Non-radioactive targeting of multiple classes of protein post

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					                                                               Non-radioactive targeting of multiple classes of protein post-translational modifications (PTMs) with click chemistry
                                                               Xiao-Dong Qian, Courtenay Hart, Tamara Nyberg and Brian Agnew
                                                               Molecular Probes® – a part of Life Technologies™ • Labeling and Detection Group • 29851 Willow Creek Road • Eugene, Oregon 97402 • USA

                       Introduction                                                                                          A Universal Approach to PTM Analysis                                                                  Figure 3. 3T3-L1 Adipocyte stimulation with insulin                                                                                                   Optimization of PTM protein enrichment                                                                                               Figure 8. Cleavage of model O-GlcNAc
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              modified peptide from click resin
     Characterizing changes in multiple classes of protein post-translational                                                                        •Feed cells azide/alkyne PTM metabolic precursors                                      Con Glc Far GG                            Myr Pal ODA                                                                                      GlcNAz labeled
                                                                                                                                                                                                                                                                                                                              Western blot analysis of insulin
                                                                                                                                                                                                                                   Insulin: + - + - + - + -                           + - + - + -                                                                                          GlcNAz labeled
     modifications (PTMs) is key to deciphering the complex signaling                                                                                                                                                                                                                                                                                                                  Jurkat cell lysate                                          Supernatant
                                                                                                                                                     •Treat cells with or without activation reagents                                                                                                                         stimulated and unstimulated 3T3-                             Jurkat cell lysate
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         An O-GlcNAc-modified peptide was
     networks involved in cellular regulation. PTMs directly affect protein                                                                                                                                                         AKT                                                                                       L1      adipocytes    that      were                          click resin
                                                                                                                                                                                                                                                                                                                                                                                       plusPlus Clickresin                                                          TAMRA alkyne               Unbound O-GlcNAz
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     TAMRA-alkyne        Unbound O-GlcNAc                                                  MPBA2-N3                                      azide-labeled with the Click-iT® O-
     activities and orchestrate signal transduction processes by regulating                                                                                                                                                                                                                                                   metabolically labeled with PTM                                                                                                                                    modified
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         modified proteins proteins                                                                                      GlcNAc Enzymatic Labeling System
     both spatial and temporal protein expression parameters. Disruption of                                                                                                                                                        pAKT                                                                                       analogs.      Detection of Glut-4                                                                                                                                                                                                                                          (Invitrogen C33368). 4 nmol of azido-
     these regulatory processes often leads to disease states. In the past,                                                                          Same cells, same treatment, multiple analyses                                                                                                                            confirms differentiation of 3T3-L1                                                                                                                                                                                                                                         peptide (MPBA2-N3) was reacted with
     radioactive metabolic precursors have been used to label, track, and                                                                                                                                                                                                                                                     cells into adipocytes. Detection of
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Bound and digested                                                                           Click
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Click loadload            20 μL of cleavable click resin in the
                                                                                                                                                                                                                                   IRS-1                                                                                                                                                                                                                                                O-GlcNAc modified digested
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Bound and
     detect changes in protein PTMs, however the use of radioactivity has                                                                                                                                                                                                                                                     phospho-AKT        (Ser473)      and                                                                                                  Reduce and Alkylate
                                                                                                                                  Protein Labeling                    Cell Imaging                       Protein Enrichment                                                                                                                                                                                                                                       Reduce and Alkylate proteins
                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Lys-C/Trypsin             O-GlcNAz modified                                                                                          presence of 2 mM Cu(I) for 30
     many shortcomings. It is labor-intensive, hazardous, and expensive to                                                                                                                                                                                                                                                    phospho-IRS-1 (Ser636/639) in                                                                                                                                                                                                Depleted supernatant minutes. The resin was washed then
     dispose of. Although the method is quite sensitive, it is not compatible                                                                                                                                                      pIRS-1                                                                                                                                                                                                                Resin      Lys-C/Trypsin                  proteins                                                                    Depleted supernatant
                                                                                                                        • Global proteomic studies           • Multiplex with antibodies            • Identify PTM site peptides                                                                                              treated cells confirms insulin                                                                                             Resin                                                                                                                                           incubated with 40 μL of cleavage
     with in-gel protein detection or cellular microscopy, and X-ray film                                                                                                                                                                                                                                                     stimulation in all but azido                                                                                                                                                                                                                                               reagent for 15 minutes. The cleavage
     exposure times can be painstakingly long. Here we provide an                                                       • 1-D or 2-D electrophoresis         • Spatial/temporal studies             • Shotgun MS of PTM proteins   Glut-4                                                                                     farnesyl alcohol labeled cells.                                                                                                                                                                                                            Cleaved peptide step was performed 3 times.
     alternative non-radioactive approach to the labeling and detection of                                                followed by MS analysis                                                                                                                                                                                                                                                                                                                                                                                                                              Cleaved peptide
                                                                                                                                                             • Endocytosis/phagocytosis             • Differential PTM analyses                                                                                                                                                                                                                                                                                                                                                                          Normalized volumes of the load,
     multiple types of post-translationally modified proteins using a two-
                                                                                                                        • Multiplexed Western blots                                                                                                                                                                                                                                      Figure 6. Determination of O-GlcNAc protein binding                                                                          6.50        17.00        17.50        18.00  18.50 19.00  19.50  20.00 20.50 21.00 supernatant and pooled cleavage
     step labeling(1-4) and detection(5,6) platform that employs a “toolbox” of                                                                              • Cellular transport                   • Combine with iTRAQ or ICAT
     metabolic PTM protein labeling compounds. We demonstrate dynamic                                                                                                                                                              Figure 4. ZOOM® IEF fractionation of treated adipocytes                                                                                               efficiency to click enrichment resin                                                                                                                              Minutes
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         fractions were analyzed by reversed
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1; 214nm; Acq 5/19/2009 4:24:44 PM PDT ; Vol = 40; A= 0.1% T FA; LC11B 0 40%AB 40min flow03; Atlantis2mm 3um

