Clickable and High Sensitivity Metal-Containing Tags for Mass

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/bc

Clickable and High-Sensitivity Metal-Containing Tags for Mass Cytometry Bedilu Allo,* Xudong Lou, Alexandre Bouzekri, and Olga Ornatsky Fluidigm Canada Inc., Markham, Ontario L3R 4G5, Canada S Supporting Information *

ABSTRACT: Mass cytometry is a highly multiplexed singlecell analysis platform that uses metal-tagged reagents to identify multiple cellular biomarkers. The current metal-tagged reagent preparation employs thiol−maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues have been challenging to label using this method. This inherent limitation led us to develop a new conjugation strategy for labeling a wide range of biomolecules and affinity reagents. In this report, we present a metal tagging approach using a new class of azide- or transcyclooctene-terminated MCPs with copper(I)-free strain-promoted alkyne−azide cycloaddition or tetrazine−alkene click chemistry reactions, in which biomolecules with -NH2 functional groups are selectively activated with a dibenzocyclooctyne or tetrazine moiety, respectively. This approach enabled us to generate highly sensitive and specific metal-tagged IgGs, IgMs, small peptides, and lectins for applications in immunophenotyping and glycobiology. We also created dual-tagged reagents for simultaneous detection of markers by immunofluorescence, mass cytometry, and imaging mass cytometry using a two-step conjugation process. The Helios mass cytometer was used to test the functionality of reagents on suspension human leukemia cell lines and primary cells. The dual-tagged Abs, metal-tagged lectins, and phalloidin staining reagent were used to visualize target proteins and glycans on adherent cell lines and frozen/FFPE tissue sections using the Hyperion Imaging System. In some instances, reagents produced by click conjugation showed superior sensitivity and specificity compared to those of reagents produced by thiol-maleimide chemistry. In general, the click chemistry-based conjugation with new MCPs could be instrumental in developing a wide range of highly sensitive metal-containing reagents for proteomics and glycomics applications.

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INTRODUCTION Since the introduction of mass cytometry, high-dimensional single-cell analysis has been transforming the understanding of biological mechanisms driving human diseases and healthy tissue development.1−4 Mass cytometry uses inductively coupled plasma (ICP) to vaporize, atomize, and ionize individual cells that have been probed using DNA-binding metallo-intercalators and target-specific antibodies (Abs) tagged with stable metal isotopes of high purity. The elemental composition of individual cells is recorded by a time-of-flight mass spectrometer designed specifically for high-speed analysis of the short, transient ion signals generated by each cell event. The detector provides well-resolved atomic fingerprints of many elemental tags with little overlap of neighboring signals and a wide dynamic range both for a single antigen and between antigens.2−4 Recently, two different technologies based on the use of time-of-flight mass spectrometry detection of metal-containing reagents, multiplexed ion beam imaging (MIBI)5 and imaging mass cytometry (IMC),6 were developed. Both are able to generate highly multiplexed images for quantitation of proteins and mRNAs from frozen/formalinfixed, paraffin-embedded (FFPE) tissue sections or cultured © XXXX American Chemical Society

cells mounted on glass slides labeled with metal-tagged probes.6−10 The current generation of commercially available metalchelating polymers (MCPs) in the Maxpar X8 Antibody Labeling Kits (Fluidigm) uses the thiol−maleimide conjugation method to covalently couple MCPs to antibodies (Abs). Several versions of MCPs suited for the thiol−maleimide conjugation method were also developed by different researchers.11,12 A maleimide end group on the polymer permits selective conjugation to reactive thiol groups in the hinge region of the immunoglobulin heavy chain under partial interchain disulfide bond reduction conditions. Because of the dependence of this method on the presence of cysteine residues and an Ab reduction step, some classes of Abs (for example, IgMs and IgAs) as well as biomolecules lacking cysteine residues (for example, peptides, lectins, and hormones) cannot be efficiently labeled using this method. This inherent limitation and the lack of flexibility in the preparation of Received: April 3, 2018 Revised: May 2, 2018 Published: May 7, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00239 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Figure 1. Applications of the Cu(I)-free click chemistry-based conjugation method for developing a wide range of protein-, peptide-, and fluorescence-tagged reagents for use in mass cytometry.

tetraazacylcododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) per polymer chain. Biomolecules were metal-tagged through the sequential addition of DBCO or a tetrazine moiety and MCPs with azide or TCO end groups, respectively. These reagents react specifically and efficiently with primary amines (e.g., side chain of lysine residues) at pH 7−9 to form a covalent bond. In general, the mild reaction conditions and flexibility of the click chemistry approach would allow generation of highly sensitive affinity reagents for different applications, including proteomics, glycomics, and transcriptomics, thereby expanding the utility of mass cytometry for understanding of complex biological phenomena with an extended panel of new reagents adapted to click chemistry MCP tagging.

