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Mar 2021

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Enrichment of Tyrosine Phosphorylated Peptides for Quantitative Mass Spectrometry Analysis of RTK Signaling Dynamics
丰富酪氨酸磷酸化肽用于RTK信号动力学的定量质谱分析   

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Abstract

Cells sense and respond to mitogens by activating a cascade of signaling events, primarily mediated by tyrosine phosphorylation (pY). Because of its key roles in cellular homeostasis, deregulation of this signaling is often linked to oncogenesis. To understand the mechanisms underlying these signaling pathway aberrations, it is necessary to quantify tyrosine phosphorylation on a global scale in cancer cell models. However, the majority of the protein phosphorylation events occur on serine (86%) and threonine (12%) residues, whereas only 2% of phosphorylation events occur on tyrosine residues (Olsen et al., 2006). The low stoichiometry of tyrosine phosphorylation renders it difficult to quantify cellular pY events comprehensively with high mass accuracy and reproducibility. Here, we describe a detailed protocol for isolating and quantifying tyrosine phosphorylated peptides from drug-perturbed, growth factor-stimulated cancer cells, using immunoaffinity purification and tandem mass tags (TMT) coupled with mass spectrometry.


Keywords: Tyrosine phosphorylation (酪氨酸磷酸化), Receptor tyrosine kinases (受体酪氨酸激酶), Signal transduction (信号转导), Phosphoproteomics (磷酸蛋白质组学), Phosphotyrosine enrichment (磷酸酪氨酸富集), Tandem mass tag labeling (串联质量标签), Growth factor stimulation (生长因子刺激)

Background

Tyrosine phosphorylation (pY)-mediated signaling networks regulate important cellular processes like cell growth, migration, differentiation, and aging. Given the importance of this post-translational modification, it is not surprising that nearly half of all protein tyrosine kinases (PTKs) are deregulated in cancer (Del Rosario and White, 2010). Although the function of PTKs as oncogenes has been well established, protein tyrosine phosphatases (PTPs) can have either positive or negative effects on cell proliferation. These deregulated enzymes can modulate the tyrosine phosphorylation landscape of cells, resulting in aberrant cellular signaling and cancer. Therefore, research over the past decade has focused on the development of target-based therapeutics that inhibit these enzymes.


Receptor tyrosine kinases (RTKs) are a sub-class of PTKs that mediate cellular responses to growth factors. RTKs can be aberrantly activated by gain-of-function mutations, genomic amplification, chromosomal rearrangements, or autocrine signaling (Du and Lovly, 2018). These activated RTKs can initiate a wide range of downstream signaling pathways, such as RAS/MAPK or PI3K/AKT signaling, which elicit oncogenic responses in cells (Du and Lovly, 2018). Understanding these complex dynamic signaling networks and identifying ways to attenuate this aberrant signaling has important implications for cancer treatment.


Phosphoproteomics is a powerful method that can be used to measure the global tyrosine phosphorylation status of cancer cells in a site-specific manner. It can be used to characterize novel small molecule inhibitors and improve our understanding of signaling networks in disease contexts that aid in the translation of these findings into clinical benefit. Recently, the effects of a SHP2 allosteric inhibitor (SHP099) on the pY network in response to cell stimulation with epidermal growth factor (EGF) identified two classes of phosphosites – sites that are dephosphorylated by SHP2 and sites that are protected by the SH2 domains of SHP2 from dephosphorylation by other PTPs (Vemulapalli et al., 2021). These findings explain how SHP2 has both positive and negative effects on signaling. Here, we present a detailed workflow on sample preparation, enrichment, and quantitative profiling of phospho-tyrosine peptides, via immobilized metal affinity chromatography (IMAC) and tandem mass tag (TMT) labeling, followed by a final immunoaffinity purification of the phospho-tyrosine proteome using a pY monoclonal antibody, adapted from Vemulapalli et al. (2021).

Materials and Reagents

  1. PCR tubes (Denville Scientific, catalog number: C18064)

  2. Chloroform-resistant 15 mL centrifuge tubes (Falcon, catalog number: 352196)

  3. Low protein binding tubes (Thermo Fisher Scientific, catalog number: 90410)

  4. 50 mL reagent reservoir (Corning, catalog number: 4870)

  5. 96-well U-bottom assay plate (Falcon, catalog number: 353910)

  6. C18 47 mm Extraction Disc (Empore, catalog number: 2215-C18)

  7. Sep-Pak C18 50 mg sorbent cartridges (Waters, catalog number: WAT054960)

  8. SOLA HRP 10 mg Sep-Pak cartridges (Thermo-Fisher, catalog number: 03-150-391)

  9. Autoradiography films (Ece Scientific, catalog number: E3018)

  10. Plunger for preparing StageTip (Hamilton, catalog number: 1122-01)

  11. mColorpHast pH (2.0-9.0) test strips (MilliporeSigma, catalog number: 109584)

  12. Amersham Hybond 0.45 µm PVDF (GE, catalog number: 10600023)

  13. 35 cm Sepax GP-C18 resin (1.8 µm, 150 A, Thermo Fisher Scientific)

  14. MDA-MB-468 cells (ATCC, catalog number: HTB-132)

  15. Liquid Nitrogen

  16. 10× Tris Buffered Saline (Boston BioProducts, catalog number: BM-300)

  17. EPPS (Sigma, catalog number: E9502)

  18. Sodium dodecyl sulfate (Sigma, catalog number: L3771)

  19. NaCl (Fisher chemical, catalog number: S671)

  20. 1 M Tris-HCl buffer, pH 7.5 (ThermoFisher, catalog number: 15567027)

  21. Tris base (Fisher Chemical, catalog number: BP152-500)

  22. Glycine (Millipore-Sigma, catalog number: 56-40-6)

  23. MOPS (Sigma, catalog number: M5162)

  24. Sodium hydroxide (VWR, catalog number: BDH7225)

  25. Sodium phosphate, dibasic (Sigma, catalog number: S9763

  26. Ni-NTA Magnetic Agarose beads (Qiagen, catalog number: 36113)

  27. Leibovitz's L-15 Medium (Gibco, catalog number: 11415064)

  28. TC treated 150 mm dishes (Thermo Fisher Scientific, catalog number: 168381)

  29. 1× HBSS (Gibco, catalog number: 14025092)

  30. 1× PBS (Corning, catalog number: 21-031-CV)

  31. 1× Trypsin-EDTA, 0.25% phenol red (Corning, catalog number: 25-050-Cl)

  32. Fetal bovine serum (GeminiBio, catalog number: 100-106)

  33. Penicillin-streptomycin 100× solution (Gibco, catalog number: 15140-122)

  34. Gibco Trypan blue solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061)

  35. SHP099 (DC chemicals, catalog number: DC9737)

  36. DMSO (Corning, catalog number: 25-950-CQC)

  37. EGF (Gibco, catalog number: PHG0311)

  38. Phospho-Tyr-1000 antibody (Cell Signaling Technology, catalog number: 8954)

  39. Protease inhibitor cocktail (Roche, catalog number: 04693159001)

  40. PhosSTOP (Roche, catalog number: 04906837001)

  41. Sodium orthovanadate (NEB, catalog number: P0758L)

  42. Dithiothreitol (DTT) (Thermo Fisher Scientific, catalog number: R0861)

  43. Iodoacetamide (IAA) (Thermo Fisher Scientific, catalog number: A39271)

  44. Bicinchoninic acid assay kit (Pierce, catalog number: 23225)

  45. Water, HPLC-grade (MilliporeSigma, catalog number: 270733)

  46. Methanol, HPLC-grade (MilliporeSigma, catalog number: 34860)

  47. Chloroform, HPLC-grade (MilliporeSigma, catalog number: 528730)

  48. Urea (MilliporeSigma, catalog number: U4883)

  49. HEPES (MilliporeSigma, catalog number: H3375)

  50. Lysyl Endopeptidase, Mass Spectrometry Grade (FUJIFILM Wako, catalog number: 125-05061)

  51. Trypsin, sequencing-grade (Promega, catalog number: V511C)

  52. Acetonitrile, LiChrosolv® (MilliporeSigma, catalog number: 103725)

  53. Formic acid, LiChroPurTM (MilliporeSigma, catalog number: 543804)

  54. Trifluoroacetic acid (TFA), HPLC-grade (MilliporeSigma, catalog number: 302031)

  55. Quantitative Colorimetric Peptide Assay (Pierce, catalog number: 23275)

  56. Ammonium hydroxide, LiChroPurTM (MilliporeSigma, catalog number: 543830)

  57. EDTA, LiChroPurTM (MilliporeSigma, catalog number: 79884)

  58. FeCl3 (MilliporeSigma, catalog number: 451649)

  59. Ethanol, HPLC-grade (MilliporeSigma, catalog number: 09-0851)

  60. TMT11plex isobaric label reagents (Thermo Fisher Scientific, catalog number: A37725)

  61. 50% Hydroxylamine (MilliporeSigma, catalog number: 159417)

  62. Protein A agarose (MilliporeSigma, Roche, catalog number: 11134515001)

  63. Phospho-Thr202/Tyr204-Erk1/2 antibody (Cell Signaling Technology, catalog number: 9101)

  64. Erk1/2 antibody (Cell Signaling Technology, catalog number: 9102)

  65. β-actin antibody (Sigma-Aldrich, catalog number: A1978)

