Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry

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Letter

Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry for Quantitative Proteomics Arne H. Smits, Annika Borrmann, Mark Roosjen, Jan C.M. van Hest, and Michiel Vermeulen ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00520 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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ACS Chemical Biology

Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry for Quantitative Proteomics

Arne H. Smits1,*, Annika Borrmann2,*, Mark Roosjen1,2, Jan C.M. van Hest2,# and Michiel Vermeulen1,#

1

Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences,

Radboud University Nijmegen, Nijmegen, The Netherlands 2

Department of Bio-organic Chemistry, Faculty of Science, Institute for Molecules and Materials,

Radboud University Nijmegen, Nijmegen, The Netherlands

ACS Paragon Plus Environment


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Abstract Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS that combines the power of site-specific UAA incorporation, bioorthogonal chemistry and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-Lphenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.


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Peptide and protein tags have become invaluable tools to study the function of a protein of interest (POI).1 Protein tagging is a necessity because high-quality antibodies targeting endogenous proteins are of limited availability2 and often exhibit cross reactivity.3 Green fluorescent protein (GFP) is among the most used tags and has been utilized for the visualization of a POI inside cells, but also for the selective enrichment of the POI.4 Additionally, peptides have been used for the selective enrichment of a POI.5 Although peptide tags are smaller than protein tags, peptides can be structurally disordered possibly leading to adverse effects such as aggregation and degradation. The usage of both protein and peptides tags necessitates their fusion to the N- or C-terminus of the POI, which can interfere with the structure and/or function of the POI, as was reported for Myef26 and Smad3.7 A small reactive handle that can be placed at any position inside a protein could overcome these technical problems. The amber suppression technology provides a powerful method to siteselectively incorporate an unnatural amino acid (UAA) into a POI.8 In this approach, an amber stop codon is introduced into the POI and a 21st orthogonal tRNA and aminoacyl-tRNA synthetase (aa-RS) pair is added to the endogenous translational machinery of the host organism. Among a variety of different UAAs, an azido-functionalized UAA has also been added to the genetic code.9 The azide is a small reactive handle that is bioorthogonal, meaning it does not show any undesired cross-reactivity with naturally occurring functional groups.10, 11 It reacts selectively with alkynes in, amongst others, the strain-promoted azide-alkyne cycloaddition (scheme S1) which uses ring strain to activate the alkyne.11 Here, we combine the power of amber suppression technology, bioorthogonal chemistry and quantitative mass spectrometry-based proteomics to develop a novel workflow, named Click-MS, for the selective enrichment of a single POI based on covalent capture of the POI by copper-free click chemistry. We demonstrate selective enrichment for two different proteins using Click-MS, the signal transducer and activator of transcription 1 (STAT1)12 and the methyl-CpG-binding domain protein 3



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(MBD3).13,

14

Furthermore, MBD3 is known to assemble in the Nucleosome Remodeling and

Deacetylase (NuRD) complex and via this minimally invasive labeling technique, we were able to demonstrate co-enrichment of two known NuRD-subunits.

Results and Discussion We selected a tRNA and aaRS pair that enables the incorporation of an azido-moiety into the growing peptide chain of a POI in response to the amber (TAG) stop codon (Figure S1).15 To test the incorporation of the UAA p-azido-L-phenylalanine (azF), we selected an mCherry-GFP (mCherryGFPTAG) construct that contains an amber stop codon in its linker region.16 HEK293T cells were transiently co-transfected with three plasmids each carrying the tRNA, aaRS or mCherry-GFPTAG genes and cultured for 36-48h in the presence of 250 µM azF. Analysis by confocal microscopy showed that green fluorescence was only detected when cells were grown in the presence of azF (Figure 1A). Flow cytometry analysis further revealed that approximately 60-70% of all cells were successfully transfected using optimized conditions with the mCherry-GFPTAG construct and half of these cells showed green fluorescence (Figure 1B and S2). Taken together, these data indicate the successful incorporation of azF in response to the amber stop codon. Although the incorporation of an UAA leads to minimal perturbance of the POI, it still requires the cellular introduction of an additional tRNA, aaRS and UAA of choice. Since the UAG stop codon is a naturally occurring stop codon (≈20% of all genes)17, we wanted to investigate whether the selected tRNA/aaRS pair and azF have an effect on the proteome of the cell. The effects of amber suppression were recently studied by phenotypical examination of Drosophila melanogaster18 and by transcriptome analysis of mouse embryonic fibroblasts19 and both reported only minimal effects. To the best of our knowledge, in-depth analysis of the effect of amber suppression technology on the cellular proteome has not been reported yet. We performed a stable isotope labeling of amino acids in cell culture (SILAC) experiment, in which we incubated cells grown in ‘heavy’ medium