metal conjugates led us to develop new MCPs and conjugation strategies for labeling a wide range of biomolecules and affinity reagents used in different biological applications.13 Since the concept was introduced by Sharpless in 2001,14,15 click chemistry has played a pivotal role in advancing bioconjugate chemistry applications by providing high chemical specificity and reaction efficiency amenable to both biomolecules and small molecule probes such as fluorophores, toxins, and therapeutics.16 To date, several known reactions have been developed to meet the requirements of click chemistry, such as copper(I)-catalyzed azide−alkyne cycloaddition,17 thiol−ene coupling,18 and Diels−Alder reactions.19 However, because of stringent requirements such as nontoxic aqueous reaction conditions for cells and organisms, only a few click reactions became candidates for use in biology.20 Bertozzi and coworkers developed strain-promoted copper-free azide−alkyne [3+2] cycloaddition chemistry (SPAAC), which they applied in living systems.21 Because of its fast, robust, and efficient chemistry, it was quickly adopted for synthesis of a wide range of conjugates, including peptide conjugates with polymers, particles, and substrates.22 This also led to several new applications, as well as improvements on its shortcomings, such as cycloaddition rate and aqueous solubility of the cyclooctyne.23−26 In SPAAC, cyclooctynes such as difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne (DBCO) are used to react with azide-functionalized molecules. The reaction is performed under physiological conditions and has no adverse effects on biomolecules such as Abs. Recently, the tetrazinestrained alkene [4+2] inverse electron demand Diels−Alder cycloaddition reaction was introduced for bioorthogonal applications.27,28 Extremely fast reaction rates and the high selectivity of trans-cyclooctene (TCO) with tetrazines (210− 30000 L mol−1 s−1)29 have become another attractive choice for bioorthogonal labeling of biological molecules. In this report, we describe the applications of SPAAC and TCO−tetrazine ligation chemistry for development of reagents, including preparation of MCP−Ab, MCP−peptide, MCP− lectin, and dual-labeled (fluorescence and metal tag) conjugates for suspension and IMC applications (Figure 1). We synthesized two new MCPs with azide and TCO end groups containing ≤50 copies of the chelating ligand 1,4,7,10-

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RESULTS AND DISCUSSION Polymer Synthesis. TCO−MCP was prepared by reaction of the thiol-end DOTA-containing polymer with TCO− PEG3−maleimide at room temperature, as described in Experimental Procedures. The apparent number-average molecular weight (Mn) for the final polymer was 11000 g mol−1 with a narrow polydispersity (Mw/Mn = 1.20). Polymers were characterized by 1H nuclear magnetic resonance (NMR) spectroscopy in D2O (Figure 2). The peak at δ 0.9 can be

Figure 2. 1H NMR spectrum of RAFT TCO−MCP in D2O. B


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Figure 3. (A) PBMCs were stained with Maxpar Abs against major human PBMC markers. (B) Azide−MCP-conjugated CD4−145Nd, CD14−160Gd, and CD16−165Ho Abs at 1−2 μg/mL and CD57(HCD57)−172Yb (IgM class) replaced the respective Abs in the phenotyping panel. Panels A and B show the gating strategy from live single cells. (C) Bar graph comparing mean intensities [normalized to EQ Four Element Calibration Beads (Fluidigm)] for Maxpar-labeled Abs vs azide−MCP-conjugated Abs. (D) Signal distribution histogram for CD57+CD4-CD3+ cells for Maxpar-labeled (red) and azide−MCP-labeled (green) cells. The inset shows the mean intensity values for Maxpar- and azide−MCPconjugated CD57 Abs.

assigned to the protons on the tert-butyl end group. The broad peak at δ 1.2−2.2 can be assigned to the protons of the methylene groups on the copolymer main chain. The broad peaks at δ 5.6−5.8 are due to protons from the TCO end group. For azide−MCP, the ultraviolet−visible (UV−vis) spectrum was acquired after reaction with DBCO-Cy3. The presence of the peak at 553 nm from dye Cy3 shows that the azide group is covalently attached to the polymer (Figure S1). Azide−MCP Polymers for Mass Cytometry Application. The conventional Ab conjugation uses either the surfaceexposed lysine or interchain cysteine residues as an attachment site for reporter molecules. In the case of the Maxpar antibody labeling strategy, the interchain cysteine residue is used as a conjugation site, where the coupling of MCPs to Abs occurs after partial reduction of the interchain disulfide bonds located in the hinge region. The necessity of the Ab reduction step and

the requirement to have a cysteine residue restricted the utility of Maxpar labeling for generating affinity reagents with no disulfide group and/or sensitivity to reduction steps. On the other hand, the click chemistry approach discussed in this report utilizes a two-step conjugation method to generate metal-tagged Abs of different classes (e.g., IgG and IgM): (1) activation of Abs using a water-soluble and -NH2 reactive bifunctional linker molecule to introduce DBCO or tetrazine moieties and (2) reaction of azide or TCO end group MCPs with activated Abs. To determine the successful attachment of MCPs to the Ab molecule, we used ICP-MS to measure the number of attached metal atoms and MCPs per Ab. Also, we optimized the degree of labeling by using various molar equivalents of DBCO−PEG5−NHS, monitored the number of MCPs per Ab after reaction, and assessed the immune reactivity of the resultant metal-tagged Ab. We found that the optimal C