  66. Rabbit IgG, HRP-linked whole Ab (from donkey) (GE Healthcare, catalog number: NA934V)

  67. Mouse IgG, HRP-linked whole Ab (from sheep) (GE Healthcare, catalog number: NXA931V)

  68. Novex WedgeWell 12% Tris Glycine gels (Thermo Fisher Scientific, catalog number: XP00122BOX)

  69. 6× Laemmli sample buffer (Boston BioProducts, catalog number: BP-111R)

  70. Precision Plus protein dual color standards (Bio-Rad, catalog number: 1610374)

  71. Bovine Serum Albumin Fraction V (MilliporeSigma, Roche, catalog number: 10735094001)

  72. SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34580)

  73. PTMScan IAP buffer 10× (Cell Signaling Technology, catalog number: 9993)

  74. Complete L-15 media (see Recipes)

  75. Serum-free L-15 media (see Recipes)

  76. Lysis buffer (see Recipes)

  77. 1× SDS-PAGE Running buffer (see Recipes)

  78. 1× Transfer buffer (see Recipes)

  79. Blocking buffer (see Recipes)

  80. IAP buffer (see Recipes)

  81. Solvent A (see Recipes)

  82. Solvent B (see Recipes)

  83. Solvent C (see Recipes)

  84. Solvent D (see Recipes)

  85. Solvent E (see Recipes)

  86. Solvent F (see Recipes)

  87. Solvent G (see Recipes)

  88. Solvent H (see Recipes)

  89. Solvent I (see Recipes)

  90. Solvent J (see Recipes)

  91. Solvent K (see Recipes)

  92. Solvent L (see Recipes)

  93. Solvent M (see Recipes)

  94. Solvent N (see Recipes)

  95. Solvent O (see Recipes)

  96. 1× TBST (see Recipes)

  97. Digestion buffer (see Recipes)

Equipment

  1. -80°C freezer

  2. End-over-end tube rotator/shaker (Fisher Scientific, catalog number: 13-687-12Q)

  3. Cell scrapers (VWR, catalog number: 734-2602)

  4. Biosafety Cabinet

  5. Nikon Eclipse TS100 microscope

  6. 37°C water bath

  7. 37°C, 0% CO2 tissue culture incubator

  8. 25°C and 37°C shakers

  9. 1490 Reichert Bright-Line Hemocytometer (Hausser Scientific)

  10. Refrigerated and room temperature microcentrifuges

  11. XCell SureLock Mini-Cell Electrophoresis System (Invitrogen)

  12. XCell IITM Blot Module (Invitrogen)

  13. PowerPac Power supply (Bio-Rad)

  14. X-ray film cassette

  15. X-ray film processor

  16. SpectraMax M5 plate reader (Molecular Devices)

  17. Branson Digital Sonifier 250 with Tapered Titanium Microtip

  18. Vacuum concentrator centrifuge (Thermo Fisher)

  19. 20-position Extraction Manifold (Waters)

  20. Mass spectrometer (Thermo Fisher)

Software

  1. SoftMax Pro (Molecular Devices)

  2. Microsoft Excel

Procedure

  1. Ligand stimulation of MDA-MB-468 cells

    SHP2 facilitates the full activation of RAS/MAPK signaling upon induction of EGFR or other receptor tyrosine kinases. It functions by interacting with tyrosine phosphorylated proteins and by dephosphorylating various substrates throughout the RTK signaling network. To capture the system-wide phosphorylation events regulated by SHP2, we allosterically inhibited SHP2 in a serum-starved breast cancer cell line, which was harboring EGFR amplification and stimulated with EGF ligand for various time periods.

    1. Seed MDA-MB-468 cells in eleven 150 mm dishes (10 million cells in 20 mL media per dish) in pre-warmed complete L-15 media.

    2. Grow the cells in a humidified tissue culture incubator at 37°C and 0% CO2 until they reach 80% confluence (~48 h).

      Note: The base medium for MDA-MB-468 cells is ATCC-formulated Leibovitz's L-15 Medium supplemented with 10% fetal bovine serum. L-15 medium is formulated for use in carbon dioxide (CO2) – free systems, hence these cells were cultured in 0% CO2.

    3. Aspirate the media and wash the cells twice with 15 mL of pre-warmed 1× HBSS buffer. Aspirate the HBSS buffer, add pre-warmed serum-free L-15 media (20 mL per dish) and incubate the cells at 37°C for 24 h.

    4. The following day, treat the cells with DMSO carrier or 10 µM SHP099, and incubate at 37°C for 2 h.

    5. Stimulate the cells with 10 nM EGF for 5, 10, or 30 min (as shown in Figure 1B, Vemulapalli et al., 2021).

    6. To terminate ligand stimulation, gently wash the cells with 15 mL of ice-cold PBS. Aspirate PBS.

    7. Using a cell scraper, scrape the cells off the dish in 1 mL of ice-cold PBS and transfer them to a 1.5 mL microcentrifuge tube.

    8. Centrifuge the cells at 1,000 × g for 5 min at 4°C and aspirate the supernatant. Flash-freeze the cell pellets, by placing the tubes in a bath of liquid nitrogen for 10 s. Store the tubes at -80°C until further use.


  2. Preparation of cell lysates

    Whole cell extracts prepared from EGF-stimulated cells served as starting materials for enrichment of tyrosine phosphorylated peptides for mass spectrometry.

    1. Thaw the cell pellets on ice and resuspend them in 1 mL of lysis buffer.

    2. Sonicate the cell lysates on ice at 30% amplitude for a total of 30 s (three 10 s pulses), to shear DNA and reduce sample viscosity.

    3. Centrifuge the cell extracts at 24,000 × g for 20 min at 4°C. Transfer the supernatant into new 1.5 mL tubes and discard the pellets (insoluble cell debris).

    4. Perform a bicinchoninic acid (BCA) protein assay according to the manufacturer’s instructions. Absorbance of the lysates and BSA standards were measured in a SpectraMax microplate reader, using SoftMax Pro software. A BSA standard curve was created using Microsoft Excel and used to normalize lysates to a concentration of 1 mg/mL.

    5. Aliquot 50 µL from the normalized samples, boil them in 1× Laemmli sample buffer at 95°C for 5 min, and store them at -80°C until further use.


    Notes:

    1. These samples will be used for testing the ERK phosphorylation response of MDA-MB-468 cells to SHP099 and EGF treatments by western blot.

    2. ERK is a serine/threonine kinase whose dual phosphorylation at T202/Y204 is used as a readout for RTK activation.


    1. Add dithiothreitol (DTT) to the 950 µL of normalized cell lysate, to a final concentration of 5 mM. Incubate at 55°C for 30 min to reduce disulfide bonds. Allow the samples to cool to room temperature.

    2. Add iodoacetamide across all samples, to a final concentration of 14 mM. Incubate at room temperature for 45 min in the dark to alkylate reduced cysteines.

    3. Add DTT to a final concentration of 10 mM. Incubate in the dark at room temperature for 15 min to quench excess iodoacetamide.

    4. Freeze samples at -80°C until further use.


  1. Western blotting

    To confirm the cellular response to drug/ligand treatment, western blotting was performed. As expected, EGF stimulation induced an ERK1/2 phosphorylation response in MDA-MB-468 cells and SHP099 treatment inhibited this (Figure 1A, Vemulapalli et al., 2021).

    1. Load the samples (20-30 µg/lane) in 12% tris glycine gels, along with 5 µL of protein ladder.

    2. Run the gels in 1× Tris-Glycine SDS buffer at 100 V. Stop the electrophoresis when the dye front reaches the bottom of the gel.

    3. Transfer the proteins to a 0.45 µm PVDF membrane using XCell II Blot module following the manufacturer’s instructions.

    4. Incubate the membrane in 10 mL of blocking buffer for 1 h at room temperature with gentle agitation.

    5. Decant the blocking buffer and incubate the membrane in 5-10 mL of primary antibody [Phospho-Thr202/Tyr204-Erk1/2 (1:2000 dilution), Erk1/2 (1:4000 dilution), and β-actin (1:10,000 dilution)] diluted in blocking buffer with gentle agitation overnight at 4°C.

    6. Wash the membrane three times for 10 min each with 15 mL of 1× TBST.

    7. Incubate the Phospho-Thr202/Tyr204-Erk1/2 and Erk1/2 membranes in 10 mL of rabbit IgG secondary antibody, and the β-actin membrane in 10 mL of mouse IgG secondary antibody, both diluted in blocking buffer at 1:5,000 dilution for 1 h at room temperature with gentle agitation.

    8. Wash the membrane with 15 mL of 1× TBST for 10 min three times.

    9. Incubate the membrane in 3 mL of a solution containing the SuperSignal West Pico PLUS Luminol/Enhancer and SuperSignal West Pico PLUS peroxide (1:1 dilution) for 5 min at room temperature with gentle agitation. Place the membrane in a plastic sheet protector in a film cassette and expose it to X-ray film (optimal exposure times vary for each primary antibody) and image using an X-ray film processor (Figure 1A, Vemulapalli et al., 2021).

    10. After confirming appropriate cellular response to drug and/or ligand treatments, proceed to sample preparation for mass spectrometry.


  2. Protein precipitation and digestion

    After cell lysis, proteins were precipitated by the methanol-chloroform method to remove unwanted cellular material such as lipids, genomic DNA, and detergents present in the lysis buffer, which can interfere with downstream steps, including enzymatic digestion. The protein extract is enzymatically digested using proteases, such as trypsin and/or LysC, to generate peptides that are suitable for analysis via bottom-up proteomics and mass spectrometry.

    1. Thaw and transfer the 950 µL of cell lysates to chloroform-resistant 15 mL Falcon tubes (see Materials and Reagents).

    2. Add 4 volumes (3.80 mL) of methanol to the cell lysate, vortex for 5 s, and centrifuge at 2,000 × g for 30 s at room temperature. Keep the pellet and remove the supernatant.

