Article pubs.acs.org/ac
Single Cell Chemical Proteomics with Membrane-Permeable ActivityBased Probe for Identification of Functional Proteins in Lysosome of Tumors Dongjuan Chen,†,‡ Fengkai Fan,†,§ Xingfu Zhao,†,‡ Fei Xu,‡ Peng Chen,‡ Jie Wang,‡ Lin Ban,‡ Zhihua Liu,‡ Xiaojun Feng,‡ Yuhui Zhang,*,‡ and Bi-Feng Liu*,‡ ‡
Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics−Hubei Bioinformatics and Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China § Hubei Key Laboratory of Purification and Application of Plant Anti-Cancer Ingredients, College of Chemistry and Life Science, Hubei University of Education, Wuhan, 430205, China S Supporting Information *
ABSTRACT: Proteomics at single-cell resolution can help to identify the heterogeneity among cell populations, shows more and more significance in current chemistry and biology. In this work, we demonstrated a new single cell chemical proteomic (SCCP) strategy with a membrane-permeable activity-based probe (ABP) to characterize the functional proteins in lysosome located in the cytosol. The ABP targeted to the cysteine cathepsin family protein, CpFABP-G, was designed for cysteine cathepsins labeling. The labeled HeLa cell of a cancer cell line was injected into a capillary and was lysed by SDS solution with heating. The lysate was then online readout by capillary electrophoresis-laser-induced fluorescence method. Due to the employment of highly specified ABP kicking out the uncorrelated proteins, the expression of cysteine cathepsins in individual HeLa cells was easily detected, and heterogeneity among those HeLa cells was readily discriminated. Further work was concentrated on SCCP analysis of the mouse leukemia cell of monocyte macrophage (RAW264.7). It was for the first time identifying two expression modes of cysteine cathepsins in RAW264.7, which could be undermined by the analysis of cell populations. We believed that SCCP would be one of the powerful alternatives for proteomics at single-cell resolution.
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uncorrelated proteins at the cost of tedious antibody-binding procedures, but disregards the heterogeneity among single cells. Recently, Shi et al.19 developed a new microchip-based SCP strategy, providing a comprehensive picture of protein signaling networks of tumor cells. Herr’s group20,21 constructed a brand new microfluidic SCP approach for high-throughput single cell Western blotting, which overcame the limitation of antibody fidelity and constituted a versatile tool for the study of complex cell populations at single-cell resolution. More recently, we proposed a novel single cell chemical proteomic (SCCP) strategy7 with an activity-based probe (ABP),22−26 which has been successfully applied for identifying GPCR receptors in a single primary neuron. In SCCP, the first step was the labeling of the targeted functional proteins with ABP, which is usually consisting of three molecular domains, a “warhead” sharing the similar pharmacophore with the receptor antagonists for targeting the active domain, a photolabile
ith emerging biology systems, qualitative and quantitative profiling of nucleic acids, proteins, metabolites, and their interactions has become one of the major tasks to unravel the chemical basis of life. Due to the heterogeneity in cellular events, such investigations have recently been scaled down to the single cell level1 known as single cell genomics,2 single cell transcriptomics,3,4 single cell proteomics (SCP),5−7 single cell metabolomics,8 and so on, which represent the new challenges in chemistry and biology. Since 2004, Dovichi5 and Nolan6 first proposed the concept of SCP, great advancements have been achieved either with chemical cytometry9−13 or with flow cytometry.14,15 Chemical cytometry is performed by two-dimensional capillary electrophoresis for globe proteome profiling of single cells.16 The heterogeneity among individual cells can be readily discriminated, which is significantly useful for stem cell development, neuroscience, or cancer biology, where cells show high heterogeneity. In the meantime, an approach with flow cytometer shows great potential in mapping cellular signal transduction networks and investigating molecular mechanisms of diseases.17,18 It avoids interference from a large amount of © 2016 American Chemical Society
Received: December 8, 2015 Accepted: January 25, 2016 Published: January 25, 2016 2466
DOI: 10.1021/acs.analchem.5b04645 Anal. Chem. 2016, 88, 2466−2471