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The other application area that we identified for azide−MCP click chemistry conjugation is generation of metal-tagged affinity reagents for glycobiology. Previously, it was shown that lectins can be modified with thiol-end MCP on surface lysine residues through a bifunctional linker such as SulfoSMCC.30 However, efficient labeling kits for lectins have yet to be commercialized for mass cytometry applications. In the work presented here, we used wheat germ agglutinin (WGA) isolated from Triticum vulgaris as a model molecule for direct conjugation of MCPs using the click chemistry approach. WGA is a lectin that can bind oligosaccharides containing terminal N-acetylglucosamine or chitobiose, structures that are common to many serum and membrane glycoproteins. WGA interacts with sialic acid and N-acetylglucosamine residues, ubiquitously present in plasma membranes of cells.31−33 In solution, its activity can be inhibited using a NAG sugar molecule.34 We tested WGA−174Yb conjugates for staining plasma membranes of Ramos, KG1a, and CCRF-CEM cells (Figure 4A−D). First, cells were stained with specific surface markers CD20−147Sm, CD34−148Nd, and CD4−145Nd in a separate tube. Cells were then incubated with WGA−174Yb. To evaluate the specificity of WGA staining, Ramos cells were stained with WGA−174Yb preincubated with N-acetylglucosamine (NAG) at a molar excess. Some of the stained cells were used for CyTOF analysis, and the remaining stained cells were attached to a slide to visualize the membrane staining by IMC. Figure 4A shows a representative IMC pseudocolor overlay image of Ramos cells stained with WGA−174Yb, CD45−154Sm, and a 191/193Ir−DNA intercalator, wherein WGA (green) staining was visible on the cell periphery at the cell membrane. A similar membrane staining pattern was observed for A431 cells (Figure 4D). Images of mixed Ramos, CCRF-CEM, and KG1a cells analyzed by IMC are presented in panels B and C of Figure 4. The results demonstrate the specificity of WGA to NAG expressed on the plasma membrane of cells. The CyTOF analysis also confirmed the IMC result. Figure 4D shows that Ramos cells stained with WGA−174Yb registered high intensity (mean intensity of ∼6000) compared to that of cells stained with NAG-inhibited WGA−174Yb (mean intensity of ∼15). As indicated in Figure 4E, we observed a change in mean intensity values for different cells stained with the same amount of WGA−174Yb, which could be related to the size of each cell type.33 Stern et al.33 also reported that WGA staining intensity is correlated to cell size in a mass cytometry assay. We stained human whole blood using 18 panel Abs against surface markers to identify major immune cell types (Table S1) and then stained cells with WGA−174Yb. Figure 5A shows a twodimensional viSNE representation of whole blood with clustering of major cell populations. Figure 5B shows the distribution of WGA intensity across immune cell subtypes. In agreement with the previous report, we saw that the relative WGA intensity was consistent within specific immune cell subsets but varied across the cell types. We further quantified the metal-tagged WGA mean intensities for gated cell populations and observed (Figure 5C) a correlation between cell size and WGA intensity for each immune cell subset, an observation that agrees with published data.33,35 The click chemistry conjugation strategy has also been used to modify small peptides for tagging or targeting purposes in different biological systems.22,36−39 However, no study has used MCPs to label small peptides for mass cytometry applications. In this study, we employed azide−MCP to label phalloidin as a

number of molar equivalents for DBCO−PEG5−NHS to Ab was 10 (∼6 DBCO per Ab), which resulted in 3 or 4 MCPs or 150−200 metal atoms per Ab. This finding agrees with a published report by Gong and his co-workers on the number of DBCO per molecule.26 We used polyclonal goat anti-mouse IgG Fc secondary Abs (GAM) as a model for Ab conjugation protocol optimization. It was coupled with 169Tm-loaded MCPs using DBCO−azide reaction. The functionality test was performed on human Ramos (positive cell line) and KG1a (negative cell line) cells stained with a primary mouse antihuman monoclonal CD20 (2H7) Ab (5 μg/mL) followed by 169 Tm-tagged GAM (1.25 μg/mL). From CyTOF analysis results, we determined that click chemistry-conjugated 169Tm− GAM showed high sensitivity and low background compared to those of Maxpar-labeled 169Tm−GAM (Figure S2). Monoclonal Abs of IgG and IgM isotypes were conjugated to MCPs according to this click chemistry protocol and tested for their functionality in mass cytometry assays. In Figure 3, results for human PBMC stained with Maxpar metal-tagged Abs (Table 1) Table 1. Maxpar Metal-Tagged and Unconjugated Antibodies reagent Maxpar antibodies

antibodies for click conjugation

Fluidigm cell identification

clone

catalog no.

vendor

CD3−170Er

name-metal

UCHT1

3170001B

Fluidigm

CD44−166Er CD45−154Sm CD14−160Gd CD20−147Sm CD4−145Nd CD8a−146Nd CD16−148Nd CD34−166Er CD57−172Yb CD107a−151Eu LEAF CD3

BJ18 HI30 M5E2 2H7 RPA-T4 RPA-T8 3G8 581 HCD57 H4A3 UCHT1

3166001B 3154001B 3160001B 3147001B 3145001B 3146001B 3148004B 3166012B 3172009B 3151002B 300414

Fluidigm Fluidigm Fluidigm Fluidigm Fluidigm Fluidigm Fluidigm Fluidigm Fluidigm Fluidigm BioLegend

LEAF CD14 LEAF CD16 LEAF CD4 LEAF CD57 CD3 intercalator−Rh (103Rh) intercalator−Ir (191Ir/193Ir)

M5E2 3G8 RPA-T4 HCD57 UCHT1 −

301810 302014 300516 322325 300443 201103A

BioLegend BioLegend BioLegend BioLegend BioLegend Fluidigm

−

201192A

Fluidigm

are shown. In a separate experiment, azide−MCP-conjugated CD4−145Nd, CD14−160Gd, and CD16−165Ho Abs at 1−2 μg/ mL and CD57(HC57)−172Yb (IgM class) replaced the respective Maxpar Abs in the phenotyping panel to assess the effect of click conjugation in a multiplex assay format. The CyTOF analysis revealed that the new conjugates detected similar cell phenotypes (Figure 3A,B) and demonstrated superior sensitivity (2−3-fold mean signal increase) with a lower background signal (Figure 3C,D and Figure S3). The improved sensitivity of the click chemistry-conjugated Abs is mainly attributed to the higher number of amine groups that can be accessed for attachment in each Ab molecule. In general, the azide−MCP shows good labeling efficiency for IgM isotype monoclonal Ab conjugation and for generating new Ab-based affinity reagents for probing low-expression level markers. D


