discard water. Rock 2 min in 5 to 10 ml TBS at room temperature.

31.Discard TBS and add 5 to 10 ml blocking buffer. Rock 1 hr at room temperature in the dark.

32.Dilute primary antibody in 4 to 6 ml (depending on the size of the container) of 1:1 blocking buffer:primary diluent.

33.Discard blocking buffer, and add diluted primary antibody. Mix overnight at 4°C.

34.Remove and store diluted primary antibody. Wash membrane three times for 5 min each with TBST.

35.Dilute secondary antibody in 1:1 blocking buffer:secondary diluent.

36.Incubate 1 hr at room temperature.

37.Wash three times for 5 min each with TBST.

38.Wash 2 min with TBS to remove residual Tween-20.

39.Dry membrane between two sheets of clean filter paper.

40.Image membrane protein-side down using a near-infrared imager.

Quantification of total protein by densitometry

41.Select appropriate fluorescence channel in the right-hand Display tab of Image Studio Lite.

42.In the Analysis tab, select Draw Rectangle. Draw a rectangle around the entire lane of interest (test darkest lane first). Rotate or resize box using the graph in the right-hand Profile tab. Move box to the left-most lane, duplicate, and drag boxes to the other lanes. Adjust each box, if necessary, using the Profile tab.

43.In the Background pane, select User Defined for background quantification.

44.Draw a small box in between two lanes with representative background fluorescence. In the Background tab, select Assign Shape to apply this box for background subtraction.

45.Export data from the Shapes tab into Microsoft Excel or other spreadsheet manager. Use the background-corrected “Signal” column for data normalization and graphing.

Quantification of immunoblots (repeat for each protein of interest)

46.Select appropriate fluorescence channel in the right-hand Display tab of Image Studio Lite.

47.In the Analysis tab, select Add Rectangle. Place a box on the image by clicking near the darkest band of interest. Rotate or resize the box using the graph in the right-hand Profile tab. Move box to the left-most lane, duplicate, and drag boxes to the other lanes. Adjust each box, if necessary, using the Profile tab.

48.In the Background pane, select Median for background quantification. Adjust borders to top/bottom or right/left, and choose the background box size.

49.Export data from the Shapes tab into Microsoft Excel or other spreadsheet manager. Use the background-corrected “Signal” column for data normalization and graphing.

Horseradish peroxidase (HRP)-conjugated antibodies in conjunction with chemiluminescence imaging is another common approach to visualizing immunoblots. In contrast to direct dye conjugation in near-infrared imaging, HRP-adsorbed blots are developed by addition of an HRP substrate (a luminol/enhancer mixture) that generates a chemiluminescent signal from HRP-conjugated secondary antibodies. Although this method can in some cases be quantitative, the dynamic range is typically narrower than in near-infrared imaging, and it is easy to over- or under-produce signal in this indirect method. To achieve the best signal, gel loading, antibody dose, and substrate choice should be optimized. An advantage of this approach is that the HRP enzyme can be efficiently inactivated and the blot reprobed with a different set of antibodies.

Additional materials (also see Basic Protocol 1)

• HRP-conjugated secondary antibody (e.g., Southern Biotech)

• SuperSignal West Femto Maximum Sensitivity Substrate (e.g., Thermo Scientific, 34096)

• Plastic wrap

• Luminescence imager

1.Complete steps 1 to 22 and steps 28 to 38 of Basic Protocol 1 using HRP-conjugated secondary antibody for step 35.

2.Prepare substrate working solution by combining equal amounts of peroxide and enhancer solutions (from SuperSignal kit). Place membrane on a piece of plastic wrap, and pipet a minimum volume of substrate working solution onto the surface of the blot. Tilt membrane to thoroughly coat, and watch for bands to develop, following manufacturer's instructions.

3.Cover membrane in clear plastic, and smooth to remove bubbles. Image using a luminescence imaging system.

First described in the 1970s (Kessler, 1975), immunoprecipitation is a common method for separating proteins from cell lysates in denaturing or nondenaturing conditions. It has been further refined for protein purification, enrichment of low-abundance species, and identification of protein complexes (co-immunoprecipitation). As a tool for studying cell signaling, immunoprecipitation typically starts with preparation of an antibody–bead complex (noncovalent in this protocol, covalent in the Support Protocol). These antibody-coated beads are then mixed with cell lysates and gently tumbled under conditions that maximize protein capture but minimize protein degradation and further post-translational modification. The protein–antibody–bead complex is then collected, and the (co-)immunoprecipitated proteins are eluted for analysis (Fig. 2).

This protocol describes immunoprecipitation of a target protein from cell lysate. In the absence of the usual loading controls available in whole-cell lysate (described in Basic Protocol 1), a protein content normalization step is essential for quantitative analysis. The immunoprecipitation time, detergent, and salt content will determine the extent of interacting protein co-immunoprecipitation. The composition of the immunoprecipitate can then be probed by blotting for pan-phosphotyrosine, specific phosphorylation sites, total protein, or other epitopes to reveal protein–protein interactions and post-translational modifications that follow cell perturbation (as in Basic Protocol 1). The immunoprecipitates can also be subjected to phosphoproteomics to identify unknown proteins or targeted mass spectrometry to quantify specific peptides and post-translational modifications. A targeted approach is described in Basic Protocol 3.