    3. Add 1 volume (950 µL) of chloroform, vortex for 5 s, and centrifuge at 2,000 × g for 30 s at room temperature. Keep the pellet and remove the supernatant.

    4. Add 3 volumes (2.85 mL) of water and vortex for 5 s.

      Note: The solution will appear milky; this indicates protein precipitation.

    5. Centrifuge the tubes at 4,000 × g for 20 min at room temperature.

      Note: A protein pellet will appear at the interphase between the bottom chloroform layer and the top aqueous layer.

    6. Aspirate the top aqueous layer carefully with a 1 mL micropipette to waste, and add 4 volumes of ice-cold methanol to wash the protein pellet.

    7. Vortex the pellet for 10 s and centrifuge at 4,000 × g for 20 min at room temperature.

      Note: The pellet settles to the bottom of the tube. Aspirate the supernatant using a micropipette to waste.

    8. Add 4 volumes of ice-cold methanol, vortex for 10 s, and centrifuge at 4,000 × g for 20 min at room temperature. Repeat for a total of three methanol washes.

    9. With the final methanol wash, aspirate the supernatant with a 1 mL micropipette to waste.

      Note: Remove most of the methanol without touching the pellet and allowing it to dry completely.

    10. Add 1 mL of digestion buffer: 8 M urea diluted in 25 mM HEPES, pH 8.5 to the pellet.

      Note: Always prepare fresh urea solution.

    11. Resolubilize the pellet with physical disruption, such as sonication (40% amplitude, 5 s pulses, three times).

    12. Dilute lysates to a final 4 M of urea concentration, with the addition of 1 mL of 25 mM HEPES pH 8.5.

    13. Digest the protein sample in Lysyl endopeptidase (Lys-C), at an enzyme-to-protein ratio of 1:100 at 37°C for 2 h with shaking.

    14. Dilute the 4 M urea to a final concentration of 2 M urea, by the addition of 2 mL of 25 mM HEPES pH 8.5.

    15. Add Lys-C at an enzyme-to-substrate ratio of 1:50 and digest overnight while shaking at room temperature, to ensure that 100% of the protein is cleaved by Lys-C.

    16. The following day, dilute the sample to 1 M urea, by the addition of 4 mL of 25 mM HEPES pH 8.5.

    17. Continue protein digestion with trypsin added at an enzyme-to-substrate ratio of 1:100, and incubate at 37°C for 6-8 h with shaking.


  3. Assessment of missed cleavages

    Verify the completeness of protein digestion to ensure that the majority of identified peptides are suitable for analysis and peptide sequencing via mass spectrometry.

    1. To assess the percentage of missed cleavages, de-salt a small fraction (~2-3 µg) of peptides from a few representative samples using C18 STAGE Tips, following Rappsilber et al. (2003).

    2. Following the desalting steps, dry peptides via vacuum centrifugation.

    3. Reconstitute the dried peptides in ~6 µL of Solvent A to analyze by LC-MS/MS.

      Note: A missed cleavage rate of less than 10% is acceptable. If the missed cleavage rate is >10%, repeat the tryptic digestion at 1 M urea.

    4. After ensuring that the cleavage rate is >90%, aliquot 5 µL from each of the 11 samples to measure their corresponding peptide concentrations, using the Pierce quantitative colorimetric peptide assay, according to the manufacturer’s instructions.

    5. Store the peptide samples at -80°C until further use, or directly proceed to the next step.


  4. De-salting and clean-up of peptide samples

    De-salting by C18 Solid-Phase Extraction (SPE) will remove DNA, RNA, metabolites, and undigested protein from the peptide mixture.

    1. Thaw and acidify samples to a final concentration of 1% TFA (v/v; pH ~2) and incubate on ice for 15 min.

      Note: Acidification will improve peptide interaction with the stationary phase of the SPE cartridges.

    2. Centrifuge the samples at 1,780 × g for 15 min at room temperature, and transfer the supernatant to new 15 mL tubes.

    3. Label and mount 50 mg C18 Sep-Pak cartridges to a vacuum manifold connected to a waste collection vessel. Turn on the vacuum.

      Note: Use 50 mg sorbent per 1 mg of protein per sample.

    4. Activate the C18 Sep-Pak with 1 mL of Solvent B.

    5. Equilibrate the Sep-Pak with 1 mL of Solvent C.

    6. Wash the Sep-Pak three times with 1 mL of Solvent D.

    7. Load the acidified peptides diluted in Solvent D onto the cartridge. Apply vacuum starting at the lowest setting and gradually increase, to allow a dropwise flow rate of the sample.

      Note: A slower flow rate allows adequate interaction between the sample and the sorbent.

    8. Perform four 1 mL washes with 1 mL of Solvent D.

    9. Turn off the vacuum, discard the waste liquid, insert clean labeled 2 mL collection tubes, and replace the manifold cover.

    10. Elute the peptides with 450 µL of Solvent E.

    11. Perform a second elution in the same collection tubes, using 300 µL of Solvent F.

    12. Dry the desalted peptides by vacuum centrifugation overnight. Store peptides at -80°C until further use, or directly proceed to next step.


  5. Immobilized Metal Affinity Chromatography (IMAC)

    IMAC is the most commonly method for phosphopeptide enrichment. It works on the principle that negatively charged phosphate groups on peptides have affinity towards positively charged metal ions. Here, we used agarose beads that have strong metal-chelating nitrilotriacetic acid (NTA) groups to chelate with Fe3+ ions and enriched phosphorylated peptides from the peptide mixture.

    1. Fill 2 mL tubes with Solvent H to reduce peptide-to-surface binding and set tubes aside until the elution step.

    2. Prepare Ni-NTA magnetic agarose beads (Qiagen):

      1. Transfer 500 µL (500 µL/mg protein) of bead slurry into a 1.5 mL tube.

      2. Wash the beads with 1 mL of water, centrifuge briefly, and aspirate supernatant into waste. Wash beads twice more with 1 mL of water to remove all traces of ethanol.

      3. Add 1 mL of 40 mM EDTA to the beads and incubate at room temperature for 30 min with shaking.

      4. Wash the beads with 1 mL of water thrice and aspirate supernatant into waste.

      5. Chelate the beads with 1 mL of 100 mM FeCl3 and incubate at room temperature for 30 min with shaking.

      6. Wash the beads with 1 mL of Solvent G and aspirate supernatant into waste. Repeat this step for a total of four washes.

    3. Reconstitute the peptides in Solvent G to achieve a final concentration of 1 mg/mL. Adjust the Solvent G volumes based on the concentrations determined from the previous colorimetric peptide assay. Transfer the peptides to the tubes containing the beads. Incubate the peptide bead mixture via end-over-end rotation for 30 min at room temp.

      Note: Prior to sample adsorption, ensure that the pH of the peptide samples is acidic.

    4. After 30 min, briefly centrifuge the peptide-bead slurry and collect the supernatant containing non-phosphorylated peptides into new 2 mL tubes. Do not disturb or touch the magnetic bound phosphopeptides. Dry the non-phosphorylated peptides by vacuum centrifugation and store at -80°C until needed for proteomic analyses.

    5. Wash the phosphopeptide bound-bead slurry with 1 mL of Solvent G three times and aspirate the supernatant to waste.

    6. Remove Solvent H from the 2 mL elution tubes from the previous step 1 into waste. Add 36 µL of Solvent I to a final concentration of 2% formic acid of your total elution volume (below step) in these same elution tube(s).

    7. Add 300 µL of Solvent J to the phosphopeptide bound-bead slurry and shake for 3 min at room temperature. Centrifuge briefly and transfer the phosphopeptide eluate (supernatant) to the acidified 2 mL elution tubes from step 6.

    8. Repeat the above elution step twice to ensure that all phosphopeptides have eluted off the IMAC beads. All three eluates (900 µL) should be collected in a single acidified 2 mL tube(s) at 2% formic acid.

      Note: The phosphopeptide yield can vary from on average ~2-4% of the initial protein input concentration. For example, using these protocols, 1 mg of digested peptides can yield ~20 µg of enriched phosphopeptides.

    9. Dry the phosphopeptides via vacuum centrifugation and solubilize the phosphopeptides in 500 µL of Solvent D.

    10. Desalt the phosphopeptides using SOLA HRP 10 mg cartridges (Thermo Fisher):

      1. Activate with 500 µL of Solvent H.

      2. Equilibrate with 500 µL of Solvent K.

      3. Wash with 500 µL of Solvent D three times.

      4. Load 500 µL of acidified phosphopeptides (ensure pH <4).

      5. Wash with 500 µL of Solvent D three times.

      6. Elute phosphopeptides twice with 500 µL of Solvent F, collect the eluate in a single 2 mL tube, and dry using vacuum centrifugation.

      7. Store desalted phosphopeptides at -80°C until further use, or directly proceed to TMT labeling.


  6. Tandem Mass Tag (TMT) labeling

    To enable relative quantification of phosphopeptides from the eleven EGF/SHP099 treatments simultaneously, samples were labeled with 11 TMT reagents, to allow for multiplexed quantitative measurements via mass spectrometry.

    1. Reconstitute desalted phosphopeptides in 200 mM EPPS pH 8.5 at room temperature.

    2. Perform isobaric labelling of the enriched phosphopeptides using TMT reagents (0.8 mg) that are dissolved in 40 µL of Solvent H, for a final concentration of 20 µg/µL. Label each phosphopeptide sample with one TMT reagent at a ratio of 4:1 (TMT:phosphopeptide) by mass, to assure efficient labeling of all phosphopeptides (see below for labeling efficiency criteria). For example, 30 µg of phosphopeptides will use 6 µL of TMT reagent.