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Figure 4. (A) Representative IMC pseudocolor image of Ramos cells stained with WGA−174Yb, CD45−154Sm, and a 191/193Ir−DNA intercalator. Panels B and C are representative IMC images of mixed KG1a, CCRF-CEM, and Ramos cells. (B) Ramos cells (blue) are identified by 147Sm− CD20, KG1a cells (green) by CD34−148Nd, and CCRF-CEM cells (red) by CD4−145Nd Abs prior to lectin staining. (C) Untreated WGA−174Yb is used to stain KG1a (green) and CCRF-CEM (red) cells. WGA−174Yb treated with N-acetylglucosamine (NAG) is used to stain Ramos cells (blue) as a negative control. (D) Representative IMC pseudocolor image of A431 cells stained with WGA−174Yb, CD44−166Er, and a 191/193Ir−DNA intercalator. (E) Representative CyTOF histograms of Ramos cells stained with NAG-treated WGA−174Yb (blue) and stained with WGA−174Yb (red) without NAG treatment. (F) Mean signal intensity (normalized to EQ beads) values of human KG1a, CCRF-CEM, and Ramos (NAG+ or NAG−) cells stained with WGA−174Yb as analyzed by CyTOF. The scale bar is 100 μm.

metal tags, respectively. The functionality assay on Ramos (CD20+, CD3−) and Jurkat (CD3+, CD20−) cells showed high specificity and low background intensity (Figure S5). On the basis of the findings from this study, we developed the TCO-end MCP using the Fluidigm polymer for labeling the anti-CD3(UCHT1) monoclonal Ab stored in phosphatebuffered saline (PBS) with or without sodium azide. The Ab was first modified with a tetrazine moiety and then coupled with TCO−MCP to generate metal-tagged Abs. To test the degree of labeling of this conjugation strategy, we used azideand career-free (LEAF) anti-CD3 Abs and tagged them with 170 Er metal using Maxpar, SPAAC, and TCO−tetrazine conjugation methods. The functionality test of these three conjugates on human Jurkat and Ramos cells showed comparable sensitivity and low background signal among the three conjugation chemistries (Figure 7A). In a separate experiment, we used the affinity chromatography-purified anti-human CD3 Ab containing 0.09% sodium azide and EDTA for conjugation with TCO−tetrazine and Maxpar labeling strategy. From the conjugation experiment, we observed no interference from the azide molecule during the coupling process. The number of MCP tags per Ab was similar (3 or 4 tags per Ab) to previous conjugations. The functionality test on Jurkat and Ramos cells also showed a reproducible

model peptide for mass cytometry applications. Phalloidin is a highly selective bicyclic peptide that is used for staining actin filaments (also known as F-actin) in cells and tissue sections. A metal-tagged phalloidin reagent can be very attractive for visualizing or quantifying actin filaments in formaldehyde-fixed and permeabilized tissue sections and cells. Our tagging protocol was optimized to attach one azide−MCP per phalloidin molecule, which resulted in bright actin filament staining without saturation in IMC images of HeLa cells and FFPE mouse colon sections. Results presented in Figure 6 demonstrate staining with phalloidin−165Ho, metal-tagged Abs, and DNA-binding metal intercalators. TCO−Tetrazine Ligation Chemistry for Mass Cytometry Application. As sodium azide is the most common preservative used for biomolecule storage, it is noteworthy to develop a conjugation strategy that can be performed in the presence of azide molecules. One advantage of the TCO− tetrazine ligation method over the SPAAC conjugation method is its tolerance to azide in the reaction mixture.40 The fast reaction kinetics make TCO−tetrazine ligation a preferable option for preparing MCP-conjugated biomolecules.27,40 The initial evaluation of this conjugation method was performed using a RAFT polymer, which was then used to label azide- and career-free (LEAF) anti-human CD3(UCHT1) and antihuman CD20(2H7) monoclonal Abs with 170Er and 147Sm E


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Figure 6. F-Actin staining of HeLa cells and mouse colon. (A) Pseudocolor IMC images of HeLa cells. Lysosomes are visualized using CD107a−151Eu (red) staining, actin filamentswith phalloidin−165Ho (green), and cell nuclei (blue) with Ir−DNA intercalator staining. (B) Pseudocolor IMC images of FFPE sections of mouse colon. Actin filaments (red) are visualized using phalloidin−165Ho and nuclei (blue) with Ir−DNA intercalator staining, and WGA−174Yb are used to stain the mucus of goblet cells (blue). The scale bar is 100 μm.

using mass cytometry. However, the reproducibility of these approaches for different Ab targets remains a challenge. In this work, we successfully generated dual-tagged conjugates with a controlled reaction using conventional thiol−maleimide conjugation strategy for fluorophores followed by TCO−tetrazine ligation chemistry for metal tagging. One of the challenges that was commonly encountered during the preparation of duallabeled reagents was controlling the optimal number of fluorophore molecules and metal tags. We tested three different molar equivalents (10, 20, and 30) of Alexa Fluor 594 dye to Ab, which yielded 4, 5, and 7 dyes per Ab molecule, respectively. The UV−vis measurement showed a distinct peak at 594 nm for attachment of Alexa Fluor 594 to the Ab (Figure S4). From the ICP-MS analysis, the TCO−MCP click chemistry conjugation yields approximately 3.5 tags (∼175 atoms/Ab) per Ab. All three conjugates were tested on Jurkat and Ramos cells by mass cytometry. The result revealed that all the click chemistry conjugates showed a high positive mean intensity and a low background signal compared to those of the Maxpar control (Figure 7C). One possible application that we envisaged for dual-labeled conjugates is use in IMC and IF systems, either for platform comparison or for prescanning of tissue sections for identification of regions of interest. Erik et al. have recently reported multiplex protein detection on circulating tumor cells (CTC) from liquid biopsies using IMC.44 In the study, a two-step procedure was employed, first staining with fluorophore-tagged Abs to locate rare CTCs using high-speed automated fluorescence scanners and then applying a cocktail of metal-tagged Abs for single-cell deep profiling with IMC. Having both types of tags, fluorophore and metal, attached to the same Ab molecule would significantly increase the accuracy and speed of analysis. Figure 7D and 7E show the representative IMC and IF images of frozen human tonsillar tissue sections labeled with dual-tagged CD3−170Er−Alexa Fluor 594 mAb prepared via a two-step (thiol−maleimide fluorophore tagging and TCO−MCP tagging) conjugation method. As can be seen, the distribution of CD3-positive T cells in the IMC image is consistent with what is observed in the IF image. This result proves the potential of dual-tagged Abs for applications that require cross-platform comparison between fluorescence- and mass cytometry-based methods and