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(excitation line: 488 nm, Model Shanghai, China) were used as the excitation source for the injection process and LIF detection, respectively. A cube with a 460−490 nm band-pass filter, a 505 nm dichroic mirror, and a 510−550 nm band-pass filter (Olympus, Japan) were used for LIF detection. Fluorescent signal was collected under 60× objective (NA 0.7) by a PMT (Hamamatsu R928) with 20 Hz sampling rate and filtered by a current preamplified filter (SR575, Stanford Research System, U.S.A.), A LabVIEW TM (National Instruments, U.S.A.) program was written to collect filtered signal via a PCI controller card (PCI6035E, National Instruments, U.S.A.). Microwell Holder and Capillary Rider Fabrication. We designed and fabricated a microwell holder and a bench-shaped capillary rider for single cell injection with poly(dimethylsiloxane) (PDMS) as the structural materials. Microwell holder was made following standard soft-lithography procedure. For capillary rider, PDMS was cured in a Petri dish and was then cut into one 5 cm × 5 cm × 0.6 cm piece and four 1 cm × 1 cm × 0.6 cm pieces. The four smaller pieces were bonded to the larger piece after being treated with O2 plasma. A hole of 0.2 mm diameter was perpendicularly punched where the capillary would go through. Capillary Pretreatment. The inner wall of bare fused capillary was coated by polyacrylamide to resist protein adsorption and minimize the EOF. After silylation with γMAPS, the polymerized acrylamide were attached to the inner wall by free ethylene groups on γ-MAPS molecules. The −NH2 groups in the free end of polyacrylamide were cross-linked by overnight reaction with 37% formaldehyde. The inner-wallcoated capillary was filled with deionized water for preservation. Cell Culture. The cultured cell lines such as HeLa and RAW264.7 were maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal calf serum (NCS, Gibco) and 100 U/mL penicillin and streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Cells were harvested by treatment of 0.2% trypsin-EDTA solution (Gibco) and suspended in phosphate buffered saline (PBS) at the concentration of 105 cells per mL for single cell injection. Probe Synthesis. The activity-based probe (ABP), CpFABP, as shown in Figure S1 for cysteine cathepsin family proteins was synthesized with standard solid-phase peptide synthesis on 2-chlorotrityl-chloride resin using standard deprotection and coupling procedures, as reported previously.28 5(6)-Carboxyfluorescein (FAM) was used as fluorophores of CpFABP-G and cell-penetrating octa-arginine peptide (rRrRrRRR, r: D-Arg, R: L-Arg) was employed to enhance the cellular permeability of CpFABPs. To cleave products from the resin, a cleavage cocktail solution containing triisopropylsilane (TIS, 2%) and trifluoroacetic acid (TFA, anhydrous, 98%) was added to the resin. Then probes were purified by preparative high-performance liquid chromatography (HPLC) to reach a purity greater than 90%, and the mass was confirmed by electrospray ionization-mass spectrometry. Cell Labeling. Cells were seeded at a density of 1.0 × 104 cells/well in growth media (DMEM supplemented with 10% (v/v) FBS) and were grown at 37 °C under CO2 (5%, v/v) overnight. Washed with PBS (pH 7.4) twice and incubated with the probes CpFABP-G (15 μM, 300 μL) in PBS for 30 min, the medium was discarded, and the cells were rinsed with PBS twice. After a post-labeling incubation in DMEM (300 μL) containing 10% FBS for 4 h, the cells were analyzed with a
diazirine group that can effectively generate a covalent, irreversible linkage between the probe and the receptors after UV irradiation, and a fluorescent tag for detection. Afterward, the functionally labeled single cells were subsequently encapsulated in buffer droplets stored in a PDMS chip holder for further capillary electrophoresis−laser-induced fluorescence (CE-LIF) detection. In this scenario, the employment of ABP was crucial. It significantly reduced the number of uncorrelated proteins, thus, greatly simplifying the CE-LIF readout. However, it was only applicable for cellular membrane proteins because the ABP molecules usually could not enter into the cytosol. In this work, we further demonstrated a SCCP approach with membrane-permeable ABP for identifying functional proteins in the cytosol. For testing the membrane-permeable capability of the ABP, cysteine cathepsins in lysosome located in the cytosol were chosen as the targeted proteins, which play important roles in tumor biology and are emerging as promising targets for diagnosis and therapy.27 It meant that the ABP molecules would undergo two consecutive transmembrane procedures to reach the targets, that was, ABP trafficking in plasma membrane and lysosome membrane. The labeled cell was then injected into capillary for cell lysis, and subsequent CE-LIF readout. With this SCCP strategy, cysteine cathepsin family proteins were successfully identified for two kinds of tumor cells at single cell resolution.