Materials

• Protein G (or A or other functionalized) Sepharose beads (e.g., GE Healthcare, 17061801)

• PBS (e.g., Corning, MT21031CV)

• Immunoprecipitation antibody

• Protease and phosphatase inhibitor (e.g., Sigma-Aldrich, MSSAFE-5VL)

• n-Dodecyl β-D-maltoside (lauryl maltoside) lysis buffer (see recipe)

• Cells of interest (see Basic Protocol 1)

• Normal serum (e.g., Jackson ImmunoResearch)

• Immunoprecipitation elution buffer (see recipe)

• Bicinchoninic acid (BCA) assay kit (e.g., Thermo Scientific, 23225)

• NP-40 alternative wash buffer (see recipe)

• Refrigerated microcentrifuge (e.g., Eppendorf, 5415R)

• Vortex

• Tube rotator

• Cell scraper (e.g., Corning, 353085)

• 1.5-ml LoBind (low-adsorption) microcentrifuge tubes (e.g., Eppendorf, 022431081)

• Sonicator (e.g., Diagenode Bioruptor Pico or other small-capacity bath or probe sonicator)

• Micro-Bio spin chromatography column (e.g., Bio-Rad, 732-6204)

Cell stimulation and lysis

1.Collect 4 to 5 µl protein G Sepharose beads per 10⁶ cells by microcentrifuging 2 min at 500 × g , room temperature. Carefully remove supernatant, and replace with PBS. Pulse vortex, collect beads, and repeat wash two times.

2.Prebind immunoprecipitation antibody to beads by incubating 1 to 2 µg antibody per 40 × 10⁶ cells with beads. Rotate beads at least 2 hr at room temperature prior to use.

3.Add protease and phosphatase inhibitors to an aliquot of chilled lauryl maltoside lysis buffer. Protect from light and keep on ice until use.

4.Prepare and treat cells as described in steps 1 to 9 of Basic Protocol 1.

5.Quench signaling by washing cells two times with ice-cold PBS and placing plates on ice.

6.Lyse cells by adding 50 µl lauryl maltoside lysis buffer per 10⁶ cells.

7.Scrape plates to lift cells, and collect in sterile 1.5-ml LoBind tubes.

8.Sonicate cells (5 min total, 50% duty cycle in a Diagenode Bioruptor Pico) to disrupt membranes, break up protein aggregates, and shear DNA.

9.Centrifuge lysate 15 min at 15,000 × g in a chilled microcentrifuge to remove insoluble material.

Immunoprecipitation

10.Preclear samples by adding 50 µl protein G Sepharose beads and 10 µl normal serum per 1 ml lysate. Rotate 30 min at 4°C.

11.Collect beads by centrifuging 2 min at 500 × g in a chilled microcentrifuge, and transfer precleared supernatant to new tube. Keep all tubes on ice.

12.Mix 50 µl whole-cell lysate with 50 µl immunoprecipitation elution buffer for later immunoblot.

13.Remove 50 µl lysate for BCA assay (see manufacturer's instructions). Calculate protein concentration in each sample, and aliquot corrected volumes of lysate into LoBind tubes.

14.Wash antibody–beads two times with PBS, discarding supernatant. Resuspend antibody–beads in lauryl maltoside lysis buffer, and portion evenly among lysate samples.

15.Tumble lysates and beads 2 hr at 4°C to immunoprecipitate protein of interest.

16.Apply samples to spin columns. Centrifuge 2 min at 450 × g , 4°C. Mix 50 µl flow-through with 50 µl immunoprecipitation elution buffer for immunodepleted blotting samples.

17.Wash beads and column five times with 1 ml NP-40 alternative wash buffer, centrifuging 2 min at 450 × g , 4°C, and discarding flow-through between washes.

18.Elute immunoprecipitated protein by applying enough immunoprecipitation elution buffer to cover the beads in the spin column. Incubate 15 min at room temperature. Elute 5 min at 10,000 × g , 4°C, in a microcentrifuge.

19.Incubate lysate and immunoprecipitate samples 5 min at ≥99°C. Centrifuge samples 1 min at 10,000 × g , 4°C. Handle and store gel samples as described in step 12 of Basic Protocol 1.

20.To assess the efficiency of immunoprecipitation and the general stoichiometry of co-immunoprecipitated protein binding, run immunoblots with whole-cell and immunodepleted lysates, as described in steps 13 to 46 of Basic Protocol 1.Assess immunoprecipitates by immunoblot, skipping the total protein stain in steps 22 to 26 of Basic Protocol 1.

In some cases, it is best to conjugate immunoprecipitation antibodies covalently to immunoprecipitation beads rather than co-eluting antibodies with the immunoprecipitate samples. For immunoblotting analysis (Basic Protocol 1), covalently conjugated antibody–beads complexes produce cleaner images, facilitating visualization and quantification of proteins comigrating with the heavy and light chains. Covalent conjugation is also ideal for subsequent mass spectrometry analysis (Basic Protocol 3) in that resulting immunoprecipitates can be desalted and run directly without further purification of proteins or detergents that would otherwise harm the mass spectrometer. In spite of these advantages, covalent conjugation tends to be used selectively because of the increased investment of time and reagents.

Materials

• Protein G (or A or other functionalized) Sepharose beads (e.g., GE Healthcare, 17061801)

• PBS (e.g., Corning, MT21031CV)

• Immunoprecipitation antibody

• Dimethyl pimelimidate (DMP; e.g., Thermo Scientific, 21666)

• 0.15 M sodium borate, pH 9.0 (e.g., Millipore, SX03551)

• 0.2 M ethanolamine, pH 8.0 (e.g., Fisher Scientific, M251-1)

• 1 M glycine, pH 3.0 (e.g., Fisher Scientific, BP381-1)

• NaN₃

• 15-ml conical tubes (e.g., Falcon, 14-959-70C)

• Centrifuge

• Tube rotator

NOTE : If this protocol will be followed by mass spectrometry analysis, wear a face mask and gloves for all steps to minimize keratin contamination.

1.Collect enough protein G Sepharose (or alternative) beads for each conjugation.

2.Wash beads twice with PBS by centrifuging 30 s at 1000 × g , room temperature.

3.Resuspend in PBS.

4.Add immunoprecipitation antibody, and tumble 1 hr at room temperature.

5.Prepare 10 ml of 20 mM DMP in 0.15 M sodium borate, pH 9.0, per 1 ml beads.

6.Wash beads twice with 10 ml of 0.15 M sodium borate, pH 9.0.