                                                                                                                        • Immunoprecipitation                                                                                                                                                                                                                                                                                                                                                                                                                                                            phase HPLC.
     changes in protein farnesylation, geranylgeranylation, palmitoylation,                                                                                  • Pulse-chase experiments              • ID secreted PTM proteins     with and without insulin stimulation
     myristoylation, O-GlcNAc modification using cellular models for                                                                      Seamless integration of proteomics with cell biology                                                                                                                                                                                                                                          Unbound O-GlcNAc modified proteins
     apoptosis in HeLa cells and insulin stimulation in 3T3-L1 adipocytes.
                                                                                                                                                                                                                                                                                     TAMRA -alkyne
                                                                                                                                                                                                                                                                                                                                 SYPRO Ruby Protein Stain                                                                              TAMRA-alkyne                  SYPRO® Ruby gel stain                                         Results and Conclusions
                                                                                                                             Figure 1. Monitoring changes in multiple PTMs in HeLa cells                                                      Click-labeled




 What is click chemistry?                                                                                                                                                                                                                    protein samples




                                                                                                                             during induction of apoptosis with staurosporine







                                                                                                                                                                                                                                                                  Insulin: + - + - + - + - + -                                  Insulin:   + - + - + - + - + -                                                                                                                                                                • Metabolic labeling combined with click chemistry detection is a fast,
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                sensitive, and radioactivity-free method for detecting changes in cell
  Click chemistry is a concept first introduced by K. Barry Sharpless in                                                                                      TAMRA -alkyne              SYPRO® Ruby Protein Stain                                                                                                                                                                                                                                                                                                              signaling upon cellular activation or insult.
  2001 that describes a set of chemical reactions for use in chemical                                                                                       Con Glc   GG Far   Pal Myr       Con Glc GG Far   Pal Myr