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(supplemented with heavy lysine (13C615N21H1416O2) and heavy arginine (13C615N41H1416O2)) with 250 µM azF and cells grown in ‘light’ medium (supplemented with normal lysine and arginine) were left untreated (Figure 1C). We also performed a ‘reverse’ experiment by incubating light-labeled cells with azF and leaving heavy-labeled cells untreated. In SILAC-based analyses, each peptide identified by mass-spectrometry has a heavy-to-light ratio (H/L), which reflects its relative abundance in the heavy and light samples, respectively. Proteins that are affected by azF in the ‘forward’ experiment have a high H/L ratio in case of protein up-regulation or a low H/L ratio in case of down-regulation (Figure 1C) and these ratios will be reversed in the reverse experiment. Using the MaxQuant software20, we quantified ratios for all identified peptides and corresponding proteins to investigate the effect of azF incubation on global protein expression. As shown in figure 1D, no substantial changes in protein expression were observed when cells were treated with azF. Interestingly, analysis of the SILAC samples that were transfected with the tRNA and aaRS carrying plasmids revealed a small upregulation of four histone proteins (Figure 1E) and subsequent addition of azF resulted in upregulation of 6 proteins (Figure 1F). To increase the depth of the proteome, we applied strong anion exchange (SAX) fractionation (Figure S3). In total, 4600 proteins were quantified of which only 5 proteins showed an upregulation of more than 1.5 fold. None of the identified regulated proteins in all experimental conditions showed an H/L ratio greater than three. Notably, these proteins are not directly involved in cellular pathways involving stress responses or cell cycle regulation and do not contain an amber stop codon. These data suggest that the addition of the tRNA/aaRS pair and azF is well tolerated by cells and does not significantly affect the expression of other proteins. Next, we examined whether we could label the incorporated azide using copper-free click chemistry (scheme S1). We used an aza-dibenzocyclooctyne (DBCO)-conjugated fluorescent dye that was incubated with cell lysates. In-gel fluorescence analysis showed a fluorescent signal at the expected height (Figure 2B and S4). However, other proteins were also fluorescently labeled indicating azide-



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independent labeling of proteins by the DBCO-dye. Since cyclooctynes are also known to react with thiols21, preincubation with iodoacetamide (IAA) showed a strong reduction in background fluorescence (Figure 2B). A fluorescent band was visible at the expected height of the mCherryGFPTAG (≈70 kDa), demonstrating successful labeling with the DBCO-dye. Notably, three species of lower molecular weight also showed fluorescent labeling, which are likely truncated forms of the mCherry-GFPTAG fusion protein. Immunoblot analysis for GFP showed the same pattern as was observed for the fluorescent labeling (Figure 2A) strongly suggesting that all three protein species contain azides. These data confirm that the copper-free click reaction can be utilized for the specific labeling of azido-containing proteins in complex protein mixtures, as was shown before.21-23 Next, we examined whether we could selectively enrich the azido-mCherry-GFPTAG from cell lysate. We designed a 1-step and a 2-step enrichment workflowusing DBCO-functionalized agarose beads and using a DBCO-SS-biotin linker and streptavidin beads, respectively (Figure 2C). The 1-step protocol allowed for direct covalent binding of the POI to beads and consequently its depletion from lysate. Incubation with DBCO-modified beads indeed showed depletion of mCherry-GFPTAG from lysate (≈80% efficiency) (Figure 2D), whereas incubation with unmodified agarose beads did not show a reduction. These results indicate that azido-mCherry-GFPTAG could be selectively enriched from cell lysates. The 2-step workflow might be advantageous because the smaller DBCO-SS-biotin linker likely has better accessibility to the azide-moiety within the protein. Additionally, the linker harbors a cleavable disulfide bond, which can be used to elute the protein by reduction (Figure 2C). The DBCO-SS-linker was incubated with cell lysates, subsequently enriched using streptavidin-beads and reduced in Laemmli buffer for immunoblot analysis (Fig. 3D). A specific enrichment of mCherry-GFPTAG was observed (Figure 2D), whereas no enrichment was seen with unmodified agarose beads. Notably, the biotin binding-capacity of streptavidin beads is limited, which explains the rather limited depletion of the highly expressed mCherry-GFPTAG in the 2-step workflow. In summary, these experiments