7.Resuspend beads in 20 mM DMP in 0.15 M sodium borate, pH 9.0.

8.Mix beads and DMP 30 min at room temperature on a rotator.

9.Collect beads and remove DMP solution. Quench by adding 10 ml of 0.2 M ethanolamine, pH 8.0. Incubate 2 hr at room temperature with gentle mixing.

10.Spin beads down by briefly centrifuging and remove ethanolamine. Elute unbound antibody by incubating two times for 10 min each with 1 M glycine, pH 3.0, at room temperature.

11.Wash beads with PBS.

12.Resuspend beads in PBS with 0.02% (w/v) NaN₃.

Commercial antibodies are not available for every protein epitope and post-translational modification. Antibodies may be raised against custom sequences, but this process is costly and at times problematic. Mass spectrometry is a valuable tool for detecting changes in protein homeostasis and identifying novel sites of modification prior to investing in antibody generation. We present a protocol for quantifying phosphorylation on an immunoprecipitated protein via targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS), using parallel reaction monitoring (PRM) on a high-resolution mass spectrometer. Traditional, data-dependent acquisition triggers fragmentation of the top N ions in a MS1 scan (the first component of MS/MS), effectively surveying the most abundant populations of ions. PRM instead uses precursor ion selection to trigger fragmentation of modified peptides of interest and creating full-scan MS2 spectra, improving selectivity, sensitivity, and signal-to-noise ratios (Rauniyar, 2015). This protocol may be adapted for other epitopes or modifications and for kinase (or other) activity assays for probing modification of a target site in cells, lysates, or recombinant proteins.

We describe steps to ensure accurate peptide identification and quantification using a heavy isotope–labeled internal standard. Prior to beginning proteolytic in-gel digestion, a BCA assay is used to quantify the total protein concentration in cell lysates, a critical step for normalizing phosphopeptide levels across samples. Subsequently, protein concentrations are determined using a standard curve of titrated BSA, quantified by densitometry after SDS-PAGE. This ensures that phosphopeptide quantification can be expressed as a concentration ratio relative to the amount of protein subjected to tryptic digest. Finally, a stable isotope–labeled reference peptide is spiked into the immunoprecipitate prior to proteolytic digestion. Peptide concentrations can then be definitively identified and placed on an absolute scale via a peptide standard curve (Fig. 3).

Materials

• Immunoprecipitated samples (see Basic Protocol 2)

• SDS sample buffer (see recipe)

• BCA protein assay kit (e.g., Thermo Scientific, 23225)

• Tris-acetate protein gel (e.g., Fisher Scientific, EA03585BOX)

• SimplyBlue SafeStain (e.g., Invitrogen, LC6065)

• 100 mM ammonium bicarbonate (e.g., JT Baker, 300301)

• Acetonitrile, HPLC grade (e.g., Fisher Scientific, A955-4)

• DTT (e.g., Fisher Scientific, BP172)

• Iodoacetamide (e.g., Sigma-Aldrich, I1149)

• Trypsin digest solution (see recipe)

• Reference peptides (e.g., Sigma-Aldrich)

• Custom-synthesized ¹³C,¹⁵N-heavy isotope amino acid–labeled reference peptide corresponding to digested phosphopeptide of interest

• Custom-synthesized unlabeled phosphorylated peptide standard corresponding to digested phosphopeptide of interest

• CaCl₂ (e.g., Honeywell-Fluka, C1016100G)

• Formic acid (e.g., Fisher Scientific, A117-50)

• Water, HPLC grade (e.g., Fisher Scientific, W6-4)

• Desalting wash solvent (see recipe)

• Trifluoroacetic acid

• Desalting wetting solvent (see recipe)

• Desalting elution solvent (see recipe)

• Calibration curve buffer (see recipe)

• HPLC buffer A (see recipe)

• HPLC buffer B (see recipe)

• Centrifugal filter column (e.g., Millipore, UFC500324)

• Electrophoresis system (e.g., Invitrogen, EI0002)

• Near-infrared imaging system (e.g., LI-COR Odyssey)

• Razor blade

• 1.5-ml LoBind microcentrifuge tubes (e.g., Eppendorf, 022431081)

• Vortex

• Microcentrifuge

• Variable temperature incubator

• Vacuum concentrator (e.g., SpeedVac; Thermo Scientific, SPD140DDA)

• C18 reverse-phase extraction disk (e.g., 3M, 2240/2340)

• 18-G needle

• HPLC system (e.g., Thermo Easy-nLC 1000)

• Silica PicoTip Emitter Column, 100-µm ID, 75-cm final length (e.g., New Objective)

• ReproSil-Pur C18 AQ LC column (packed in-house)

• Orbitrap Fusion Tribrid Mass Spectrometer (e.g., Thermo Fisher)

• Computer running Skyline Targeted Mass Spec software (see Internet Resources)

Quantification of protein content

1.Perform an immunoblot (Basic Protocol 1) with immunoprecipitated samples (Basic Protocol 2). Serially dilute samples in SDS sample buffer (e.g., 1:1, 1:2, 1:10).

2.Quantify amount of immunoprecipitated protein for each sample via densitometry (Basic Protocol 1). Create a standard curve using signals from the serially diluted lanes to calculate the relative amount of precipitated protein in each sample.

3.Concentrate equal amounts of immunoprecipitated protein sample with as much lysate as possible to ensure detection of potentially rare peptides via LC-MS/MS and using centrifugal filter columns according to manufacturer's instructions. Store eluent at −80°C indefinitely.

Gel electrophoresis and quantification

For the following steps, wear a face mask and gloves to minimize keratin contamination during gel loading, excision, reduction, and alkylation.

4.Prepare BSA protein quantification standards at 0.05 to 20 µg total per lane.

5.Load gel with immunoprecipitated samples and BSA standards. Resolve samples by SDS-PAGE (see Basic Protocol 1).

6.Stain gel with SimplyBlue SafeStain in a clean container according to the manufacturer's instructions.