    3. Allow the TMT reaction to proceed at room temperature for ~1.5 h prior to performing a quality control check, often referred to as a TMT labeling ‘ratio check’, to assess labeling efficiency and phosphopeptide enrichment efficiency. Aliquot approximately 2 µL of each phosphopeptide-TMT reaction into a combined mixture and desalt using a C18 StageTip, as described previously for the digestion and missed cleavage check (section E). Freeze the stock phosphopeptide labeling reactions at -80°C, until the ratio check has been acquired and analyzed.

    4. If the labeling efficiency is >98%, the labeling reactions can be quenched individually with Solvent L to a final concentration of 0.5% hydroxylamine for 15 min, acidified, and all TMT labeled peptides combined into a single mixture of TMT labeled phosphopeptides to be dried by vacuum centrifugation.

    5. Desalt the combined TMT labeled phosphopeptide mix with a 50 mg Sep-Pak, using the method previously discussed for unenriched peptides (section F).

    6. Lyophilize the final desalted TMT labeled phosphopeptide mix for up to ~48 h, to ensure that all traces of trifluoroacetic acid have been removed from the phosphopeptides. Store the phosphopeptides at -80 °C until further use, or directly proceed to next step.


  7. Immunoaffinity purification (IAP) of tyrosine phosphorylated peptides

    Phosphopeptide mixtures enriched by IMAC majorly contain phospho-serine and phospho-threonine peptides and very low levels of phosphotyrosine peptides. Since SHP2 is an SH2 domain-containing protein tyrosine phosphatase that binds and dephosphorylates phospho-tyrosine (pY) residues, we enriched pY peptides by immunoprecipitating the IMAC-purified peptides using a phospho-tyrosine antibody.

    1. Aliquot 60 µL of protein A-agarose bead slurry (which is equal to 30 µL of packed beads) using a precut 1 mL micropipette tip into a clean 1.5 mL tube.

      Note: Invert the bottle well before pipetting beads.

    2. Wash the beads with 1 mL of ice-cold 1× PBS, vortex briefly, centrifuge at 2,000 × g for 30 s, and aspirate supernatant. Repeat the wash for a total of three times.

    3. Conjugate 30 µL of phospho-tyrosine rabbit antibody (P-Tyr-1000) (equivalent to 100 µg antibody) with the pre-washed protein A-agarose beads and 1× PBS to a final volume of 1.5 mL.

      Note: Couple the phospho-tyrosine rabbit antibody (P-Tyr-1000; Cell Signaling Technology) to protein A agarose beads one day prior to the phosphotyrosine peptide enrichment.

    4. Seal the tube with parafilm and incubate by end-over-end rotation overnight at 4°C.

    5. Centrifuge the antibody-conjugated beads at 2,000 × g for 30 s, aspirate and discard supernatant.

    6. Wash the antibody-conjugated beads thrice with 1 mL of ice-cold 1× PBS, aspirate and discard supernatant.

    7. Resuspend antibody-conjugated beads in 40 µL of ice-cold 1× PBS and use immediately.

    8. Reconstitute lyophilized TMT-labeled phosphopeptides in 500 µL of 1× IAP buffer.

      Note: Pipette gently using a 1 mL micropipette to avoid introducing air bubbles.

    9. Ensure that the sample has a neutral pH (~7.4). If the sample is acidic (pH <6), add 5-10 µL of 1 M Tris buffer pH 8.0.

    10. Centrifuge the sample at 10,000 × g for 5 min at 4°C.

    11. Transfer the supernatant (TMT labelled phosphopeptides) directly atop the antibody-conjugated beads, to ensure immediate mixing while avoiding the introduction of air bubbles.

    12. Seal the tube with parafilm and incubate for 2 h at 4°C.

    13. Centrifuge at 2,000 × g for 30 s. Transfer the supernatant to a clean tube and store at -80°C.

      Note: The supernatant contains phospho-serine and phospho-threonine peptides which can be analyzed by mass spectrometry if desired.

    14. Add 1 mL of 1× IAP buffer to the antibody-conjugated beads, gently invert the tube five times, centrifuge at 2,000 × g for 30 s at 4°C, and discard supernatant.

    15. Add 400 µL of ice-cold water to the beads, transfer contents to a 0.2 µM filter spin column, and centrifuge at 1,000 × g for 30 s at 4°C. Discard the flow-through. Use 400 µL of ice-cold water to wash, collect any residual beads from the initial tube, and transfer them to the filter spin column with the previously transferred antibody-conjugated beads. Centrifuge at 2,000 × g for 30 s at 4°C and discard the flow-through.

    16. Place the spin column in a new 1.5 mL collection tube, add 75 µL of 100 mM formic acid to elute the pY peptides from the antibody-conjugated beads, and incubate for 10 min at 25°C.

      Note: Gently tap/flick the tube every 5 min for mixing. Do not vortex. Centrifuge at 1,000 × g for 30 s.

    17. Perform a second elution in the same collection tube. Add 75 µL of 100 mM formic acid to the beads, incubate for 10 min gently tapping the tube every 5 min, and centrifuge at 2,000 × g for 30 s.

    18. Desalt the acidified eluate containing phospho-tyrosine peptides using a C18 StageTip as previously described. Elute the pY peptides with 25 µL of Solvent M, successively with 50 µL of Solvent N into a glass vial suitable for LC-MS/MS, and dry by vacuum centrifugation. Reconstitute the phospho-tyrosine peptides in 4.5 µL of Solvent O for analysis via liquid chromatography-mass spectrometry.

    Recipes

    1. Complete L-15 media

      Leibowitz’s L-15 media

      10% FBS

      1× Penicillin-streptomycin

    2. Serum-free L-15 media

      Leibowitz’s L-15 media

      1× Penicillin-streptomycin

    3. Lysis buffer

      2% SDS

      150 mM NaCl

      50 mM Tris (pH 8.5-8.8)

      Protease inhibitor cocktail (1 tablet per 20 mL)

      PhosSTOP (2 tablets per 20 mL)

      2 mM Sodium orthovanadate

    4. 1× SDS-PAGE Running buffer

      25 mM Tris base

      192 mM Glycine

      0.1% SDS

    5. 1× Transfer buffer

      25 mM Tris base

      192 mM Glycine

      20% Methanol

    6. Blocking buffer

      5% BSA

      1× TBST

    7. IAP buffer

      50 mM MOPS/NaOH pH 7.2

      10 mM Na2HPO4

      50 mM NaCl

    8. Solvent A

      5% acetonitrile

      5% formic acid

      90% water

    9. Solvent B

      100% methanol

    10. Solvent C

      50% acetonitrile

      0.1% trifluoroacetic acid

      49.9% water

    11. Solvent D

      0.1% trifluoroacetic acid

      99.9% water

    12. Solvent E

      40% acetonitrile

      0.1% trifluoroacetic acid

      59.9% water

    13. Solvent F

      70% acetonitrile

      0.1% trifluoroacetic acid

      29.9% water

    14. Solvent G

      80% acetonitrile

      0.15% trifluoroacetic acid

      19.85% water

    15. Solvent H

      100% acetonitrile

    16. Solvent I

      50% formic acid

      50% water

    17. Solvent J

      50% acetonitrile

      0.7% ammonium hydroxide

      49.3% water

    18. Solvent K

      70% acetonitrile

      29% water

      1% trifluoroacetic acid

    19. Solvent L

      50% hydroxylamine

      50% water

    20. Solvent M

      40% acetonitrile

      0.1% formic acid

      59.9% water

    21. Solvent N

      70% acetonitrile

      0.1% formic acid

      29.9% water

    22. Solvent O

      3% acetonitrile

      0.5% formic acid

      96.5% water

    23. 1× TBST

      For 1 L of 1× TBST, add:

      100 mL of 10× Tris Buffered Saline

      900 mL of water

      1 mL of 100% Tween 20

    24. Digestion buffer

              8 M urea diluted in 25 mM HEPES pH 8.5

Acknowledgments

We thank Jon Aster, Michael Acker, Jonathan LaRochelle and members of the Gygi and Blacklow laboratories for helpful discussions. We thank Cell Signaling Technologies for providing the Phospho-Tyr-1000 antibody for the immunoaffinity purification experiments. This protocol is adapted from Vemulapalli et al. (2021; DOI: 10.7554/eLife.64251).

Funding: This work was supported by NIH grant R35 CA220340 (SCB), the Dana Farber-Novartis Translational Drug Development Program (SCB) and GM97645 (SPG).

Competing interests

SCB received research funding for this project from Novartis, is a member of the SAB of Erasca, Inc., is an advisor to MPM Capital, and is a consultant on unrelated projects for IFM, Scorpion Therapeutics, Odyssey Therapeutics, and Ayala Therapeutics.

References

  1. Del Rosario, A. M. and White, F. M. (2010). Quantifying oncogenic phosphotyrosine signaling networks through systems biology. Curr Opin Genet Dev 20(1): 23-30.
  2. Du, Z. and Lovly, C. M. (2018). Mechanisms of receptor tyrosine kinase activation in cancer. Mol Cancer 17(1): 58.
  3. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P. and Mann, M. (2006). Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127(3): 635-648.
  4. Rappsilber, J., Ishihama, Y. and Mann, M. (2003). Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75(3): 663-670.
  5. Vemulapalli, V., Chylek, L. A., Erickson, A., Pfeiffer, A., Gabriel, K. H., LaRochelle, J., Subramanian, K., Cao, R., Stegmaier, K., Mohseni, M., et al. (2021). Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling. Elife 10: e64251.