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demonstrate that a single POI can be selectively enriched from crude lysates by copper-free click chemistry using either the 1-step and 2-step protocols. To study the feasibility of click chemistry-based protein enrichment of biologically relevant proteins, we chose to study the nuclear protein MBD3, which assembles in the NuRD complex,13, 14 and the cytoplasmic protein STAT1, which is involved in signal-transduction of growth factors.12 We developed two different amber mutants for MBD3, one containing the amber stop codon in the Cterminally fused GFP (tyrosine 340 corresponding to the regularly used Y39 amber in GFP)24 and the other at threonine 211 (Figure 3A). For STAT1 asparagine N530 was mutated into an amber stop codon. Sites were selected to be outside highly conserved regions and functional domains as well as to be inside predicted hydrophilic amino acid stretches and loops, in order to ensure that both the function of the POI was not disturbed and the reactive group was accessible. To directly validate successful incorporation of azF into the proteins, we used C-terminal GFP fusion proteins. All amber mutants showed green fluorescence and the full-length protein was detected for all amber mutants upon azF addition by western blot analysis (Figure 3B). The amber mutants were expected to be expressed higher than endogenous levels, which was confirmed for MBD3T211 by western blot analysis (Figure S5). The MBD3 amber mutant harboring azF at position 340 (MBD3Y340) functioned as a technical control, because the azF incorporation at this position does not influence the MBD3 protein and has also previously been used as a position for unnatural amino acid incorporation.24 Therefore, MBD3Y340 was expected to be amendable for protein enrichment. Indeed, 1-step depletion of MBD3Y340 proved succesful (Figure 3C) and 2-step enrichment and elution showed a clear and specific elution of MBD3Y340 (Figure 3D). Interestingly, MBD3T211 which harbors the azide-moiety within the body of MBD3 was strongly enriched using 1- and 2-step workflows (Figure 3C and 4D). This indicates that the azide group in MBD3T211 is highly accessible and demonstrates the power of amber suppression for internal tagging, i.e. tagging that is not limited to the N- or C-terminus. 1- and 2-step enrichment of



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STAT1N530 showed clear depletion of STAT1 in the supernatants and strong elution in the 2-step workflow (Figure 3C and 3D). This extends the use of our click chemistry-based enrichment workflow to biologically relevant proteins within different cellular compartments. The successful immobilization and enrichment of MBD3 and STAT1 amber mutants using copper-free click chemistry prompted us to incorporate this enrichment strategy in our mass spectrometry-based quantitative proteomics analysis.25,