Figure 5. Human whole blood cell population analysis based on surface staining of 29 biomarkers. (A) Two-dimensional viSNE representation of major cell populations. (B) Metal-tagged WGA staining intensity of gated populations. (C) Quantitation of metaltagged WGA mean intensities in gated cell populations for cell size discrimination.

result, a high positive mean intensity, and a low background (Figure 7B). TCO−Tetrazine Ligation Chemistry for Dual-Tagged Fluorophore and Metal-Tagged Reagent Preparation. Developing dual-labeled Ab conjugates for cross-platform comparison between fluorescence-based systems and mass cytometry has recently become of major interest for various researchers.41−43 They demonstrated dual-labeled Ab binding to cells in flow and mass cytometry and enrichment of rare cell populations using conventional FACS before deep profiling F


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Figure 7. TCO−MCP-based click chemistry conjugation for mass cytometry application. Jurkat and Ramos cells were stained with Maxpar and TCO−MCP anti-CD3(UCHT1) (2.5 μg/mL) Abs and analyzed by CyTOF. Representative histograms show comparisons between (A) Maxparand click chemistry-conjugated (azide−MCP and TCO−MCP) LEAF anti-CD3(UCHT1) Abs, (B) Maxpar- and click chemistry-conjugated (TCO−MCP) purified anti-CD3(UCHT1) Abs, and (C) Maxpar- and click chemistry-conjugated (TCO−MCP) dual-tagged (170Er and Alexa Fluor 594) purified anti-CD3(UCHT1) Abs. Representative (D) IMC and (E) IF images of frozen tonsillar tissue sections labeled with the dual-tagged CD3−170Er−Alexa Fluor 594 (1:20) Ab prepared via a two-step (thiol−maleimide conjugation for fluorophore tagging and click chemistry for MCP tagging) conjugation method. Intercalator 191Ir (IMC) and DAPI (IF) were used for nuclear staining. The scale bar is 500 μm.

pre-enrichment or rapid scanning using fluorescence microscopy and deep profiling using IMC.

and allowed us to prepare highly sensitive and specific metaltagged conjugates from a wide range of primary aminecontaining biomolecules (for example, IgG, IgM, enzymes, lectins, peptides, and oligonucleotides) for mass cytometry and IMC applications. Functionality tests showed that some Abs generated by click chemistry demonstrated a high sensitivity and a low background signal compared with those of metal-tagged reagents prepared by thiol−maleimide conjugation chemistry. We also demonstrated the applicability of the click chemistry conjugation method to generate dual-labeled reagents for immunofluorescence and mass cytometry application that can be utilized for cross-platform comparison and enrichment of rare populations using conventional fluorescence systems before deep-profiling using mass cytometry. Overall, introduc-

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CONCLUSIONS Currently available metal-tagged affinity reagents for mass cytometry are mainly prepared by reacting maleimide-functionalized MCPs with biomolecules that contain reactive thiol groups. Thus far, no other MCP with the capability to produce metal-tagged conjugates from biomolecules lacking cysteine residues has been developed. To address this issue, we have successfully synthesized a new class of MCPs with azide and TCO end groups used for Cu(I)-free click reaction-based conjugation methods. In this work, we have demonstrated that the new class of MCPs (azide−MCP and TCO−MCP) can overcome the limitations of maleimide-functionalized MCPs G


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Bioconjugate Chemistry ing the Cu(I)-free click chemistry-based conjugation toolbox for the preparation of metal-tagged reagents holds enormous potential for the development of a wide range of new affinity reagents for different areas of suspension mass cytometry and IMC applications.

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out all the free dye. The UV−vis spectrum of the resulting polymer was measured in PBS. TCO End Group MCP. TCO and MCP were prepared by reaction with TCO−PEG3−maleimide at room temperature in PBS for 2 h. The polymer was purified by being passed through Amicon Ultra-15 centrifugal filter units with a molecular weight cutoff of 3 kDa to remove low-molecular weight impurities. The Mn of the polymer was 11000 with an Mw/Mn of 1.20, measured by aqueous GPC. The polymer was characterized by proton NMR using D2O as a solvent. The peaks between 5.6 and 5.8 ppm were assigned to the TCO end groups. Preparation of Metal-Tagged Conjugates. DBCO− PEG5−NHS or tetrazine−PEG5−NHS bifunctional linkers were used to modify amines of biomolecules with the desired number of DBCO or tetrazine groups. The activated biomolecules were then reacted with azide- or TCO-modified polymers under aqueous conditions without Cu(I) to form stable conjugates following previously published protocols with minor modification.26 In brief, 100 μg of protein was incubated with DBCO−PEG5−NHS with 10 molar equivalents in PBS for 120 min at room temperature. Unreacted DBCO−PEG5− NHS was removed from higher-molecular weight biomolecules (Abs and lectins) using the Amicon Ultra-0.5 NMWL 10 kDa centrifugal filters, whereas small molecule conjugation (e.g., phalloidin) was directly used in the metal tagging procedure. A similar approach was followed for activation of biomolecules with a tetrazine moiety. Click reaction of purified DBCO or tetrazine-functionalized biomolecules with azide- or TCOmodified MCPs was conducted at 37 °C for 60−90 min. The unreacted polymers were removed using the Amicon Ultra-0.5 NMWL 100 kDa centrifugal filters. For the dual-labeled Ab preparation, carrier protein-free purified anti-CD3 (UCHT1) was first labeled with Alexa Fluor 594 at molar equivalents of 1:10, 1:20, and 1:30 using thiol− maleimide chemistry according to the manufacturer’s instructions. The purified Alexa Fluor 594-tagged anti-CD3 Abs were conjugated with MCPs using tetrazine−TCO ligation chemistry. The degree of labeling and the concentration of Ab conjugates were determined photometrically at 280 and 594 nm using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Finally, the concentration of conjugates was adjusted to 0.25 mg/mL using the PBS-based Ab stabilizer (CANDOR Bioscience, Wangen, Germany). Instrumentation. Characterization of MCPs. The Mn and Mw/Mn of all anionic, water-soluble samples were measured with a Viscotek gel-permeation chromatograph equipped with a Viscotek VE3580 refractive index detector and PolyAnalytik AquaGel PAA-203 and PAA-204 columns (kept at room temperature). The flow rate was maintained at 0.7 mL/min using a Viscotek VE1122 solvent delivery system and a VE7510 GPC degasser. An eluent of 0.2 M KNO3, with 200 ppm of NaN3, was used. The system was calibrated with poly(ethylene glycol) standards. 1H NMR (400 MHz) spectra were recorded on a Varian Hg 400 or a Varian VNMRS 400 spectrometer with a 45° pulse width at 25 °C. All water-soluble polymers were dissolved in D2O, with chemical shifts referenced to the HDO peak at 4.77 ppm. Polymers were analyzed with 512 transients and a delay time of 10 s. Characterization of Conjugates. The number of DBCO molecules per protein was determined by measuring the absorbance at 309 and 280 nm using a NanoDrop spectrophotometer. The obtained absorbance values and the