                                                                                                                      Con: Control             Staurosporine: - + - + - + - + - + - +        - + - + - +- +   - + - +
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Both 1-D and 2-D gel analyses can be combined with cell imaging, using
  library synthesis. However the name stuck to one reaction in particular:
                                                                                                                      Glc: GlcNAz                                                                                                      ZOOM® IEF Fractionator                                                                                                                                                                                                                                                                   the same cells in the same experiments, enabling the seamless
                                                                                                                      GG: Geranylgeranyl azide alcohol                                                                                                                                                                                                                                                                                                                                                                          correlation of proteomic results with cell biological results.
            The copper(I)-catalyzed azide-alkyne cycloaddition
                                                                                                                      Far: Farnesyl azide alcohol
                            (CuAAC) reaction                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • HeLa cell protein PTM labeling shows distinct changes in response to
                                                                                                                      Pal: Palmitic acid azide
                      N+                                                                                                                                                                                                                                                                                                                                                                                                        +Cu(I)        —Cu(I)                      +Cu(I)     —Cu(I)                                     staurosporine treatment as shown by gel electrophoresis and
                           N-   R''                                               N                                   Myr: Myristic acid azide
                 N                             Cu(I)                                                                                                                                                                                                                                                                                                                                                                                                                                                                            fluorescence imaging.

                                                                              N       N R''
                                      Room Temperature                                                                                                                                                                                                                                                                                                                                   GlcNaz-labeled Jurkat cells were lysed in 10 mM Tris-HCl pH 7.5 and 400 mM NaCl by
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 3T3-L1 adipocytes were stimulated upon insulin treatment as shown by
                 R'                                                      R'                                                                                                                                                                                                                                                                                                              probe tip sonication. The lysate was centrifuged at 47,000 x g at 4° C for 50 minutes and
                                                                                                                             1D gel analysis of staurosporine treated (3 hr, 0.5 μM) and untreated HeLa cells that were                                                                                                                                                                                                                                                                                                         Western analysis of phosphorylated Akt and IRS-1.
                                                                                                                                                                                                                                           Run 1-D or narrow pH                                                                                                                          the supernatant (3 mg/mL protein) was frozen at -80° C for later use. 1 mL of GlcNAz
                                                                                                                             metabolically labeled with PTM analogs. TAMRA detection shows distinct patterns of                               range 2-D gels
                                                                                                                                                                                                                                                                                                                                                                                         soluble lysate was diluted 1:1 with click buffer and incubated with 50 μL of click resin                                             • Azide-labeled proteins can be efficiently enriched from complex cellular
                                                                                                                             labeling for each PTM. Boxes indicate examples of differences between treated and                                                                                                                                                                           overnight at room temperature in the presence or absence of Cu(I). Resin supernatants                                                  extracts.
  A toolbox of metabolic labeling compounds for                                                                              untreated samples.                                                                                                                                                                                                                                          were clicked with TAMRA alkyne and analyzed by 1-D gel electrophoresis to assess
  the study of cellular signaling pathways                                                                                                                                                                                            IEF fractionation of GlcNAz and azido-GG-OH analog-fed adipocytes shows enrichment                                                                                                                                                                                                      • Demonstrated feasibility of cleaving PTM site peptides using a model O-
                                                                                                                                                                                                                                                                                                                                                                                         depletion of GlcNAz-labeled proteins from the lysate. TAMRA signal was imaged and the
                                                                                                                                                                                                                                      of low abundant labeled proteins.                                                                                                                                                                                                                                                         GlcNAz-modified peptide.
                                                                                                                             Figure 2. Imaging click-labeled HeLa cells after metabolic                                                                                                                                                                                                  gels were post-stained with SYPRO® Ruby total protein gel stain. Reactions containing
                                                                                                                                                                                                                                                                                                                                                                                         Cu(I) show nearly complete removal of “clickable” GlcNAz proteins from the lysates
                                           O                                                                                 labeling with PTM analogs during apoptosis                                                            Figure 5. Imaging of 3T3L1 adipocytes treated with                                                                                                    compared to the copper-free reactions (see data graph below).
N3                                             OH         N3                                                                                                                                                                       isoprenyl and fatty acid azide precursors
                                                                                                        OH                                                                                                                                                                                                                                                                                                                             120%
                                                                                                                                     Control unfed                    Azido-Farnesyl-OH                       Azido-GG-OH