7.Remove stain by washing two times for 1 hr each with distilled water.

8.Image gel on an appropriate imaging system.

9.Generate a standard curve via densitometry analysis of the BSA bands, as described in Basic Protocol 1 and shown in Figure 4.Use this curve to quantify the amount of experimental sample in each lane.

10.Using a fresh, clean razor blade for each sample, excise a sample of gel corresponding to the desired protein (molecular weight range), and place into 1.5-ml LoBind tubes.

Reduction and alkylation

11.Cut gel samples into small (∼2 mm) pieces. Wash gel fragments three times for 15 min each by submerging in ∼100 µl (depending on gel fragment size) of a 1:1 mixture of 100 mM aqueous ammonium bicarbonate:acetonitrile. Mix by vortexing prior to each incubation.

12.Remove final wash, and incubate 1 min in 100% acetonitrile, until gel pieces turn opaque. Collect fragments by briefly centrifuging in a microcentrifuge and discard acetonitrile.

13.Submerge gel fragments in an aqueous solution of 10 mM DTT/50 mM ammonium bicarbonate. Incubate 1 hr at 56°C. Pulse spin in a microcentrifuge and remove supernatant.

14.Submerge fragments in an aqueous solution of 55 mM iodoacetamide/50 mM ammonium bicarbonate. Incubate 30 min at room temperature in the dark. Pulse spin in a microcentrifuge and remove supernatant.

15.Wash gel fragments twice with a 1:1 mixture of 100 mM ammonium bicarbonate:acetonitrile.

16.Remove solution and dry fragments by incubating 1 min in 100% acetonitrile.

In-gel protease digest

17.Remove acetonitrile and cover with trypsin digest solution (see Shevchenko, Wilm, Vorm, & Mann, 1996) spiked with ¹³C,¹⁵N-heavy isotope amino acid–labeled reference peptides. Incubate 15 min on ice.

18.Remove excess trypsin digest solution, and cover gel fragments with an aqueous solution of 50 mM ammonium bicarbonate/5 mM CaCl₂. Digest samples 16 hr at 37°C.

Peptide extraction

19.Collect gel fragments in a microcentrifuge by pulse spinning. Remove supernatant and place in a new LoBind tube.

20.Extract peptides from gel fragments in a minimum volume of 50% (v/v) acetonitrile/0.3% (v/v) formic acid in HPLC-grade water. Pulse vortex and incubate 15 min at room temperature.

21.Transfer peptide-containing supernatant to the LoBind tube from step 19.

22.Submerge fragments in 80% (v/v) acetonitrile/0.3% (v/v) formic acid in HPLC-grade water. Pulse vortex and incubate 15 min at room temperature.

23.Pool second extraction in the LoBind tube with the previous supernatant. Store at −80°C indefinitely.

24.Remove solvent by vacuum concentration (e.g., SpeedVac), and store at −80°C indefinitely.

Peptide desalting

25.Assemble desalting tips by punching two holes from C18 reverse-phase extraction material with an 18-G needle and expelling into a 200-μl pipet tip using a clean capillary tube (Rappsilber, Ishihama, & Mann, 2003; Rappsilber, Mann, & Ishihama, 2007).

26.Add 60 µl desalting wash solvent to vacuum-dried peptide samples. Vortex 45 s and centrifuge 1 min at 3000 × g , room temperature. Add more trifluoroacetic acid if necessary to adjust pH ≤3.

27.Wet stage tips with 60 µl desalting wetting solvent. Centrifuge 2 min at 450 × g , room temperature.

28.Discard solvent and apply acidified samples to stage tip. Centrifuge 2 min at 450 × g , room temperature.

29.Wash stage tip two times with 60 µl desalting wash solvent. Centrifuge 2 min at 450 × g , room temperature.

30.Place stage tip into new LoBind tube, and elute peptides with 60 µl desalting elution solvent. Centrifuge 2 min at 450 × g , room temperature.

31.Remove solvent by vacuum concentration (e.g., SpeedVac), and store at −80°C indefinitely.

Preparation of calibration curve samples

32.Dilute 1000 fmol heavy isotope–labeled phosphorylated peptide standard in calibration curve buffer into several LoBind tubes.

33.To create a calibration curve to quantify the amount phosphorylated peptide, add increasing concentrations of unlabeled phosphorylated peptide standard so that the molar ratio of unlabeled phosphorylated peptide standard:heavy-labeled phosphorylated peptide standard spans 0.1 to 1.5.

34.Dilute 1000 fmol phosphorylated peptide standard in calibration curve buffer into several LoBind tubes.

35.To create a calibration curve to quantify the ratio of phosphorylated and unphosphorylated peptide in each sample, add increasing amounts of unphosphorylated peptide standard such that the molar ratio ranges from 0.05 to 1.5.

36.Submit calibration curve samples and in-gel digested samples for LC-MS/MS (steps 37 to 42).

LC-MS/MS

37.Load a 75-cm × 100-µm silica PicoTip Emitter column for nanospray with ReproSil-Pur 1.9-mm C18 AQ.

38.Mount loaded PicoTip Emitter column in a nanospray source in line with an Orbitrap Fusion with 2.1 kV spray voltage in the positive mode and heated capillary maintained at 275°C.

39.Set up a tripartite peptide elution program decreasing the fraction of HPLC buffer A and increasing the fraction of HPLC buffer B with a 300 nl/min flow rate:

• 5% to 10% HPLC buffer B over 5 min
• 10% to 16% HPLC buffer B over 40 min
• 16% to 26% HPLC buffer B over 5 min.

40.Define an acquisition method comprising a full scan and PRM to detect singly, doubly, and triply charged precursor ions without scheduling. Set the full scan event to employ a 380 to 1500 m/z selection, an Orbitrap resolution of 60,000 (at m/z 200), a target automatic gain control (AGC) value of 4 × 10⁵, and maximum ion injection time of 50 ms. Set the PRM scan to employ an Orbitrap resolution of 30,000 (at m/z 200) and a target AGC value of 5 × 10⁴ and/or maximum ion injection time of 54 ms to ensure that enough fragment ions are captured for MS/MS detection.