EXPERIMENTAL PROCEDURES

Materials. Azido−PEG3−maleimide, TCO−PEG3−maleimide, DBCO−PEG5−NHS, and TCO−PEG5−NHS were purchased from Click Chemistry Tools (Scottsdale, AZ). 1,4,7,10-Tetraazacylcododecane-1,4,7,10-tetraacetic acid (DOTA) was purchased from Macrocyclics, Inc. (Plano, TX). tert-Butyl 2-aminoethylcarbamate, trifluoroacetic acid, Alexa Fluor 594 C5 maleimide, and wheat germ agglutinin (WGA) lectin (Triticum vulgaris, ∼38 kDa) were purchased from SigmaAldrich Canada (Oakville, ON). N-Acetylglucosamine (NAG) was purchased from MJS BioLynx Inc. (Brockville, ON). Amicon Ultra-15 NMWL 3 kDa centrifugal filters were purchased from Millipore Canada (Etobicoke, ON). Amicon Ultra-0.5 NMWL 10 kDa and Amicon Ultra-0.5 NMWL 100 kDa centrifugal filter units were purchased from EMD Millipore (Billerica, MA). All Abs were purchased from BioLegend (San Diego, CA) unless otherwise noted. Phalloidin amine (∼902 Da) was purchased from AAT Bioquest (Sunnyvale, CA). Human leukemia cell lines (Ramos, Jurkat, CCRF-CEM, THP1, and KG1a) were obtained from ATCC (American Type Culture Collection, Manassas, VA). Cells were cultured within 15 passages in RPMI-1640 (ATCC catalog no. 30-2001) supplemented with 10% heat-inactivated bovine serum, 2 mM L-glutamine, and penicillin/streptomycin at 37 °C with 5% CO2. Cells were passaged every 2−3 days to maintain exponential growth between 1 × 106 and 2 × 106 cells/mL. Cryopreserved peripheral blood mononuclear cells (PBMC, catalog no. CTL-UP1) and anti-aggregate wash supplement 20× (catalog no. CTL-AA-001) were purchased from Cellular Technology (Shaker Heights, OH). For all mass cytometry experiments, the cell viability was typically >90% as determined by a trypan blue exclusion assay. Snap-frozen tissue sections from human tonsil were purchased from AMSBIO LLC (Cambridge, MA). Polymer Synthesis. Thiol-End Metal-Chelating Polymers. A proprietary polymer (Fluidigm) and a thiol-end polymer synthesized by reversible addition−fragmentation chain transfer polymerization with DOTA chelating groups (RAFT polymer)11,45 were used in this study to generate clickable MCP. Azide End Group MCP. Azide−MCP was prepared by reaction of the Fluidigm polymer with azido−PEG3− maleimide at room temperature in PBS for 2 h. The polymer was purified by washing through Amicon Ultra-15 centrifugal filter units with a molecular weight cutoff of 3 kDa to remove low-molecular weight impurities. The number-average molecular weight (Mn) of the polymer was 11000 with a polydispersity index (Mw/Mn) of 1.20, measured by aqueous gel-permeation chromatography (GPC). We first characterized azide−MCP by reacting it with DBCO-Cy3 (Sigma-Aldrich) and then measuring its UV−vis spectrum. DBCO-Cy3 is a versatile dye for detection of azide-containing compounds with UV−vis absorption at 553 nm. Two milligrams of azide−MCP was dissolved in 200 μL of PBS; 1.0 mg of DBCO-Cy3 in 50 μL of DMF was added, and the mixture was incubated at room temperature for 2 h. The resulting product was washed with PBS through Amicon NMWL 3 kDa centrifugal filters to wash H