简介

[摘要]细胞通过激活主要由酪氨酸磷酸化 (pY) 介导的级联信号事件来感知和响应有丝分裂原。由于其在细胞稳态中的关键作用,这种信号传导的失调通常与肿瘤发生有关。为了了解这些信号通路异常的机制,有必要在癌细胞模型中在全球范围内量化酪氨酸磷酸化。然而,大多数蛋白质磷酸化事件发生在丝氨酸 (86%) 和苏氨酸 (12%) 残基上,而只有 2% 的磷酸化事件发生在酪氨酸残基上 ( Olsen et al. , 2006 )。酪氨酸磷酸化的低化学计量使得难以以高质量准确度和重现性全面量化细胞 pY 事件。在这里,我们描述了一个详细的协议,用于从药物扰动、生长因子刺激的癌细胞中分离和量化酪氨酸磷酸化肽,使用免疫亲和纯化和串联质量标签 (TMT) 与质谱法相结合。

[背景]酪氨酸磷酸化 (pY) 介导的信号网络调节重要的细胞过程,如细胞生长、迁移、分化和衰老。鉴于这种翻译后修饰的重要性,几乎一半的蛋白质酪氨酸激酶 (PTK) 在癌症中失调也就不足为奇了 ( Del Rosario 和 White, 2010 )。尽管 PTKs 作为癌基因的功能已得到充分证实,但蛋白酪氨酸磷酸酶 (PTPs) 对细胞增殖可能具有积极或消极的影响。这些失调的酶可以调节细胞的酪氨酸磷酸化景观,导致异常的细胞信号传导和癌症。因此,过去十年的研究集中在开发抑制这些酶的靶向治疗药物。
受体酪氨酸激酶 (RTK) 是 PTK 的一个亚类,可介导细胞对生长因子的反应。 RTK 可能被功能获得性突变、基因组扩增、染色体重排或自分泌信号异常激活( Du 和 Lovly,2018 年)。这些激活的 RTK 可以启动广泛的下游信号通路,例如 RAS/MAPK 或 PI3K/AKT 信号通路,从而在细胞中引发致癌反应( Du 和 Lovly,2018 年)。了解这些复杂的动态信号网络并确定减弱这种异常信号的方法对癌症治疗具有重要意义。
磷酸蛋白质组学是一种强大的方法,可用于以位点特异性方式测量癌细胞的整体酪氨酸磷酸化状态。它可用于表征新型小分子抑制剂,并提高我们对疾病背景下信号网络的理解,有助于将这些发现转化为临床益处。最近,SHP2 变构抑制剂 (SHP099) 对 pY 网络响应表皮生长因子 (EGF) 细胞刺激的作用确定了两类磷酸化位点——被 SHP2 去磷酸化的位点和受 SH2 结构域保护的位点SHP2 被其他 PTP 去磷酸化( Vemulapalli等人,2021 年)。这些发现解释了 SHP2 如何对信号传导产生积极和消极的影响。在这里,我们通过固定化金属亲和层析 (IMAC) 和串联质量标签 (TMT) 标记,介绍了磷酸化酪氨酸肽的样品制备、富集和定量分析的详细工作流程,然后对磷酸化酪氨酸进行最终免疫亲和纯化蛋白质组使用 pY 单克隆抗体,改编自 Vemulapalli等人。 (2021 年)。

关键字:酪氨酸磷酸化, 受体酪氨酸激酶, 信号转导, 磷酸蛋白质组学, 磷酸酪氨酸富集, 串联质量标签, 生长因子刺激

材料和试剂


1. PCR管(Denville Scientific,目录号:C18064

2. 耐氯仿的15 mL离心管(Falcon,目录号:352196

3. 低蛋白结合管(Thermo Fisher Scientific,目录号:90410

4. 50 mL 试剂容器(Corning,目录号:4870

5. 96U型底测定板(Falcon,目录号:353910

6. C18 47 mm提取盘( Empore,目录号:2215-C18

7. Sep-Pak C18 50 mg 吸附剂小柱(Waters,目录号:WAT054960

8. SOLA HRP 10 mg Sep-Pak 药筒(Thermo-Fisher,目录号:03-150-391

9. 放射自显影胶片(Ece Scientific,目录号:E3018

10. 用于制备 StageTip 的柱塞(Hamilton,目录号:1122-01

11. mColorpHast pH2.0-9.0)测试条(MilliporeSigma,目录号:109584

12. Amersham Hybond 0.45 µm PVDFGE,目录号:10600023

13. 35 cm Sepax GP-C18 树脂(1.8 µm150 AThermo Fisher Scientific

14. MDA-MB-468 细胞(ATCC,目录号:HTB-132

15. 液氮

16. 10 × Tris缓冲盐水(Boston BioProducts,目录号:BM-300

17. EPPSSigma,目录号:E9502

18. 十二烷基硫酸钠(Sigma,目录号:L3771

19. NaClFisher Chemical,目录号:S671

20. 1 M Tris-HCl 缓冲液,pH 7.5ThermoFisher,目录号:15567027

21. Tris碱(Fisher Chemical,目录号:BP152-500

22. 甘氨酸(Millipore-Sigma,目录号:56-40-6

23. MOPSSigma,目录号:M5162

24. 氢氧化钠(VWR,目录号:BDH7225

25. 磷酸氢二钠(Sigma,目录号:S9763

26. Ni-NTA磁性琼脂糖珠(Qiagen,目录号:36113

27. Leibovitz L-15 培养基(Gibco,目录号:11415064

28. TC 处理的 150 mm 培养皿(Thermo Fisher Scientific,目录号:168381

29. 1 × HBSSGibco,目录号:14025092

30. 1 × PBS(康宁,目录号:21-031-CV

31. 1 ×胰蛋白酶-EDTA0.25%酚红(Corning,目录号:25-050-Cl

32. 胎牛血清(GeminiBio,目录号:100-106

33. 青霉素-链霉素100 ×溶液(Gibco,目录号:15140-122

34. Gibco 台盼蓝溶液,0.4%Thermo Fisher Scientific,目录号:15250061

35. SHP099DC化学品,目录号:DC9737

36. DMSO(康宁,目录号:25-950-CQC

37. EGFGibco,目录号:PHG0311

38. Phospho-Tyr-1000 抗体(Cell Signaling Technology,目录号:8954

39. 蛋白酶抑制剂混合物(Roche,目录号:04693159001

40. PhosSTOPRoche,目录号:04906837001

41. 原钒酸钠(NEB,目录号:P0758L

42. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,目录号:R0861

43. 碘乙酰胺(IAA)(Thermo Fisher Scientific,目录号:A39271

44. 二辛可宁酸测定试剂盒(Pierce,目录号:23225

45. 水,HPLC级(MilliporeSigma,目录号:270733

46. 甲醇,HPLC级(MilliporeSigma,目录号:34860

47. 氯仿,HPLC级(MilliporeSigma,目录号:528730

48. 尿素(MilliporeSigma,目录号:U4883

49. HEPESMilliporeSigma,目录号:H3375

50. 赖氨酰内肽酶,质谱级( FUJIFILM Wako,目录号:125-05061

51. 胰蛋白酶,测序级(Promega,目录号:V511C

52. 乙腈, LiChrosolv® MilliporeSigma,目录号: 103725

53. 甲酸,LiChroPur TM MilliporeSigma,目录号:543804

54. 三氟乙酸(TFA),HPLC级(MilliporeSigma,目录号:302031

55. 定量比色肽测定(Pierce目录号:23275

56. 氢氧化铵,LiChroPur TM MilliporeSigma,目录号:543830

57. EDTALiChroPur TM MilliporeSigma,目录号:79884

58. FeCl 3 MilliporeSigma,目录号:451649

59. 乙醇,HPLC级(MilliporeSigma,目录号:09-0851

60. TMT11plex 等压标记试剂(Thermo Fisher Scientific,目录号:A37725

61. 50%羟胺(MilliporeSigma,目录号:159417

62. 蛋白 A 琼脂糖(MilliporeSigmaRoche,目录号:11134515001

63. Phospho-Thr202/Tyr204-Erk1/2 抗体(Cell Signaling Technology,目录号:9101

64. Erk1/2 抗体(Cell Signaling Technology,目录号:9102

65. β-肌动蛋白抗体(Sigma-Aldrich,目录号:A1978

66.  IgGHRP 连接的全抗体(来自驴)(GE Healthcare,目录号:NA934V

67. 小鼠 IgGHRP 连接的全抗体(来自绵羊)(GE Healthcare,目录号:NXA931V

68. Novex WedgeWell 12Tris甘氨酸凝胶(Thermo Fisher Scientific,目录号:XP00122BOX

69. 6 × Laemmli 样品缓冲液(Boston BioProducts,目录号:BP-111R

70. Precision Plus 蛋白质双色标准品(Bio-Rad,目录号:1610374

71. 牛血清白蛋白组分VMilliporeSigmaRoche,目录号:10735094001

72. SuperSignal West Pico PLUS 化学发光底物(Thermo Fisher Scientific,目录号:34580

73. PTMScan IAP 缓冲液 10 × Cell Signaling Technology,目录号:9993

74. 完整的 L-15 培养基(见配方)

75. 无血清 L-15 培养基(见配方)

76. 裂解缓冲液(见配方)

77. 1 × SDS-PAGE 运行缓冲液(参见配方)