41.Set the MS2 quadrupole isolation window to 1.6 m/z. Perform fragmentation with a higher-energy collision-induced dissociation (HCD) of 30%, and collect an MS2 scan from 100 to 1000 m/z.

42.Collect PRM data in centroid mode, and export for quantification.

Data analysis using Skyline

Configuration

43.Analyze data in the Skyline Targeted Mass Spec program (see Internet Resources; MacLean et al., 2010; Pino et al., 2020). Open the Skyline Start Page, and select Blank Document and Save As.

44.Select the Settings tab, and locate Peptide Settings. Input parameters to reflect the experimental settings.

45.In the Settings tab locate Transition Settings.

46.Navigate to the Edit tab, then Insert and Peptides. Enter the phosphorylated peptide sequences and select Insert.

Importing and inspecting standard raw data

47.Import raw mass spectrometer files into Skyline by navigating to File, Import, and Results. Choose Add single injection replicates in files and select OK, which will prompt the Import Results Files to show raw standard curve data files. Upload the selected files by choosing Open, followed by Do Not Remove when the option to remove the naming prefix appears. Confirm and close the window by selecting OK.

48.Using raw files generated from standards (e.g., heavy isotope and light isotope phosphopeptides), inspect the chromatographic traces for quality control.

49.Manually inspect each peptide-extracted product ion chromatogram.

Analyzing PRM data from samples

50.Import raw sample files into Skyline as described in step 47.

51.Inspect chromatographic traces, retention times, and fragmentation patterns of heavy and light phosphorylated peptides in each sample.

Generating a calibration curve

52.Define concentrations of external standards. To do so, go to the View tab, and select Document Grid.

53.Go to the top-left Reports dropdown menu, and select Replicates from the Reports list.

54.Define the standard raw files as Sample Type Standard, and specify their Analyte Concentrations. Select Unknown for sample raw files.

55.To view the calibration curve, go to the View menu, and select Calibration Curve.

56.Access Reports from the Document Grid, and select Peptide Quantification. Prepare a report in the Export tab, enter the file name, and click OK.

57.Normalize raw quantifications for each sample using the total protein amount used for in-gel digestion.

Blocking buffer

• 3% (w/v) BSA
• 25 mM Tris base
• 125 mM NaCl
• 0.02% (w/v) NaN₃
• Adjust pH to 8.0 with NaOH
• Store at 4°C for up to 6 months

Calibration curve buffer

• 5% (v/v) acetonitrile
• 0.1% (v/v) trifluoroacetic acid
• Water, HPLC grade
• Store at 4°C for up to 1 year

Desalting elution solvent

• 40% (v/v) acetonitrile
• 0.1% (v/v) trifluoroacetic acid
• Water, HPLC grade
• Store at 4°C for up to 1 year

Desalting wash solvent

• 2% (v/v) acetonitrile
• 0.1% (v/v) trifluoroacetic acid
• Water, HPLC grade
• Store at 4°C for up to 1 year

Desalting wetting solvent

• 20% (v/v) acetonitrile
• 0.1% (v/v) trifluoroacetic acid
• Water, HPLC grade
• Store at 4°C for up to 1 year

DMEM-10

• DMEM containing 4.5 g/L glucose and glutamine (e.g., Corning, 10-017-CM)
• 10% (v/v) fetal bovine serum
• 0.11 mg/ml sodium pyruvate
• 2 mM penicillin/streptomycin
• 2 mM L-glutamine
• Store at 4°C for up to 1 year

HPLC buffer A

• 0.1% (v/v) formic acid
• Water, HPLC grade
• Store at 4°C for up to 1 year

HPLC buffer B

• 0.1% (v/v) formic acid
• 99.9% (v/v) acetonitrile, HPLC grade
• Store at 4°C for up to 1 year

Immunoprecipitation elution buffer

• 125 mM Tris base
• 10% (v/v) glycerol
• 5% (v/v) 2-mercaptoethanol
• 25% (w/v) SDS
• 0.1% (w/v) bromophenol blue
• Store at 4°C for up to 6 months

Lauryl maltoside lysis buffer

• 1% (w/v) lauryl maltoside
• 150 mM NaCl
• 0.01% (w/v) NaN₃
• Store at 4°C for up to 1 year

Immediately before use add protease and phosphatase inhibitors.

NP-40 alternative wash buffer

• 1% (v/v) NP-40 alternative (e.g., Thermo Fisher Scientific, 49-201-850ML)
• 150 mM NaCl
• 10 mM Tris·HCl, pH 7.6
• 0.01% (w/v) NaN₃
• Store at 4°C for up to 1 year

Immediately before use add protease and phosphatase inhibitors.

Primary diluent

• 1× TBS (see recipe)
• 0.2% (v/v) Tween-20
• 0.02% (w/v) NaN₃
• Store at 4°C for up to 6 months

Running buffer, 20×

• 1 M tricine
• 1 M Tris base
• 2% (w/v) SDS
• Store at 4°C for several months

Dilute to 1× working solution before use.

SDS sample buffer

• 128 mM Tris base
• 10% (v/v) glycerol
• 4% (w/v) SDS
• 0.1% (w/v) bromophenol blue
• Adjust pH to 6.8 with 1 M HCl
• Store at 4°C for up to 1 year

Immediately before use add DTT to 50 mM.

Secondary diluent

• 1× TBS (see recipe)
• 0.2% (v/v) Tween-20
• 0.04% (w/v) SDS
• 0.02% (v/v) NaN₃
• Store at 4°C for up to 6 months

TBS, 20×

• 2.5 M NaCl
• 0.5 M Tris base
• Filter sterilize
• Store at room temperature for up to 6 months

Dilute to 1× working solution before use.