                                                                                                                                                                                                                                                                                                                                                                                                                Percentage Depletion
         S-palmitoylation analog                                      Geranylgeranylation                                                                                                                                                                                                                                                                                                                                              100%
                                                                            analog                                                                                                                                                                                                          Azido-Farnesyl-OH
                                                                                                             No Treatment

N3                                                                                        OAc                                                                                                                                                                                                                                                                                                                                          60%
                                          OH                             AcO              O
                                                                          AcO                  OAc
      Farnesylation analog                                                                NH                                                                                                                                                                                                                                                                                                                                           40%

                                                                                      O                                                                                                                                                                                                                                                                                                                                                20%
                                      O                                                        N3
                                       OH                              Ac4GlcNAz                                                                                                                                                                                                                                                                                                                                                              no beads     beads + Cu(I) Beads – Cu(I)

                                                                O-GlcNAc modified proteins
      N-myristoylation analog                                                                                                                                                                                                                                                                       Azido-GG-OH                                                                                                                               5.3%               95.4%      0%

                                                                                                                                                                                                                                                                                                                                                                                         Figure 7. Lys-C/Tryptic digest of bound O-GlcNAz modified
     Materials and Methods                                                                                                                                                                                                                                                                                                                                                               proteins from click-labeled resin
Metabolic Labeling
                                                                                                                                                                                                                                                                                                                                                                                                                Bound and digested O-GlcNAz modified proteins
Ac4GlcNAz (Invitrogen C33367): 40 μM for 48 hours or 200 mM overnight                                                              Azido-palmitic acid                Azido-myristic acid                         GlcNAz
Myristic acid azide (Invitrogen C10268): 50 μM for 6-9 hours
Palmitate (Invitrogen C10265): 100 μM for 6-9 hours
                                                                                                                                                                                                                                                                                            Azido-palmitic acid                                                                                                                               SYPRO® Ruby gel stain
Farnesyl alcohol azide (Invitrogen C10248): 40 μM for 12-16 hours
                                                                                                             No Treatment

Geranylgeranyl alcohol azide (Invitrogen 10249): 40 μM for 12-16 hours
Protein Labeling and Detection
Cells were lysed and labeled with TAMRA-alkyne using the Click-iT® TAMRA Protein Analysis Detection Kit
(Invitrogen, C33370).
IEF fractionation: 2 mg of TAMRA-labeled protein was reduced and alkylated, precipitated and resolubilized
in 7 M urea, 2 M thiourea, 65 mM DTT, 2% CHAPS, 2% ASB-14, 50 mM DTT, then separated into five pH
fractions using the Zoom® IEF fractionator (Invitrogen).
1D PAGE: 10 μg samples were separated on 4-12% NuPAGE® Bis-Tris gels with MOPS buffer.
TAMRA signal was imaged on a FLA9000 scanner (FujiFILM LifeScience) using 532 nm excitation and 580 nm
long-pass emission.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                References
After TAMRA imaging, gels were post-stained with SYPRO® Ruby Protein Gel Stain (Invitrogen) and imaged                                                                                                                                                                                              Control unfed