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EXPERIMENTAL SECTION Materials and Reagents. Chemicals including γ-MAPS, NaOH, H2O, HCl, CH3OH, HCHO, and acrylamide were purchased from Tianjing Chemical Co. Ltd. (Tianjing, China) and used for surface modification of capillary inner wall to prevent the protein adsorption and suppress the electroosmotic flow (EOF). Chemicals including Dextran T2000, SDS, and HEPES were obtained from Sigma-Aldrich (MO, U.S.A.) and used for capillary sieving electrophoresis to separate proteins. Bare fused silica capillary was purchased from Reafine Chromatography Equipment Co. Ltd. (Yongnian, Hebei, China). Activity-based probe was synthesized as shown in Figure S1 according to a previous report. All reagents were of analytical grade or specified otherwise. All solutions were prepared with water purified by the Milli-Q system (Millipore, U.S.A.) and filtered with a 0.22 μm microporous membrane filter prior to use. Apparatus. There were three essential parts in our instrumental system, single cell injection subsystem, capillary electrophoresis (CE) subsystem for protein separation, and laser-induced fluorescence (LIF) subsystem for ultrasensitive protein detection. A custom-built single-channel negative pressure system was utilized to realize single cell injection. Negative pressures were generated in 1.0 L air containers by microvacuum pumps (PK5008, Ruiyi, China) and monitored by a pressure gauge. A solenoid valve (VDW250-6G-2, SMC, Japan) was used for switching pressures between negative pressure and atmosphere. An electric switch was utilized to manually control the solenoid valve for injection after the distal end of capillary aligned with the selected single cell. A high voltage supplier (±30 kV/300 mA Shanghai Institute of Applied Physics, Chinese Academy of Science, China) was employed to carry out CE separations. Single cell injection and CE-LIF analysis were carried out on an inverted microscope (IX71, Olympus, Japan). A 100 W mercury lamp (USH1030L, Olympus) and an Ar-ion laser 2467
DOI: 10.1021/acs.analchem.5b04645 Anal. Chem. 2016, 88, 2466−2471
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Analytical Chemistry confocal laser scanning microscope or resuspended for single cell injection and analysis. Gel Electrophoresis Assay. The labeled cells were washed three times with cold PBS at 4 °C. Chemical reagents for lysis of cells should be made when needed. It contains 1× complete Mini Protease Inhibitor Cocktail (Roche Applied Science) in 500 μL of RIPA buffer. The labeled cell suspension, including lysis reagents, was sonicated 100× in 1 s pulses in an ice-cold water bath. Prior to sampling, the cell lysates were centrifuged at 40000 g for 20 min at 4 °C. Subsequently, the supernatants were mixed with 5× SDS sample buffer. Samples were boiled for 5 min so that samples were denatured drastically. Then, lysates were separated by SDS-PAGE on 13.5% gels. The gels were imaged by a Typhoon 9410 laser scanning system (Amersham Biosciences). Capillary Electrophoresis. Prior to cell injection, the capillary was filled with electrophoretic running buffer (7.5% Dextran 5 mM HEPES and 5 mM SDS, pH 8.0). Droplets of cell suspension and droplets of 0.5% (w/v) SDS were deposited into a microwell array on a PDMS holder slide side by side. After a short plug of SDS injected into capillary, the bench-like rider positioned the distal end of the capillary right above a droplet of the single cell supervised under microscope. A negative pressure was then applied to drag the cell up into the capillary. Afterward, another plug of SDS was injected to form a sandwich-like injection plug convenient for cell lysis and protein denaturation. The capillary tip was heated to 95 °C for 10 min to solubilize the single cell and ensure the complete bonding of SDS and proteins. CE was performed under the electric field of 300 V/cm with an inner wall coated capillary of 50 cm × 75 μm i.d. × 365 μm o.d. (effective length: 40 cm; total length: 50 cm). Prior to each injection, the capillary was washed for 15 min with the running buffer. LC-MS/MS Analysis. After electrophoresis, the molecular weight regions of interest were cut and vacuum-dried. The separated proteins were cut into petides samples by enzyme digestion.29 The peptides were extracted from the digested gel pieces and vacuum-dried. The dried peptide samples were resuspended in 2% formic acid and 5% acetonitrile and then were identited using capillary reverse phase chromatography combined with tandem mass spectrometry LTQ-Orbitrap Velos (Thermo Finnigan, U.S.). The generated MS data were searched by SORCERER with TurboSequest v4.0.3 (SageN Research Sorcerer Enterprise) against the NCBI-RefSeq Mus musculus database (ftp://ftp.ncbi.nih.gov/refseq/M_musculus/, downloaded on November, 2011). The detailed sequence information for cathepsins was obtained from the MEROPS database for peptidases (http://merops.sanger.ac.uk). All of the identified peptides of cathepsins and their Sequest XCorr values are shown in Table S2.