TBST, 20×

• 4 M NaCl
• 0.5 M Tris base
• 1% (v/v) Tween-20
• Filter sterilize
• Store at room temperature for up to 6 months

Dilute to 1× working solution before use.

Total protein removal solution

• 0.1 M NaOH
• 30% (v/v) methanol
• Store at 4°C for up to 6 months

Total protein wash

• 6.7% (v/v) acetic acid
• 30% (v/v) methanol
• Store at 4°C for up to 6 months

Transfer buffer, 20×

• 500 mM bicine
• 500 mM Bis Tris
• 20 mM EDTA
• Apply gentle heat to dissolve
• Store at 4°C for up to several months

On the day of transfer, dilute to 1× working solution in chilled water, supplement with 10% (v/v) methanol, and keep cold.

Trypsin digest solution

• 50 mM NH₄HCO₃
• 5 mM CaCl₂
• 5 ng/µl trypsin, sequencing grade (e.g., Promega, V5111)
• Store at −80°C indefinitely

Add heavy isotope–labeled reference peptide as necessary for experiment.

Background Information

Tyrosine kinases are important regulators of immune cell activation, proliferation, and survival (Bryan & Rajapaksa, 2018). Transfer of the terminal phosphate of ATP to a tyrosine residue on a protein substrate results in changes in conformation and protein–protein interaction that act as signals to direct cellular function (Lemmon & Schlessinger, 2010). The growth, survival, and proliferation functions of tyrosine kinases are important in all cells. Immune cells employ peculiar binding motifs, alternative expression of kinase family members, and additional receptor families for additional functionalities such as phagocytosis, antigen-specific signaling, and polarization. In lymphocytes, hematopoietic SFKs initiate signaling downstream of T and B cell receptors by phosphorylating immunoreceptor tyrosine-based activation motifs (ITAMs), which leads to activation of the tandem SH2–containing tyrosine kinases Syk and Zap-70. Together, these tyrosine kinases activate FAK family tyrosine kinases (FAK, Pyk2) and Tec family tyrosine kinases (Btk, Itk, Tec; Hwang et al., 2020). Parallel pathways are activated upon Fc receptor engagement in myeloid and NK cells (Bradshaw, 2010; Cox & Greenberg, 2001; Freedman et al., 2015; Futosi & Mocsai, 2016; Lowell, 2011).

Janus kinase (JAK) activation downstream of receptor tyrosine kinases is critical for activation of signal transducer and activator of transcription (STAT) proteins that mediate growth, differentiation, and polarization (Villarino, Kanno, & O'Shea, 2017). Other receptor tyrosine kinases such as Flt3, c-Kit, and Tyro/Axl/Mer control cell survival, differentiation, and many other essential functions of immune cells (Masson & Ronnstrand, 2009; Rothlin, Carrera-Silva, Bosurgi, & Ghosh, 2015). Despite the many inputs that engage tyrosine kinases and an intense research focus on the tyrosine kinases involved in immune activation, we are still discovering elements of the interactions and dynamics of tyrosine kinases with profound effects on immune regulation (Brian et al., 2019; Courtney et al., 2017; Freedman et al., 2015; Hwang et al., 2020; Salter et al., 2018). Understanding the dynamics, kinetics, substrates, and scaffolding interactions of tyrosine kinases is critical to developing therapeutics that modulate immune function (Roschewski et al., 2020; Salter et al., 2018; Solouki, August, & Huang, 2019).

Numerous tools exist for studying the actions of tyrosine kinases in immune cells, including genetic methods such as siRNA knockdown, CRISPR/Cas9-based gene editing, small-molecule inhibitors, and chemical–genetic designer kinase–inhibitor pairs. Each approach has advantages and disadvantages with regard to specificity, temporal control, and likelihood of triggering compensatory mechanisms (Table 2).

Genetic methods are attractive options for studying kinase function because of their inherent specificity and stability. While knockout gene editing strategies are valuable because they offer complete disruption of kinase signaling, siRNAs offer inducible control over kinase signaling disruption and are especially useful when knocking out a given kinase is lethal or maturation-impairing to a cell type or animal. siRNAs and genetic knockouts are routinely used to investigate the roles of kinases in immune cells. For instance, mice in which the SFK Lyn has been knocked out have become important models of autoimmune disease after studies revealed the importance of Lyn as a negative regulator of B cell and dendritic cell activation (Brodie, Infantino, Low, & Tarlinton, 2018; Scapini, Pereira, Zhang, & Lowell, 2009). Tyrosine kinase knockouts can also be coupled to Cre -lox and FLP-FRT systems for cell-specific knockout (Lamagna, Hu, DeFranco, & Lowell, 2014; Lamagna, Scapini, van Ziffle, DeFranco, & Lowell, 2013). The advent of CRISPR-Cas9 gene editing has facilitated the substitution of specific amino acid residues in knockin models, allowing researchers to dissect novel elements of tyrosine kinase function (Harder et al., 2001). The major drawback of knockout and knockdown models for studying kinase signaling is that cells often develop compensatory mechanisms for coping with loss of the given kinase. These feedback (or, in cell lines, evolutionary) effects may mask the normal signaling contributions and scaffolding interactions of the kinase of interest (El-Brolosy & Stainier, 2017; Peng, 2019).