78. 1 ×传输缓冲器(见配方)

79. 阻塞缓冲液(见配方)

80. IAP 缓冲区(参见配方)

81. 溶剂 A(见配方)

82. 溶剂 B(见配方)

83. 溶剂 C(见配方)

84. 溶剂 D(见配方)

85. 溶剂 E(见配方)

86. 溶剂 F(见配方)

87. 溶剂 G(见配方)

88. 溶剂 H(见配方)

89. 溶剂 I(见配方)

90. 溶剂 J(见配方)

91. 溶剂 K(见配方)

92. 溶剂 L(见配方)

93. 溶剂 M(见配方)

94. 溶剂 N(见配方)

95. 溶剂 O(见配方)

96. 1 × TBST(见配方)

97. 消化缓冲液(见配方)


设备


1. -80°C 冰箱

2. 端到端管旋转器/振动器(Fisher Scientific,目录号:13-687-12Q

3. 细胞刮刀(VWR,目录号:734-2602

4. 生物安全柜

5. 尼康 Eclipse TS100 显微镜

6. 37°C水浴

7. 37°C, 0% CO 2组织培养箱

8. 25°C 37°C 摇床

9. 1490 Reichert Bright-Line 血细胞计数器(Hausser Scientific

10. 冷藏和室温微量离心机

11. XCell SureLock 微型细胞电泳系统(Invitrogen

12. XCell II TM印迹模块(Invitrogen

13. PowerPac 电源 (Bio-Rad)

14. X 光胶片暗盒

15. X射线胶片处理器

16. SpectraMax M5 读板器(Molecular Devices

17. 必能信 Digital Sonifier 250 带锥形钛微尖端

18. 真空浓缩离心机(Thermo Fisher

19. 20 位提取歧管(沃特世)

20. 质谱仪(Thermo Fisher

软件


1. SoftMax Pro(分子器件)

2. 微软Excel

 

程序


A. MDA-MB-468细胞的配体刺激

SHP2 在诱导 EGFR 或其他受体酪氨酸激酶后促进 RAS/MAPK 信号的完全激活。它通过与酪氨酸磷酸化蛋白相互作用以及通过对整个 RTK 信号网络中的各种底物去磷酸化而发挥作用。为了捕获由 SHP2 调节的全系统磷酸化事件,我们在血清饥饿的乳腺癌细胞系中变构抑制 SHP2,该细胞系具有 EGFR 扩增并用 EGF 配体刺激不同时间段。

1. 在预热的完整 L-15 培养基中将 MDA-MB-468 细胞播种在 11 150 mm 培养皿中(每道培养皿 20 mL 培养基中有 1000 万个细胞)。

2. 2的加湿组织培养箱中培养细胞,直到它们达到 80% 汇合(约 48 小时)。

注意:MDA-MB-468 细胞的基础培养基是 ATCC 配制的 Leibovitz's L-15 培养基,辅以 10% 胎牛血清。 L - 15 培养基配制用于无二氧化碳 (CO 2 )系统,因此这些细胞在 0% CO 2中培养

3.  15 mL 的预热 1 × HBSS 缓冲液吸出介质并清洗细胞两次。吸出 HBSS 缓冲液,加入预热的无血清 L-15 培养基(每盘 20 mL),并在 37°C 下孵育细胞 24 小时。

4. 第二天,用 DMSO 载体或 10 µ M SHP099 处理细胞,并在37°C 下孵育 2 小时

5.  10 nM EGF 刺激细胞 510 30 分钟(如图 1BVemulapalli等人2021 年所示)。

6. 要终止配体刺激,请用 15 mL 的冰冷 PBS 轻轻清洗细胞。吸出 PBS

7. 使用细胞刮刀,在 1 mL 的冰冷 PBS 中将细胞从盘中刮下,然后将它们转移到 1.5 mL 微量离心管中。

8. 4°C 下以 1,000 × g离心细胞5 分钟并吸出上清液。将管子放入液氮浴中 10 秒,快速冷冻细胞颗粒。将试管储存在 -80°C 直至进一步使用。

 

B. 细胞裂解物的制备

EGF 刺激的细胞制备的全细胞提取物用作用于质谱分析中富集酪氨酸磷酸化肽的起始材料。

1. 在冰上解冻细胞颗粒并将它们重新悬浮在 1 mL 的裂解缓冲液中。

2. 在冰上以 30% 的振幅对细胞裂解物进行声波处理,总共 30 秒(三个 10 秒脉冲),以剪切 DNA 并降低样品粘度。

3. 将细胞提取物在4°C 下以 24,000 × g离心20 分钟。将上清液转移到新的 1.5 mL 管中并丢弃颗粒(不溶性细胞碎片)。

4. 根据制造商的说明进行二辛可宁酸 (BCA) 蛋白测定。使用 SoftMax Pro 软件在 SpectraMax 酶标仪中测量裂解物和 BSA 标准品的吸光度。使用 Microsoft Excel 创建 BSA 标准曲线,用于将裂解物标准化至 1 mg/mL 的浓度。

5. 从标准化样品中分取 50 µL 95°C 1 × Laemmli 样品缓冲液中煮沸 5 分钟,然后将它们储存在 -80°C 直至进一步使用。

注意: _

a. 这些样品将用于通过蛋白质印迹测试 MDA-MB-468 细胞对 SHP099 EGF 处理的 ERK 磷酸化反应。

b. ERK 是一种丝氨酸/苏氨酸激酶,其 T202/Y204 的双重磷酸化用作 RTK 激活的读数。

6. 添加 二硫苏糖醇 (DTT)加入 950 μL的标准化细胞裂解液,最终浓度为 5 mM。在 55 ° C 下孵育 30 分钟以减少二硫键。让样品冷却至室温。

7. 在所有样品中添加碘乙酰胺,最终浓度为 14 mM。在黑暗中在室温下孵育 45 分钟以烷基化还原的半胱氨酸。

8.  DTT 添加到 10 mM 的最终浓度。在室温下在黑暗中孵育 15 分钟以淬灭过量的碘乙酰胺。

9. 冷冻样品直至进一步使用。

 

C. 蛋白质印迹

为了确认对药物/配体处理的细胞反应,进行了蛋白质印迹。正如预期的那样,EGF 刺激在 MDA-MB-468 细胞中诱导了 ERK1/2 磷酸化反应,而 SHP099 处理抑制了这种反应(图 1AVemulapalli等人2021)。

1. 将样品 (20-30 µg /车道) 加载到 12% tris 甘氨酸凝胶中, 以及 5 µL的蛋白质阶梯。

2.  1 × Tris-Glycine SDS 缓冲液中运行凝胶。当染料前沿到达凝胶底部时停止电泳。

3. 制造商的说明,使用 XCell II Blot 模块 HYPERLINK "https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fblotmod_pro.pdf" 将蛋白质转移到0.45 µm PVDF 膜上

4. 在室温下将膜在 10 mL 的阻塞缓冲液中孵育 1 小时,并轻轻搅拌。

5. 倒出封闭缓冲液并在 5-10 mL 的一抗 [Phospho-Thr202/Tyr204-Erk1/21:2000 稀释)、Erk1/21:4000 稀释)和β-肌动蛋白(1: 10,000 稀释度)] 在封闭缓冲液中稀释,并在 4°C 下轻轻搅拌过夜。

6. × TBST清洗膜 3 次,每次 10 分钟。

7.  10 mL IgG 二抗中孵育 Phospho-Thr202/Tyr204-Erk1/2 Erk1/2 膜,在10 mL 小鼠 IgG 二抗中孵育β-肌动蛋白膜,均在封闭缓冲液中以 1:5,000 稀释在室温下轻轻搅拌 1 小时。

8. × TBST洗涤膜10 分钟 3 次。

9. 将膜在 3 mL 的溶液中孵育,该溶液含有 SuperSignal West Pico PLUS Luminol/Enhancer SuperSignal West Pico PLUS 过氧化物(1:1 稀释),在室温下轻轻搅拌 5 分钟。将膜放在胶片盒中的塑料片保护器中,并将其暴露于 X 射线胶片(每种一抗的最佳曝光时间各不相同)和使用 X 射线胶片处理器成像(图 1AVemulapalli等人2021.