Article


Cells were then washed twice in 1 mL of CSB. Finally, cells were incubated with 25 μM Cell-ID Ir−intercalator for 30 min at room temperature for nuclear staining. The samples were rinsed twice in doubly distilled H2O (ddH2O) and air-dried before IMC analysis. Staining Procedure for Tissue Sections. Immunohistochemistry (IHC) staining of frozen and FFPE tissues with metal-labeled Abs for IMC analysis was performed according to a previously published protocol.46 Each slide with snap-frozen tissue sections with two sequential sections (cores) was fixed with 4% paraformaldehyde, washed with DPBS, and blocked with 3% BSA. One core (#1) was stained with a mix of metalconjugated Abs, and the other core (#2) was stained with dualtagged Abs (metal and fluorophore) overnight at 4 °C. Slides were washed in 0.1% Triton-X in DPBS. Core #1 was stained with 191Ir/193Ir−intercalator, washed with ddH2O, air-dried, and ablated by IMC. Core #2 was stained with a DAPI solution, washed with DPBS, covered with mounting medium and a coverslip, and imaged with an immunofluorescence microscope. For FFPE tissue sections, before being incubated with metalconjugated Abs, sections were dewaxed and dehydrated and underwent heat-induced antigen retrieval in an alkaline solution according to a previously published protocol.46 After metaltagged Ab incubation steps, the tissue sections were stained with 1 μg/mL WGA−174Yb conjugate and 75 ng/mL phalloidin−165Ho in PBS for 30 min at room temperature. Finally, sections were exposed to 25 μM Ir−intercalator for 30 min at room temperature for nuclear identification. The samples were rinsed twice in distilled water and air-dried before IMC analysis. IMC and IF Image Acquisition. IMC images were acquired according to a procedure described in ref 6. Stained and air-dried slides were inserted into the ablation chamber of the Hyperion Imaging System (Fluidigm), where a pulsed 200 Hz laser is focused to a 1 μm2 spot size and applied over a userdefined area, ablating adjacent spots in 1 μm steps as the slide moves under the laser beam. The plumes of vaporized material are subjected to a stream of inert gas with a high time fidelity and carried into the inductively coupled plasma ion source for analysis by the mass cytometer. Images of each mass channel were reconstructed by plotting intensities of elements in the laser-generated plumes in the order in which they were recorded, line scan by line scan. Data processing and visualization were performed using in-house-developed Wolfram Mathematica (version 10.3) algorithms (Figure 4)47 and MCD Viewer 1.0 (Fluidigm) (Figures 6 and 7D). RGB images were overlaid for desired channel combinations. Fluorescence images were acquired using a ZEISS Axio Imager M2 microscope equipped with both 10× and 20× EC Plan-Neofluar objectives (NA 0.3 and 0.5, respectively) and ZEISS filter sets. The images were captured with a monochrome ZEISS Axiocam 506 digital camera using ZEN Pro software.

respective molar extinction coefficients were used to calculate the molar concentrations and degree of DBCO labeling for each conjugate. Inductively coupled plasma mass spectrometry (ICP-MS) analysis employed an ELAN 9000 instrument to determine the number of MCPs per biomolecule. Metal-labeled conjugates were diluted with 2 vol % HNO3 to parts per billion concentrations. Standard lanthanide solutions were prepared by a series of 10-fold dilutions of a stock solution (1000 mg/L, 2% HNO3, PerkinElmer) containing the desired lanthanide elements. Surface Ab Staining of Cells in Suspension. Live cells (2 × 106 per tube) were collected from growth medium by centrifugation and resuspended in Maxpar Cell Staining Buffer (CSB, Fluidigm). To identify dead cells, cultures were initially incubated with a 103Rh−intercalator at a final concentration of 1 μM for 15 min. After incubation, cells were washed with CSB and stained with cocktails of metal-tagged Abs at a 1:100 dilution in 100 μL of CSB for 30 min at room temperature and then washed twice with 400 μL of CSB. For WGA staining, cells were incubated with WGA−174Yb at 1 μg/mL in 100 μL of PBS for 30 min at room temperature following surface Ab staining. For evaluating the specificity of WGA binding, WGA−174Yb at a concentration of 1 μg/mL was incubated for 30 min at room temperature in a solution containing 500 mM NAG dissolved in PBS. Cells were then incubated with 100 μL of a NAG-inhibited WGA−174Yb solution. Next, cells stained with WGA were washed twice in 400 μL of CSB and treated with 25 μM Cell-ID Ir−intercalator diluted in Maxpar Fix and Perm Buffer as recommended by the manufacturer (Fluidigm). For mass cytometry analysis, cells were dispersed in 100 μL of deionized water supplemented with a 1:10 stock bead solution (EQ Four Element Calibration Beads, Fluidigm) and filtered into 5 mL round-bottom polystyrene tubes with a 30 μm strainer cap to remove possible cell aggregates. The Helios instrument (Fluidigm) was used for data acquisition in FCS3.0 file format, and the data were processed by FlowJo software (Becton Dickinson). The Fluidigm Cytobank cloudbased analysis program was used for t-distributed stochastic neighbor embedding (t-SNE) visualization (viSNE) of mass cytometry data. Staining Procedure for Adherent Cells. HeLa human cervix tumor or A431 human squamous carcinoma cells were seeded onto a four-chamber slide (Nunc Lab-Tek II CC2) at a density of 2.5 × 105 cells/mL in 1 mL of growth medium per chamber. The cells were grown as a monolayer in DMEM, supplemented with 10% FBS (Sigma-Aldrich) for 3 days at 37 °C and 5% CO2. The medium was aspirated, and cells were rinsed in PBS and then stained with a surface metal-tagged Ab mix at a 1:100 titer in 250 μL of CSB per chamber for 90 min at room temperature. Excess metal-tagged Abs were washed from the monolayer with 1 mL of CSB per chamber, and then cells were fixed for 15 min in 400 μL of 1.6% formaldehyde/PBS per chamber. Then cells either were stained with WGA−174Yb (for A431 cells) or proceeded directly to the permeabilization step. For WGA staining, cells were incubated for 30 min at room temperature with 400 μL of WGA− 174 Yb at a final concentration of 1 μg/mL in PBS. Cells were then washed three times with 1 mL of CSB. Cells were permeabilized with 400 μL of Perm-S Buffer (Fluidigm) at room temperature for 30 min and blocked with a 1% BSA/PBS mixture for 60 min at 37 °C. Cells were stained with metal-conjugated Abs and intracellular markers (1:50 ratio) in 250 μL of CSB, and phalloidin−165Ho was added for overnight incubation at 4 °C.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00239. Supplementary table and figures (PDF) I