on a FLA9000 scanner using 473 nm excitation and 580 nm long-pass emission.
Western Blotting                                                                                                                                                                                                                                                                                                                                                                                                                                   +Cu(I)          —Cu(I)                                             1. Dube DH and Bertozzi CR; (2003) Curr. Opin. Chem. Biol. 7(5):616-625.
Blots were co-incubated with rabbit anti-AKT/mouse anti-pAKT (Ser473) or mouse anti-IRS-1/rabbit anti-                                                                                                                                                                                                                                                                                                                                                                                                                2. Chan Kim S, Kho Y, Barma D, Falck J, Zhao Y; (2006) Methods Enzymol.
pIRS-1 (Ser636/639) primary antibody pairs (all from Cell Signaling Technology®) and then co-detected with                                                                                                                                                                                                                                                                               Pelleted enrichment resins from above were washed 3 times in 6 M urea, 0.5 M NaCl and                                           407:629-37
goat anti-rabbit Qdot® 625 and goat anti-mouse Qdot® 800 (Invitrogen). Rabbit anti-Glut-4 was detected
with goat anti-rabbit Qdot® 625.     Blots were imaged on a FLA4000 imager (FujiFILM) using UV epi
                                                                                                                                                                                                                                                                                                                                                                                         100 mM Tris·HCl. The resin-bound proteins were reduced and alkylated, washed 3 times                                         3. Hang HC, Geutjes EJ, Grotenbreg G, Pollington AM, Bijlmakers MJ, Ploegh HL;
illumination and 605DF40 and 820AF50 filters.                                                                                                                                                                                                                                                                                                                                            with 6 M urea, 0.5 M NaCl, 100 mM Tris·HCl then resuspended in 100 µL of 4 M urea and                                           (2007) J. Am. Chem. Soc. 129(10):2744-5.
Cell Staining                                                                                                                                                                                                                                                                                                                                                                            50 mM Tris·HCl, pH 8.0. Resin bound proteins were digested with 0.5 µg of endoprotease                                       4. Martin BR, Cravatt BF; (2009) Nat. Methods 6(2):135-8
Following fixation and permeablization, PTMs were detected with Alexa Fluor® 488 alkyne (HeLas) or Alexa                                                                                                                                                                                                                                                                                 Lys-C overnight at 37° C. The digestion mixtures were diluted 1:1 with distilled water and                                   5. Kolb HC, Finn MG, Sharpless KB; (2001) Angew. Chem. In. Ed. 40:2004-2021.
Fluor® 594 alkyne (adipocytes) using the Click-iT® Cell Reaction Buffer Kit (Invitrogen A10267, A10275 and               HeLa cells were fed with described compounds for times indicated in the methods section.                                                                                                                                                                        post-digested with 0.5 µg of Trypsin overnight at 37° C. Digestion was monitored by gel                                      6. Tornøe CW, Christensen C, Meldal M; (2002) J. Org. Chem. May 3; 67(9):3057-
C10269). Tubulin (Helas) was detected with a mouse monoclonal anti-tubulin primary and Alexa Fluor® 488
goat anti-mouse secondary (Invitrogen A21424). Nuclei were stained with Hoechst 33342 (Invitrogen                        Following fixation and permeablization, PTMs (green) were click-labeled with Alexa Fluor®                                                                                                                                                                       electrophoresis and SYPRO® Ruby protein gel stain. Triplicate digest samples with or                                            64.
H3570).                                                                                                                  488 alkyne. Tubulin (red) was detected with mouse monoclonal anti-tubulin primary and                     Adipocytes were fed with described compounds for times indicated in the methods                                                                                                                                                                                                    7. Frost SC, Lane MD; (1985) J. Biol. Chem. 10;260(5):2646-52
                                                                                                                                                                                                                                   section. Following fixation and permeablization, PTMs (red) were click-labeled with Alexa                                                             without Cu(I) show recovery of digested peptides from the Cu(I)-containing reactions but
                                                                                                                         Alexa Fluor® 555 goat anti-mouse secondary. Nuclei (blue) were stained with Hoechst                                                                                                                                                                             not from the copper-free reactions.
                                                                                                                         33342.                                                                                                    Fluor® 594 alkyne. Nuclei (blue) were stained with Hoechst 33342. (Duplicate images)