Figure 1. Schematic of the SCCP strategy. (A) ABP labeling procedure: at the first step, the ABP molecules penetrated through cell membrane and accommodated in the cytosol; second, the ABP molecules penetrated through lysosomal membrane and entered into lysosomes; third, the ABP was applied to functionally label the targeted proteins. (B) Single cell deposited in arrayed microwells on a microchip was injected into capillary for CE-LIF analysis to study the targeted proteins functionally labeled with ABP.
In the whole protocol, the first step was the cell labeling with ABP molecules. We designed the special ABP molecules (CpFABP-G) for cysteine cathepsin family proteins as shown in Figure S1, which consisted of three main parts, a “warhead” (the expoxysuccinyl scaffold) specifically targeting the active domain of cysteine cathepsins, a fluorescent tag (FAM) for fluorescent detection and a linker poly-L-arginine (R8). Due to the existence of R8, a highly efficient transmembrane peptide, CpFABP-G molecules easily penetrated through the plasmid membrane and entered into the cytosol, as depicted in Figure 1B, and further penetrated the lysosome membrane and aggregated in the lysosome. After incubation at 37 °C, the CpFABP-G molecules would covalently attached to the cysteine cathepsins. The specificity of the warhead of CpFABP-G determined that this process was highly specified. Confocal fluorescence imaging (Figure 2A,B) recorded the evidence of the CpFABP-G molecules trafficking from the outside of the cell to the inside of the lysosome. At a time of 5 min, the most of CpFABP-G molecules finished the first transmembrane process and uniformly distributed in the cytosol, although there were a few molecules even entering into the lysosome. After about 4 h, the CpFABP-G molecules finished the second transmembrane process and accumulated in the lysosome. The labeled cell was then injected into a capillary and lysed for CE-LIF analysis. CE was an ideal platform for single cell analysis due to its compatible inner diameter with regular cells.30,31 And LIF provided the best detection sensitivity even
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RESULTS AND DISCUSSION Single Cell Chemical Proteomics (SCCP) Strategy. In SCCP approach, as described in Figure 1A, activity-based probe (ABP)-labeled single cells were encapsulated in buffer droplets and individually dispensed into PDMS microwell arrays. A PDMS rider holding the inlet of a capillary was switched among those microwells for picking up one cell at a time by applying negative pressure at the oulet of the capillary. The loaded cell was then lysed by SDS solution together with heating and further analyzed by capillary electrophoresis−laser-induced fluorescence (CE-LIF) method. 2468
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Apparently, all the HeLa cells expressed cysteine cathepsins, but the expression levels were quite different (Figure 3), suggesting
Figure 3. Heterogeneity in cysteine cathepsin expression of single HeLa cells. Peak identity: *, cathepsins H, L1, S, F, O; **, cathepsins B, Z; CE conditions: running buffer, 7.5% Dextran, 5 mM HEPES, and 5 mM SDS (pH = 8.0); applied voltage, 15 kV; capillary, 50 cm × 75 μm i.d. × 365 μm o.d., with an effective length, 40 cm.
