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
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.
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
Control unfed Azido-Farnesyl-OH Azido-GG-OH
S-palmitoylation analog Geranylgeranylation 100%
N3 OAc 60%
OH AcO O
Farnesylation analog NH 40%
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
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
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
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)