26

We dubbed this new bioorthogonal chemistry-based

proteomics workflow Click-MS. For this purpose, we generated whole cell lysates from all amber mutants and empty vector (EV) expressing cells, which expressed tRNA and aaRS and were treated with azF (Figure 4A). The protein enrichment in the 1-step workflow was followed by extensive washing and on-bead digestion, before applying the peptides to LC-MS/MS. For the 2-step enrichment, the beads were extensively washed before elution of bound proteins by DTT containing buffer, in-solution digestion and LC-MS/MS analysis. The enrichments were done in triplicate to allow label-free quantification and statistical testing, and the EV served as a control for quantitative comparison to distinguish specific enriched proteins from background binders. Raw data were analyzed using label-free quantification and the resulting quantification of all identified proteins was depicted in a volcano plot, which plots the false discovery rate (-log scale) against the observed enrichment (amber mutant/EV ratio, log2 scale) (Figure 4A). For all tested amber mutants in both 1- and 2-step workflows, we observed a 100 to 1000-fold enrichment of the azide-functionalized protein compared to the EV control (Figure 4B). The MBDY340azF enrichment contains more noise, as indicated by the enrichment of unrelated proteins. This might relate to less efficient purification of this protein as observed in immunoblot analysis (Figure 3D). For MBD3T211azF and STAT1N530azF the purifications are far more specific (Figure 4B). For MBD3T211azF, we also identified GATAD2A and GATAD2B enriched in the 1- and 2-step pulldowns (Figure 4B – MBD3T211azF). These two proteins are known interactors of MBD3 and their enrichment indicates the potential of Click-MS for the identification of protein-protein interactions. MBD3 is also


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known to interact with other members of the NuRD complex,27 which were not enriched. This is likely a consequence of the transient transfection that causes very heterogeneous expression in ≈30% of the cells (Figure 1B), leading to overexpression of MBD3 that is only partially incorporated in NuRD complexes. Although we selectively enriched the azido-containing proteins using SPAAC, we did not observe complete depletion of the azido-containing protein (Figure 2D and 3D). The combination of only partial protein incorporation in the NuRD complex and incomplete protein depletion likely contributes to the inability to co-enrich all NuRD subunits. This notion is supported by the fact that GATAD2A and B are direct interactors of MBD3.28 Additionally, azF can be reduced to aminophenylalanine in mammalian cells, leading to reduced reactivity, however it was recently shown that the formation of amino-phenylalanine is rather limited in HEK293 cells.29 Comprehensive identification of protein-protein interactions using Click-MS will likely benefit from cells stably expressing the amber suppression machinery19, faster reactions for an efficient capture such as inverse electron demand Diels-Alder reactions30 and expression of the transgene at near endogenous levels. Although further optimization is still needed, the co-purification of GATAD2B as observed by mass spectrometry was validated using immunoblotting on MBD3T211azF enriched samples (Figure S6). This highlights the feasibility of Click-MS for the co-enrichment and identification of protein-protein interactions using a minimal tag within a POI. Click-MS enables a robust, single-step purification and identification of a single POI. Since the introduced azide is very small, it only minimally, if at all, affects protein structure and therefore it is a viable alternative to epitope tagging, especially for proteins that are not N- or C-terminally taggable such as small proteins. The 100 to 1000-fold enrichment that we obtained with our pulldown strategy is comparable to enrichment of a GFP-tagged protein.27 In contrast to the antibody-protein affinity method, Click-MS protein purification relies on the formation of a covalent bond for enrichment, which allows for stringent purification and enrichment to near homogeneity. This could be beneficial for analysis techniques that are sensitive to contamination, such as comprehensive PTM identification, crosslinking-MS, and chromatin immunoprecipitation purposes. The principle of Click-



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MS protein enrichment thus has great potential for both proteomics and genomics analyses, and we envision that the technique will become widely used in many different biological contexts.

Methods Details of experimental procedures are provided in the Supporting Information.

Author information * Co-first authors #Corresponding author: M.V.: [email protected] J.C.M.H. [email protected] Notes: The authors declare no competing financial interest.