10. 在确认对药物和/或配体治疗有适当的细胞反应后,继续进行质谱样品制备。

 

D. 蛋白质沉淀和消化

细胞裂解后,通过甲醇-氯仿法沉淀蛋白质,以去除裂解缓冲液中存在的脂类、基因组 DNA 和去污剂等不需要的细胞物质,这些物质会干扰下游步骤,包括酶消化。使用蛋白酶(例如胰蛋白酶和/LysC)对蛋白质提取物进行酶消化,以产生适合通过自下而上蛋白质组学和质谱分析的肽。

1.  950 μL的细胞裂解物解冻并转移到耐氯仿的 15 mL Falcon 管中(参见材料和试剂)。

2. 加入 4 体积(3.80 mL) 甲醇,涡旋 5 秒,然后以 2,000 ×离心 g在室温下保持 30 秒。保留颗粒并去除上清液。

3. 加入 1 体积 (950 µL ) 的氯仿,涡旋 5 秒,然后以 2,000 ×离心 g在室温下保持 30 秒。保留颗粒并去除上清液。

4. 添加 3 (2.85 mL) 的水并涡旋 5 秒。

注意:溶液会呈乳白色;这表明蛋白质沉淀。

5. 在室温下以 4,000 × g离心管20 分钟。

注意:蛋白质沉淀会出现在底部氯仿层和顶部水层之间的界面处。

6.  1 mL 微量移液器小心地将顶部水层吸出,然后加入 4 体积的冰冷甲醇清洗蛋白质颗粒。

7. 在室温下以 4,000 × g离心 20 分钟。

注意:颗粒沉降到试管底部。使用微量吸管吸出上清液以浪费。

8. 加入 4 倍体积的冰冷甲醇,涡旋 10 秒,在室温下以 4,000 × g离心 20 分钟。重复总共三个甲醇洗涤。

9. 在最后的甲醇洗涤中,用 1 mL 微移液器吸出上清液以浪费。

注意:除去大部分甲醇,不要接触颗粒并使其完全干燥。

10. 添加 1 mL 的消化缓冲液:8 M 尿素在 25 mM HEPESpH 8.5 中稀释到颗粒中。

注意:始终准备新鲜的尿素溶液。

11. 用物理破坏重新溶解颗粒,例如超声处理(40% 振幅,5 s 脉冲,3 次)。

12. 将裂解物稀释至最终 4 M 的尿素浓度,加入 1 mL 25 mM HEPES pH 8.5

13.  Lysyl 内肽酶 (Lys-C) 中消化蛋白质样品,酶与蛋白质的比例为 1:100,在 37°C 下振荡 2 小时。

14. 通过添加 2 mL 25 mM HEPES pH 8.5 4 M 尿素稀释至最终浓度为 2 M 尿素。

15.  1:50 的酶与底物比添加 Lys-C,并在室温下振荡消化过夜,以确保 100% 的蛋白质被 Lys-C 切割。

16. 第二天,通过添加 4 mL 25 mM HEPES pH 8.5 将样品稀释至 1 M 尿素。

17. 继续以 1:100 的酶与底物比例添加胰蛋白酶消化蛋白质,并在 37°C 下振荡孵育 6-8 小时。

 

E. 评估遗漏的乳沟

验证蛋白质消化的完整性,以确保大多数已识别的肽段适用于质谱分析和肽段测序。

1. 为了评估遗漏裂解的百分比,按照 Rappsilber等人的方法,使用 C 18 STAGE Tips从几个代表性样品中去除一小部分(~2-3 µg )肽2003 年)

2. 在脱盐步骤之后,通过真空离心干燥肽。

3. 溶剂 A ± 6 μL重组干燥的肽,以通过 LC-MS/MS 进行分析。

注意:低于 10% 的缺失切割率是可以接受的。如果错过的切割率 > 10%,请在 1 M 尿素下重复胰蛋白酶消化。

4. 在确保切割率 > 90% ,根据制造商的说明,使用 Pierce 定量比色肽测定法从 11 个样品中的每一个中分取 5 μL以测量其相应的肽浓度

5. 将肽样品储存在 -80°C 直至进一步使用,或直接进行下一步。

 

F. 样品的脱盐和净化

通过 C18 固相萃取 (SPE) 进行脱盐将从肽混合物中去除 DNARNA、代谢物和未消化的蛋白质。

1. 将样品解冻和酸化至最终浓度为 1% TFAv/vpH ~2),并在冰上孵育 15 分钟。

注意:酸化将改善肽与 SPE 小柱固定相的相互作用。

2. 将样品在室温下以 1780 × g 离心 15 分钟,然后将上清液转移到新的 15 mL

3. 标记 50 mg C 18 Sep-Pak 墨盒并将其安装到连接到废物收集容器的真空歧管上。打开真空。

注意:每个样品每 1 mg 蛋白质使用 50 mg 吸附剂。

4.  1 mL 溶剂 B激活 C 18 Sep-Pak

5.  1 mL 的溶剂 C 平衡 Sep-Pak

6.  1 mL 溶剂 D 清洗 Sep-Pak 三次。

7. 加载到墨盒上。从最低设置开始应用真空并逐渐增加,以允许样品的逐滴流速。

注意:较慢的流速允许样品和吸附剂之间充分的相互作用。

8. 使用 1 mL 溶剂 D 执行四次 1 mL 洗涤。

9. 关闭真空,丢弃废液,插入标有清洁标签的 2 mL 收集管,然后更换歧管盖。

10. μL的溶剂 E洗脱肽

11. 使用300 µL溶剂 F在同一收集管中执行第二次洗脱。

12. 通过真空离心过夜干燥脱盐肽。将肽保存在 -80°C 直至进一步使用,或直接进行下一步。

 

G. 固定化金属亲和色谱 (IMAC)

 

IMAC 是最常用的磷酸肽富集方法。它的工作原理是肽上带负电荷的磷酸基团对带正电荷的金属离子具有亲和力。在这里,我们使用具有强金属螯合次氮基三乙酸 (NTA) 基团的琼脂糖珠与 Fe 3+离子螯合,并从肽混合物中富集磷酸化肽。

1. 用溶剂 H 填充 2 mL 管,以减少肽与表面的结合,并将管放在一边,直到洗脱步骤。

2. 准备 Ni-NTA 磁性琼脂糖珠 (Qiagen)

a.  500 μL500 μL/mg 蛋白质)的珠浆转移到 1.5 mL 管中。

b.  1 mL 水清洗珠子,短暂离心,然后将上清液吸到废物中。用 1 mL 水再清洗珠子两次,以去除所有乙醇痕迹。

c.  1 mL 40 mM EDTA 添加到珠子中,并在室温下摇动孵育 30 分钟。

d.  1 mL 水三次洗涤珠子,并将上清液吸入废物中。

e.  1 mL 100 mM FeCl 3螯合珠子,并在室温下摇动孵育 30 分钟。

f.  1 mL 的溶剂 G 清洗珠子,并将上清液吸到废物中。重复此步骤共进行四次洗涤。

3. 在溶剂 G 中重构肽以达到 1 mg/mL 的最终浓度。根据先前比色肽测定确定的浓度调整溶剂 G体积将肽转移到含有珠子的管子中。在室温下通过端到端旋转孵育肽珠混合物 3 0 分钟。

注意:在样品吸附之前,确保肽样品的 pH 值是酸性的。

4. 30 分钟后,将肽珠浆短暂离心,将含有非磷酸化肽的上清液收集到新的 2 mL 管中。不要干扰或触摸磁性结合的磷酸肽。通过真空离心干燥非磷酸化肽,并在-80°C 下储存,直到需要进行蛋白质组学分析。

5. 洗涤磷酸肽结合珠浆 3 次,然后将上清液吸出

6. 将溶剂 H从上一步 1 中的 2 mL 洗脱管中去除到废物中。在这些相同的洗脱管中,将 36 µL 溶剂 I 添加到最终浓度为总洗脱体积 2% 的甲酸中(步骤如下)。

7.  300 μL 的溶剂 J 添加到磷酸肽结合珠浆中,并在室温下摇动 3 分钟。短暂离心并将磷酸肽洗脱液(上清液)转移到步骤 6 中酸化的 2 mL 洗脱管中。

8. 重复上述洗脱步骤两次,以确保所有磷酸肽均已从 IMAC 珠子上洗脱。所有三种洗脱液 (900 µL) 应收集在一个 2% 甲酸酸化的 2 mL 管中。

注意:磷酸肽产量可能会有所不同,平均约为初始蛋白质输入浓度的 2-4%。例如,使用这些协议,1 mg 消化的肽可以产生约 20 μg 的富集磷酸肽。

9. 通过真空离心干燥磷酸肽,并将磷酸肽溶解在 500 μL的溶剂 D 中。

10. 使用SOLA HRP 10 mg 墨盒 (Thermo Fisher) 对磷酸肽进行脱盐:

a.  500 µL 溶剂 H 激活。

b.  500 μL 的溶剂 K 平衡。

c.  500 μL 的溶剂 D 洗涤3 次。

d. 加载 500 μL 的酸化磷酸肽(确保 pH <4)。

e.  500 μL 的溶剂 D 洗涤3 次。

f. 洗脱磷酸肽两次,将洗脱液收集在单个 2 mL 管中,并使用真空离心干燥。

g. 将脱盐的磷酸肽储存在 -80°C 直至进一步使用,或直接进行 TMT 标记。

 

H. 串联质量标签 (TMT) 标签

 

为了同时对来自 11 EGF/SHP099 处理的磷酸肽进行相对定量,用 11 TMT 试剂标记样品,以允许通过质谱法进行多重定量测量。

1. 室温下在 200 mM EPPS pH 8.5 中复溶脱盐磷酸肽

2. μL溶剂 H 中的TMT 试剂(0.8 毫克)对富集磷酸肽进行等压标记,最终浓度为 20 μg / μL 。用一种 TMT 试剂以质量比为 4:1TMT:磷酸肽)标记每个磷酸肽样品,以确保有效标记所有磷酸肽(有关标记效率标准,请参见下文)。例如,30 µg磷酸肽将使用 6 µL TMT 试剂。

3. 在进行质量控制检查(通常称为 TMT 标记比率检查)之前,让 TMT 反应在室温下进行约 1.5 小时,以评估标记效率和磷酸肽富集效率。使用 C18 StageTip 将每个磷酸肽-TMT 反应的大约 2 μL等分到混合混合物中并脱盐,如前文所述,用于消化和遗漏的切割检查(E 节)。在 -80°C 下冷冻储备的磷酸肽标记反应,直到获得并分析比率检查。

4. 如果标记效率 > 98%,标记反应可以单独用溶剂 L 淬灭至终浓度为 0.5% 羟胺 15 分钟,酸化,然后将所有 TMT 标记的肽组合成 TMT 标记的磷酸肽的单一混合物进行干燥通过真空离心。