Small-molecule inhibitors have facilitated the study of kinases in many aspects of immune activation. Kinase inhibitors generally function by competing with ATP for access to the active site, preventing substrate phosphorylation (Davies, Reddy, Caivano, & Cohen, 2000). Although a large number of compounds are marketed for inhibition of specific kinases, caution should be used when choosing an inhibitor and interpreting its effects on signaling. ATP binding sites are highly conserved across the kinome (Manning, Whyte, Martinez, Hunter, & Sudarsanam, 2002), and most inhibitors target multiple kinases, either within a family or in different branches of the kinome (Fabian et al., 2005). Researchers should familiarize themselves with these off-target effects and use the lowest effective concentration of inhibitor to disfavor weaker binding interactions. Furthermore, many kinase inhibitors are poorly soluble in aqueous buffers, necessitating formulation for experiments in vivo or pretreatment for experiments in vitro (Eckstein et al., 2014; Herbrink, Schellens, Beijnen, & Nuijen, 2016). A final consideration when working with ATP-mimetic inhibitors is that these inhibitors typically bind and may even induce the active conformation of the kinase. This can lead to a paradoxical increase in typical readouts of kinase activation (e.g., phosphorylation of the activation loop tyrosine) and may even ultimately promote signaling due to release of autoinhibition. Careful controls (e.g., phosphorylation of inhibitory/activating sites on the kinase and direct substrates) should be probed along with downstream readouts of cell activation. Ultimately, however, small-molecule inhibitors for many kinases are well characterized and commercially available and require little up-front investment of time or resources. Moreover, a pharmacological approach can uniquely enable the study of transient effects with high kinetic fidelity and minimal regulatory compensation. Inhibitors are thus powerful tools for dissecting kinase contribution to immune activation.

Chemical–genetic methods for studying kinase signaling in immune cells combine the specificity of gene editing with the temporal control of small-molecule inhibitors. In one approach kinases are sensitized to a bulky analog of an ATP competitive kinase inhibitor by substituting a smaller amino acid side chain for the usual aliphatic, polar, or bulky gatekeeper residue (Lopez, Kliegman, & Shokat, 2014). Since the gatekeeper is not directly involved in ATP binding, the “analog-sensitive” kinase retains kinase activity until the designer inhibitor is added (Bishop et al., 2000). This chemical–genetic approach can be used in transfected cells or incorporated into the genome of a model animal as a transgene or knockin. Since endogenous kinases have more occlusive gatekeeper residues, the engineered kinase–inhibitor pair is much more specific than traditional kinase inhibition (Fig. 4). Importantly, analog-sensitive kinase inhibition has the additional advantage over genetic or siRNA knockout approaches in that normal kinase function in the absence of inhibitor will allow direct comparison of cells pre- and post-treatment. This real-time component also minimizes the likelihood of compensatory transcriptional changes and other adaptations in primary cells or animals and selective pressure and evolution in cell lines. This approach has been used to identify the specific roles for Zap-70 in T cell activation and Csk in T cell and macrophage activation, but the approach can be applied to other kinases as well (Freedman et al., 2015; Levin, Zhang, Kadlecek, Shokat, & Weiss, 2008; Tan et al., 2014). Furthermore, although many kinase inhibitors blunt signaling, some kinases such as Csk have paradoxical negative regulatory functions. Inhibition of Csk^AS with 3-IB-PP1 leads to robust SFK activation (Freedman et al., 2015; Tan et al., 2014). Inhibition of these negative regulatory kinases can be used as potent stimuli of cellular signaling and can be combined with other kinase inhibitors to tease apart kinase contribution to cellular activation and protein dynamics (Brian et al., 2019).

Although some information can be gleaned from unbiased total protein and pan-phosphotyrosine detection methods, immunoblotting is typically most effective when applied as a targeted, relatively low-throughput process, requiring antibodies raised against unique peptide sequences or sites of post-translational modification. The best antibodies have minimal cross-reactivity with other molecules in the cell. Small-volume, higher-throughput apparatuses are available, but these systems are less amenable to combining antibodies and resolving multiple species in a single blot. Despite these caveats, immunoblotting remains a robust, sensitive, and adaptable technique (Kurien & Scofield, 2015). If epitope-specific antibodies are unavailable, immunoblotting can be combined with immunoprecipitation. For example, total protein immunoprecipitation can be followed with a pan-phosphotyrosine blot, and molecular weight can be used to infer the identity of phosphorylated protein (Freedman et al., 2015). Alternatively, cyanogen bromide fragmentation (Thofte et al., 2018) can resolve phosphorylation of individual sites on multiply phosphorylated proteins.

Freed of the requirement for specific antibodies, LC-MS/MS is an excellent exploratory technique for quantifying poorly studied proteins and sites of post-translational modification. This method is especially useful for multiply modified proteins that cannot easily be probed by blotting. Advances in LC-MS/MS have allowed researchers to quantify tyrosine phosphorylation via targeted and unbiased approaches (Dekker et al., 2018; Hu, Noble, & Wolf-Yadlin, 2016; Liu & Chance, 2014). Proteomics approaches use databases to identify enzyme-digested peptides following resolution by LC-MS/MS. Unbiased LC-MS/MS can identify novel sites of phosphorylation in a cell lysate but may miss low-abundance peptides. In contrast, targeted approaches using isotope-labeled reference peptides are highly sensitive and can be used to quantify abundance or novel sites of post-translational modification in cell lysates and in vitro kinase assays using recombinant or purified proteins.

Together, these protocols describe powerful tools for investigating tyrosine kinase and other cell modulatory signaling. The methods range from general and flexible when reagents are available (immunoblotting) to more focused (co-immunoprecipitation and LC-MS/MS). Together, they constitute a process for dissecting the kinetics and dynamics of signaling pathway activation, protein–protein interaction, and novel tyrosine phosphorylation that are essential for understanding how the many inputs that engage tyrosine kinases are involved in immune activation, allowing researchers to develop tools that modulate immune function by directing kinase signaling.

Critical Parameters

Basic Protocol 1: A million cells lysed in 400 µl lysis buffer should yield enough protein for analysis with near-infrared-conjugated secondary antibodies and an appropriate imager (e.g., LI-COR Odyssey). We have found that this ratio works well for macrophages, but the ratio may need to be adjusted (∼doubled) for smaller cells, such as primary T and B cells, Jurkat cells, and mast cells, depending on the protein being analyzed. It is possible to use fewer cells, but the lysis buffer volume should be scaled to maintain comparable protein concentrations. Before beginning, primary antibodies should be validated to ensure specificity to the desired protein being probed.