Acknowledgements We thank T. Sakmar for providing us the pSVB.Yam-tRNACUA and tylRS plasmids, E. Lemke for the mCherry-GFP plasmid. S. Naganathan and M. Kazmi are acknowledged for advice on amber suppression technology, L. Pierson from the General Instrumentation at the Radboud University and P. Jansen for technical assistance. A. Borrmann and J. van Hest acknowledge the NWO VICI grant and the Ministry of Education, Culture and Science (Gravitation program 024.001.035) for financial support. Work in the lab of M. Vermeulen is supported by the EU FP7 framework program 4DCellFate (277899), an NWO-VIDI grant (864.09.003) and by the NWO gravitation program CGC.nl (024.001.028).


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Figure Legends Figure 1. Amber suppression is well tolerated by the cell without affecting the proteome. (A) Confocal microscopy pictures of transfected cells with or without the addition of azF. Scale bar represents 50 µm. (B) FACS analysis of the transfected cells with or without the addition of azF using red and green fluorescence. The results are shown for the transfection conditions as used throughout the paper. The full set of tested plasmid concentrations can be found in Figure S3. (C) Schematic presentation of the whole proteome analysis upon azF, tRNA + aaRS or tRNA + aaRS + azF treatment. (D, E and F) Scatterplots of the protein ratios obtained in the different treatments. Upper right corner represents upregulated proteins, lower left corner the downregulated proteins and upper left contaminants. Cutoff depicted as dashed lines at 1.5 fold difference.

Figure 2. Bioorthogonal labeling and immobilization of mCherry-GFPTAG. (A) Western blot analysis of mCherry-GFPTAG. (B) Copper-free click chemistry based labeling of mCherry-GFPTAG. Arrow indicates the full length functionalized mCherry-GFPTAG. Preincubation with iodoacetamide (IAA) blocks thiols from reacting with the cyclooctyne. EV stands for empty vector (negative control). (C) Schematic depiction of the enrichment assay using 1- and 2-step workflow. (D) Western blot analysis of GFP and GAPDH for the mCherry-GFPTAG depletion in the supernatant in the 1- and 2-step workflow as well as elution in the 2-step workflow.

Figure 3. Site-specific incorporation of azF in MBD3 and STAT1. (A) Schematic representation of the mutated residues in MBD3 and STAT1. (B) Western blot analysis of the MBD3 and STAT1 amber mutants. (C and D) Western blot analysis of GFP and GAPDH for the MBD3 and STAT1 amber mutants depletion in the 1-step workflow (C) and elutions in the 2-step workflow (D).



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Figure 4. Click-MS: Mass spectrometry-based analysis of the enrichment of the azide-functionalized protein of interest. (A) Schematic representation of the label-free workflow used to enrich and quantify the different MBD3 and STAT1 ambers. (B) Volcano plots depicting the enrichment (x-axis) and significance (y-axis) of the identified proteins for MBD3Y340 (top), MBD3T211 (middle) and STAT1N530 (bottom) for both the 1-step (left) and 2-step (right) workflow. Note the enrichment of GATAD2A and GATAD2B in the Click-MS experiments for MBD3T211. The other enriched proteins are likely contaminants and are sporadically observed (MIF and SRSF1 contain the UAA stop codon and YAP1 and SPRY2 contain the amber stop codon).


Page 14 of 19

Brightfield Page 15 of 19

mCherry

B GFP ACS Chemical Biology Positive cells (%)

A

- 1azF

80 70

mCherry GFP

C

1) azF 2) tRNA + aaRS 3) tRNA + aaRS + azF

60

Light

Heavy

Intensity

-4

-4

-2

-2

0

0

2

2

4

4

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Reverse - log2(H/L) 2 0 -2 -4

2 50 Lysis, FASP & LC-MS/MS 3 40 up 4 30 5 down 20 6 + azF 10 7 0 8 +azF -azF m/z 9 D E F 10 azF tRNA + aaRS tRNA + aaRS + azF 11 Contaminants Upregulated Contaminants Upregulated Upregulated 12 Contaminants 13 H4 H3.3 H4 14 H2A H2B H3.3 H2A METAP2 H2B 15 EBNA1BP2 16 NIT2 17 18 6 out of 1961 1 out of 1992 4 out of 1761 19 ACS Paragon Plus Environment Downregulated Downregulated Downregulated 20 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 21 Forward - log2(H/L) Forward - log2(H/L)