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(12) Majonis, D., Herrera, I., Ornatsky, O., Schulze, M., Lou, X., Soleimani, M., Nitz, M., and Winnik, M. A. (2010) Synthesis of a functional metal-chelating polymer and steps toward quantitative mass cytometry bioassays. Anal. Chem. 82 (21), 8961−8969. (13) Stephanopoulos, N., and Francis, M. B. (2011) Choosing an effective protein bioconjugation strategy. Nat. Chem. Biol. 7, 876. (14) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click Chemistry: Diverse chemical function from a few good reactions. Angew. Chem., Int. Ed. 40 (11), 2004−2021. (15) Lutz, J. F. (2008) Copper-free azide-alkyne cycloadditions: new insights and perspectives. Angew. Chem., Int. Ed. 47 (12), 2182−4. (16) Pickens, C. J., Johnson, S. N., Pressnall, M. M., Leon, M. A., and Berkland, C. J. (2018) Practical considerations, challenges, and limitations of bioconjugation via azide-alkyne cycloaddition. Bioconjugate Chem. 29 (3), 686−701. (17) Meldal, M., and Tornøe, C. W. (2008) Cu-catalyzed azidealkyne cycloaddition. Chem. Rev. 108 (8), 2952−3015. (18) Kade, M. J., Burke, D. J., and Hawker, C. J. (2010) The power of thiol-ene chemistry. J. Polym. Sci., Part A: Polym. Chem. 48 (4), 743− 750. (19) Becer, C. R., Hoogenboom, R., and Schubert, U. S. (2009) Click chemistry beyond metal-catalyzed cycloaddition. Angew. Chem., Int. Ed. 48 (27), 4900−4908. (20) Manova, R. K., Pujari, S. P., Weijers, C. A. G. M., Zuilhof, H., and van Beek, T. A. (2012) Copper-free click biofunctionalization of silicon nitride surfaces via strain-promoted alkyne−azide cycloaddition reactions. Langmuir 28 (23), 8651−8663. (21) Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A strainpromoted [3 + 2] azide−alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126 (46), 15046−15047. (22) Tang, W., and Becker, M. L. (2014) ″Click″ reactions: a versatile toolbox for the synthesis of peptide-conjugates. Chem. Soc. Rev. 43 (20), 7013−39. (23) Debets, M. F., van der Doelen, C. W., Rutjes, F. P., and van Delft, F. L. (2010) Azide: a unique dipole for metal-free bioorthogonal ligations. ChemBioChem 11 (9), 1168−84. (24) Ning, X., Temming, R. P., Dommerholt, J., Guo, J., Ania, D. B., Debets, M. F., Wolfert, M. A., Boons, G.-J., and van Delft, F. L. (2010) Protein modification by strain-promoted alkyne−nitrone cycloaddition. Angew. Chem., Int. Ed. 49 (17), 3065−3068. (25) Sletten, E. M., and Bertozzi, C. R. (2008) A hydrophilic azacyclooctyne for Cu-free click chemistry. Org. Lett. 10 (14), 3097− 3099. (26) Gong, H., Holcomb, I., Ooi, A., Wang, X., Majonis, D., Unger, M. A., and Ramakrishnan, R. (2016) Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells. Bioconjugate Chem. 27 (1), 217−225. (27) Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity. J. Am. Chem. Soc. 130 (41), 13518−13519. (28) Devaraj, N. K., Upadhyay, R., Haun, J. B., Hilderbrand, S. A., and Weissleder, R. (2009) Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition. Angew. Chem., Int. Ed. 48 (38), 7013−7016. (29) Karver, M. R., Weissleder, R., and Hilderbrand, S. A. (2011) Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation. Bioconjugate Chem. 22 (11), 2263−2270. (30) Leipold, M. D., Herrera, I., Ornatsky, O., Baranov, V., and Nitz, M. (2009) ICP-MS-Based Multiplex Profiling of Glycoproteins Using Lectins Conjugated to Lanthanide-Chelating Polymers. J. Proteome. Res. 8 (2), 443−449. (31) Aub, J. C., Sanford, B. H., and Cote, M. N. (1965) Studies on reactivity of tumor and normal cells to a wheat germ agglutinin. Proc. Natl. Acad. Sci. U. S. A. 54 (2), 396−399. (32) Gonatas, N. K., and Avrameas, S. (1973) Detection of plasma membrane carbohydrates with lectin peroxidase conjugates. J. Cell Biol. 59 (2), 436−443.

AUTHOR INFORMATION

Corresponding Author

*E-mail: bedilu.allo@fluidigm.com. ORCID

Bedilu Allo: 0000-0002-6496-0671 Notes

Fluidigm, Cell-ID, Maxpar, CyTOF, EQ, Helios, Hyperion, Imaging Mass Cytometry, and IMC are trademarks and/or registered trademarks of Fluidigm Corp. in the United States and/or other countries. All other trademarks are the sole property of their respective owners. For research use only. Not for use in diagnostic procedures. The authors declare the following competing financial interest(s): B.A., X.L., A.B., and O.O. are employees of, and receive remuneration from, Fluidigm Corp.

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ACKNOWLEDGMENTS The authors thank all members of the Mass Cytometry R&D team and the Reagent Development team at Fluidigm for their contributions to this study.

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Clickable and High Sensitivity Metal-Containing Tags for Mass

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