Basic Protocol 2: For each condition a large number of cells (8–40 × 10⁶) is required to ensure immunoprecipitation of sufficient protein for subsequent analysis. Stringency of the buffer, incubation, and wash conditions should be optimized so that only specific, biologically relevant protein–protein interactions are preserved. Stringency of co-immunoprecipitation can be assessed by blotting for nonassociated and loosely associated proteins in immunoprecipitates. To increase the stringency of immunoprecipitation, researchers can screen different lysis detergents and increase the salt concentration (over the typical 150 mM) in the wash buffer. It is also critical to keep lysis buffers, wash buffers, and beads cold to prevent phosphatase and protease activity.

Basic Protocol 3: When working with gels prior to protease digest, it is critical to avoid keratin contamination. Be sure to wear gloves, a face mask, and a laboratory coat, working to limit breathing or leaning over the gel as much as possible. Surfaces, tools, and instruments should be thoroughly cleaned with tissue wipes and 70% ethanol prior to use. Iodoacetamide and DTT should be portioned and dissolved in appropriate buffers immediately before use to prevent degradation from light. Buffer conditions, acquisition method, and scan events will vary depending on the chemical properties of the target peptides and samples and will require extensive method development using synthetic peptide standards (isotope-labeled and unlabeled). Data analysis will also change based on the peptides being studied.

Troubleshooting

Basic Protocol 1: See Internet Resources (e.g., Good Westerns gone bad, LI-COR) for information on common issues and troubleshooting techniques for immunoblotting.

Basic Protocol 2: Whereas primary antibodies for immunoblotting most likely recognize denatured proteins, antibodies for immunoprecipitation must recognize proteins in their native conformation. Optimization with different antibody clones may be needed. Buffers and incubation periods should be optimized to ensure immunoprecipitation stringency is desired.

Basic Protocol 3: If no peptides are detected following LC-MS/MS, researchers should ensure the immunoprecipitation process is optimized to enrich the desired protein. If no phosphorylated peptides are detected, ensure the LC-MS/MS method is first optimized to detect both phosphorylated and unphosphorylated reference peptides. We use trypsin as a protease for in-gel digestion in this protocol; however, trypsin digestion may not yield peptides suitable for MS/MS detection, necessitating the use of other enzymes with different amino acid preferences for peptide digestion.

Understanding Results

Basic Protocol 1: Immunoblotting should reveal distinct bands for each probed protein at the correct molecular weight. Immunoblotting with optimal primary and secondary antibody dilutions with near-infrared secondary antibodies should prevent signal saturation.

Basic Protocol 2: Immunoprecipitation should enrich protein complexes of interacting proteins. The robustness of immunoprecipitation can be assessed by immunoblotting for proteins that are not anticipated to interact with the immunoprecipitated protein.

Basic Protocol 3: Targeted LC-MS/MS for protein phosphorylation should identify both phosphorylated and nonphosphorylated peptides that can be quantified and normalized to gel loading based on detection of a protein (BSA) standard curve (shown in Fig. 4) and with calibration curve samples based on the ratio of heavy isotope.

Time Considerations

Basic Protocol 1: Cell stimulations will vary by the signaling pathway and cell type of interest and can range from seconds to days. The time required for SDS-PAGE and immunoblotting will depend on the particular system being used: roughly 1 to 2 hr for SDS-PAGE and 2 hr for wet transfer systems described here. However, dry transfer systems can take substantially less time. Primary antibody incubation will depend on the particular antibody being used and usually requires 1 hr at room temperature or overnight at 4°C. Secondary antibody incubation with near-infrared antibodies lasts 1 hr plus an additional 30 min for total washing.

Basic Protocol 2: If antibody–bead conjugation is being used (Support Protocol), the process should be completed at least the day before beginning immunoprecipitation. The success of antibody–bead conjugation should be verified by a separate experiment. Antibody–bead conjugation takes roughly 4 to 5 hr. Cell stimulation and lysis prior to immunoprecipitation will vary by the signaling pathway and cell type of interest and can range from seconds to days. The BCA assay to measure protein content after lysis takes ∼1 hr. Immunoprecipitation incubations can vary by primary antibody and lysis buffers and typically range from 1 hr to overnight.

Basic Protocol 3: The entire process for Basic Protocol 3 requires several days, although there are several places in which samples can be frozen for further processing. The process can be broken up as follows:

    Days 1 and 2: Quantification of immunoprecipitated lysate protein content; samples can be frozen until the next step.    Day 3: SDS-PAGE, protein quantification, reduction and alkylation.    Days 3 and 4: In-gel protease digestion.    Day 4: Peptide extraction; samples can be frozen until next step.    Day 5: Peptide desalting; samples can be frozen until next step.    Days 6 and 7: LC-MS/MS; processing time will depend on the HPLC method and number of samples.    Days 8 and 9: Data analysis.

Acknowledgments

Protocols were developed for projects with grants from the National Institutes of Health award numbers T32DA007097 (BFB) and R01AR073966, R03AI130978, T32AR007304, and F32AI082926 and from the University of Minnesota Foundation Equipment Award E-0918-01 (TSF).

Author Contributions

Ben F. Brian 4th : Funding acquisition; investigation; methodology; validation; visualization; writing-original draft; writing-review & editing. Candace R. Guerrero : Methodology; resources; software; validation; visualization; writing-original draft; writing-review & editing. Tanya S. Freedman : Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; supervision; validation; visualization; writing-original draft; writing-review & editing.

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• https://www.licor.com/bio/image-studio-lite/

Image Studio Lite Software.

• https://imagej.nih.gov/ij/index.html

ImageJ Software.

• https://www.licor.com/documents/dq6jb8sgnkiwlas0g99b5hq0tsuvcyyz

Good Westerns gone bad.

• https://skyline.ms/project/home/software/Skyline/begin.view

Skyline software.

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Affinity chromatography. vol. 1: Antibodies.

Number of times cited according to CrossRef: 4

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