Forward - log2(H/L)

B

C workflow dye labeling ACS Chemical 1-step Biology -a zF + az F EV

az F

+

EV

-a zF

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1. Incubation with DBCO-beads 2. Centrifugation

+ IAA

N3

N O

3. Immunoblot analysis of supernatant 2-step workflow 1. Incubation with DBCO-SS-biotin 2. Incubation with Strp-beads 3. Centrifugation & washing

37

N3 N

ctrl

kDa 70

ctrl

2-step workflow Sup Elution beads

ctrl

+

beads

input

1kDa 270 355 4 5 IB: GFP 6 7 8 D 9 1-step workflow 10 Sup 11 12 kDa 13 7014 15 16 3717 18

input

-a zF

az F

- IAA

beads

A

O

S

S

HN O

N O

4. Elution with reducing agent 5. Immunoblot analysis of elution

IB: Environment ACS Paragon Plus

O

GAPDH

R

R

O

IB: GFP

SH

N N N

H N

H N

= O

O

HN H

H N 3

O

S

NH H

N530 SH2

GFP STAT1N530

kDa 130

DBCO beads

DBCO beads

input

MBD3T211

IB: GFP IB: GAPDH

EV

STAT1N530 kDa 130


Elution

35

Sup

MBD3T211

Elution

IB: GAPDH

kDa 70

MBD3Y340 DBCO beads

IB: GFP

1-step

Elution

azF

input

C

input

CC

Sup

STAT1N530 +

input

Elution

Sup

input

B MBD3T211 MBD3Y340 1 + + kDa 2 130 3 4 5 70 6 7 8 35 D9 MBD3Y340 102-step 11 12 kDa 70 13 14 15 16 35 17

STAT1 Biology Chemical

GFP

input

Y340 ACS

input

T211

MBD

Sup

A MBD317 of 19 Page

IB: GFP IB: GAPDH

Label-free LC-MS/MS (triplicates)

on-bead digestion

Page 18 of Volcano plot 19 -Log FDR

ctrl

Chemical Biology Intensity

1-step enrichment ACS

Empty vector

POI

A

Protein of interest

Intensity

ctrl

0

0

1

1

2

2

3

3

4

4

5

-Log10(FDR (t-test)) 2 4 6

5

2

0

0

0

1

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2

2

3

4

3

-Log10(FDR (t-test)) 1 2 3 4

2-step enrichment

5

4

6

0

1-step enrichment

5

6

8

6

POI

Whole Cell m/z 1 Lysate 2-step enrichment 2 3 Background 4Protein of interest elution Ratio (log2) in-solution digestion 5 m/z B6 MBD3Y340 MBD3T211 STAT1N530 7 GATAD2A 8 MIF 9 MBD3 10 STAT1 11 YAP1 GATAD2B 12 MBD3 13 14 FC > 26 FC > 23.12 FC > 25 FDR < 10-1.301 15 FDR < 10-2.27 FDR < 10-2 -5 0 5 -5 0 5 -15 -10 -5 0 5 10 15 16 17 GATAD2B SPRY2 MBD3 MBD3 18 19 GATAD2A 20 STAT1 21 SRSF1 22 23 ACS Paragon Plus Environment 24 FC > 24 FC > 21.95 FC > 25 -1.301 -1.301 FDR < 10 FDR < 10-1.301 FDR < 10 25 -10 -5 0 5 10 -5 0 5 -10 -5 0 5 10 26 Log (MBD3 / EV) Log (MBD3 / EV) Log (STAT1 / EV) 2

2

Protein enrichment

-Log FDR

Quantitative readout Page 19 of ACS 19SPAACChemical Biology using

Control

POI

m/z 1 POI 2 Background 3 ACS Paragon Plus Environment Protein of interest (POI) 4 Ratio (log2) containing azido-Phe m/z 5 Click-MS 6

Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry

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