5. 使用先前讨论的未富集肽的方法(F 节),用 50 mg Sep-Pak 对组合的 TMT 标记的磷酸肽混合物进行脱盐。

6. 将最终脱盐 TMT 标记的磷酸肽混合物冻干长达 ± 48 小时,以确保已从磷酸肽中去除所有痕量的三氟乙酸。将磷酸肽储存在-80°C,直到进一步使用,或直接进行下一步。

 

I. 酪氨酸磷酸化肽的免疫亲和纯化 (IAP)

 

通过 IMAC 富集的磷酸肽混合物主要含有磷酸丝氨酸和磷酸苏氨酸肽以及极低水平的磷酸酪氨酸肽。由于 SHP2 是一种含有 SH2 结构域的蛋白酪氨酸磷酸酶,可结合磷酸化酪氨酸 (pY) 残基并使其去磷酸化,因此我们通过使用磷酸化酪氨酸抗体对 IMAC 纯化的肽进行免疫沉淀来富集 pY 肽。

1. 使用预切的 1 mL 微移液器尖端将 60 μL的蛋白 A-琼脂糖珠浆(相当于 30 μL的包装珠)放入干净的 1.5 mL 管中。

注意:在移液珠之前将瓶子倒置。

2.  1 mL 的冰冷 1 × PBS 清洗珠子,短暂涡旋,以 2000 × g离心30 秒,然后吸出上清液。重复洗涤总共三遍。

3. 偶联 30 µL磷酸酪氨酸兔抗体 (P-Tyr-1000) (相当于 100 µg抗体)与预洗的蛋白 A-琼脂糖珠和 1 × PBS 混合至最终体积为 1.5 mL

注意:在磷酸酪氨酸肽富集前一天,将磷酸酪氨酸兔抗体(P-Tyr-1000Cell Signaling Technology)与蛋白 A 琼脂糖珠结合。

4. 用封口膜密封试管,并 4°C 下通过端对端旋转孵育过夜。

5. × g将抗体偶联珠子离心30 秒,吸出并丢弃上清液。

6.  1 mL 冰冷的 1 × PBS 清洗抗体结合珠三次,吸出并丢弃上清液。

7. μL的冰冷 1 × PBS中重悬抗体结合珠子并立即使用。

8. μL 1 × IAP 缓冲液中重组冻干 TMT 标记的磷酸肽。

9. 确保样品具有中性 pH (~7.4)。如果样品呈酸性(pH <6),则添加 5-10 µL 1 M Tris 缓冲液 pH 8.0

10.  4°C 下以 10,000 × g离心样品 5 分钟。

11. 将上清液(TMT 标记的磷酸肽)直接转移到抗体偶联珠上,以确保立即混合,同时避免引入气泡。

12. 用封口膜密封试管并在 4°C 下孵育 2 小时。

13. × g离心30 秒。将上清液转移到干净的试管中并储存在-80°C

注意:上清液含有磷酸丝氨酸和磷酸苏氨酸肽,如果需要,可以通过质谱分析。

14.  1 mL 1 × IAP 缓冲液加入到抗体偶联珠中,轻轻颠倒离心管 5 次,在 4°C 下以 2,000 × g离心30 秒,弃上清。

15. 加入 400 µL冰冷的水,将内容物转移到 0.2 µ M 过滤旋转柱中,并在 4°C 下以 1,000 × g离心 30 秒。丢弃流通。使用 400 μL的冰冷水清洗,从初始管中收集任何残留的珠子,然后将它们与先前转移的抗体结合珠子一起转移到过滤器旋转柱中。在 4°C 下以 2,000 × g离心 30 秒并丢弃流出液。

16. 将离心柱放入新的 1.5 mL 收集管中,加入 75 µL 100 mM 甲酸以从抗体结合珠中洗脱 pY 肽,并在 25°C 下孵育 10 分钟。

注意:每 5 分钟轻轻敲击/轻弹试管进行混合。不要涡旋。以 1,000 × g 离心30 秒。

17. 同一收集管中进行第二次洗脱。向珠子中加入 75 μL 100 mM 甲酸,每 5 分钟轻轻敲击试管孵育 10 分钟,并以 2000 × g离心30 秒。

18. 如前所述,使用 C 18 StageTip 含有磷酸酪氨酸肽的酸化洗脱液脱盐。用 25 μL的溶剂 M pY 肽洗脱,依次用 50 μL的溶剂 N 洗脱到适合 LC-MS/MS 的玻璃小瓶中,并通过真空离心干燥。在 4.5 μL的溶剂 O中重组磷酸酪氨酸肽,以通过液相色谱-质谱法进行分析。

 

食谱

 

1. 完整的 L-15 媒体

莱博维茨的 L-15 媒体

10% 胎牛血清

1 ×青霉素-链霉素

 

2. 无血清 L-15 培养基

莱博维茨的 L-15 媒体

1 ×青霉素-链霉素

 

3. 裂解缓冲液

2% SDS

150 毫米氯化钠

50 mM Tris (pH 8.5-8.8)

蛋白酶抑制剂混合物(每 20 毫升 1 片)

PhosSTOP(每 20 毫升 2 片)

2 mM 原钒酸钠

 

4. 1× SDS-PAGE 电泳缓冲液

25 mM Tris 碱基

192 毫米甘氨酸

0.1% SDS

 

5. 传输缓冲器

25 mM Tris 碱基

192 毫米甘氨酸

20% 甲醇

 

6. 阻塞缓冲区

5% BSA

1 × TBST

 

7. IAP 缓冲区

50 mM MOPS/NaOH pH 7.2

10 毫米钠2 HPO 4

50 毫米氯化钠

 

8. 溶剂 A

5% 乙腈

5% 甲酸

90%

 

9. 溶剂 B

100% 甲醇

 

10. 溶剂 C

50% 乙腈

0.1% 三氟乙酸

49.9%

 

11. 溶剂 D

0.1% 三氟乙酸

99.9%

 

12. 溶剂 E

40% 乙腈

0.1% 三氟乙酸

59.9%

 

13. 溶剂 F

70% 乙腈

0.1% 三氟乙酸

29.9%

 

14. 溶剂 G

80% 乙腈

0.15% 三氟乙酸

19.85%

 

15. 溶剂 H

100% 乙腈

 

16. 溶剂一

50% 甲酸

50%

 

17. 溶剂 J

50% 乙腈

0.7% 氢氧化铵

49.3%

 

18. 溶剂 K

70% 乙腈

29%

1% 三氟乙酸

 

19. 溶剂 L 

50% 羟胺

50%

 

20. 溶剂 M

40% 乙腈

0.1% 甲酸

59.9%

 

21. 溶剂氮

70% 乙腈

0.1% 甲酸

29.9%

 

22. 溶剂 O

3% 乙腈

0.5% 甲酸

96.5%

 

23. 1×TBST

对于 1 L 1 × TBST,添加:

100 mL 10 × Tris 缓冲盐水

900 毫升水

1 毫升 100% 吐温 20

 

24. 消化缓冲液

8 M 尿素稀释于 25 mM HEPES pH 8.5

 

 

致谢

 

我们感谢 Jon AsterMichael AckerJonathan LaRochelle 以及 Gygi Blacklow 实验室的成员进行有益的讨论。我们感谢 Cell Signaling Technologies 为免疫亲和纯化实验提供 Phospho-Tyr-1000 抗体。该协议改编自 Vemulapalli等人2021 年; DOI10.7554/eLife.64251 )。

资金:这项工作得到了 NIH 赠款 R35 CA220340 (S CB)Dana Farber-Novartis 转化药物开发计划 (SCB) GM97645 (SPG) 的支持。

 

 

利益争夺

 

SCB 从诺华获得了该项目的研究资金,是 Erasca, Inc. SAB 的成员,是 MPM Capital 的顾问,并且是 IFMScorpion TherapeuticsOdyssey Therapeutics Ayala Therapeutics 的不相关项目的顾问。

 

 

参考

1. 德尔罗萨里奥,上午和怀特,FM2010 年)。通过系统生物学量化致癌磷酸酪氨酸信号网络。 Curr Opin Genet Dev 201):23-30

2. Du, Z. Lovly, CM (2018)癌症中受体酪氨酸激酶活化的机制摩尔癌症171):58

3. Olsen, JV, Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P. Mann, M. (2006)。信号网络中的全局、体内和位点特异性磷酸化动力学单元格1273):635-648

4. Rappsilber, J.Ishihama, Y. Mann, M. (2003)用于蛋白质组学中基质辅助激光解吸/电离、纳米电喷雾和 LC/MS 样品预处理的即走即走提取技巧。 肛门化学753):663-670

5. Vemulapalli, V., Chylek, LA, Erickson, A., Pfeiffer, A., Gabriel, KH, LaRochelle, J., Subramanian, K., Cao, R., Stegmaier, K., Mohseni, M.. 2021 年)。时间分辨 磷酸蛋白质组学揭示了 SHP2 依赖性信号的支架和催化响应模式。生命 10e64251

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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Vemulapalli et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Vemulapalli, V., Blacklow, S. C., Gygi, S. P. and Erickson, A. (2022). Enrichment of Tyrosine Phosphorylated Peptides for Quantitative Mass Spectrometry Analysis of RTK Signaling Dynamics. Bio-protocol 12(3): e4311. DOI: 10.21769/BioProtoc.4311.
  2. Vemulapalli, V., Chylek, L. A., Erickson, A., Pfeiffer, A., Gabriel, K. H., LaRochelle, J., Subramanian, K., Cao, R., Stegmaier, K., Mohseni, M., et al. (2021). Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling. Elife 10: e64251.
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