that cysteine cathepsins expressions in HeLa cells were heterogeneous. The electropherograms of eight single HeLa cells as the representatives indicated that the concentrations of cysteine cathepsins were different due to their different electrophoretic peak heights and peak areas. It was interesting that the peak shapes were also diverse, which implied the different expression patterns among those cells. Previously, we also found this phenomena while SCCP analysis of GPCRs in neurons.7 This pattern variations might be caused either by the concentration fluctuations of each cysteine cathepsin in the group, or by different protein modifications that widely dynamically occurred for proteins. It has been previously confirmed that the activities of these proteases are usually tightly regulated by posttranslation.34 SCCP Analysis of Tumor Cells. Finally, SCCP strategy was applied for analyzing the expression of cysteine cathepsin family proteins in the tumor cell, mouse leukemia cell of monocyte macrophage (RAW264.7). Different than the HeLa cells, a new peak at a molecular weight of about 50 kDa appeared in the electropherogram, besides the two peaks at 26.6 kDa and 30 kDa. However, it was more interesting that no cell simultaneously had all the three peaks. There were two patterns, as demonstrated in Figure 4A. Pattern I had the peaks of 26.6 and 30 kDa, while Pattern II had the peaks of 30 and 50 kDa. Figure S3 showed the electropherogram of three injections of single cells of Pattern II. It was obvious that the reproducibility of migration time was high, although the peak areas and peak shapes were different due to the heterogeneity of cells protein expression. As a control, inhibition experiments were also performed with cysteine cathepsins inhibitor E64. Figure 4B showed three electropherograms of cell lysates with pretreatment of different E64 concentrations (0, 10, and 100 μM). It was obvious that the peak heights of cysteine cathepsins decreased with additions of the inhibitor E64. However, different proteins exhibited diverse responses to E64, which was also indicated by SDS-PAGE experiments (Figure S4). This result further confirmed the effectiveness of the ABP for cysteine cathepsins of RAW264.7 cells. Primary statistical analysis (n = 10) showed that 40% individual cells exhibited
Figure 2. SCCP analysis of HeLa cell. ABP labeling of HeLa cells (the starting time for cell imaging was 30 min after incubation with the ABP CpFABP-G): (A) 5 min; (B) 4 h. (C) Electropherogram (a) of molecular makers of proteins and electropherogram (b) of SCCP analysis of HeLa cell. (D) SDS-PAGE result of ABP-labeled cell populations lysate. CE conditions: running buffer, 7.5% Dextran, 5 mM HEPES, and 5 mM SDS (pH = 8.0); applied voltage, 15 kV; capillary, 50 cm × 75 μm i.d. × 365 μm o.d., with an effective length, 40 cm.
down to the single-molecule level.32,33 Owing to the use of ABP, the uncorrelated proteins were strictly kicked out, which tremendously reduced the sample complexity and only focused on the targeted proteins. We optimized the CE-LIF conditions. The baseline resolution of protein molecular weight markers could be achieved with a reproducibility of RSD < 0.1% for the migration time (Figure S2A) and a detection limit of 0.1 pM (ca. 8.55 × 10−22 mole) for fluorescein (Figure S2B). For a proof-of-concept validation, capthepsins in single HeLa cell of were analyzed. Two peaks were resolved in electropherogram (Figure 2C-b). The molecular weight suggested the peak identity with a compasion with the electropherogram of protein molecular weight stadards (Figure 2C-a). The first peak belonged to a group of cysteine cathepsin H, L1, S, F, O, and the second represent a group of cysteine cathepsin B and Z. The proteins in the group could not be further separated because of their so similar molecular weights. For further confirmation, SDS-PAGE separation of lysed sample of ABPlabeled population cells was conducted. The separated proteins were subjected to LC-MS analysis for identifying the proteins (Table S1). SCCP Revealed the Heterogeneity of HeLa Cells. Next, we compared intrinsic expression level of cysteine cathepsins in different cells in the HeLa cell line with the SCCP strategy. 2469
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Figure 4. SCCP analysis of RAW264.7 tumor cell. (A) Electropherogram (a, b) of SCCP analysis of RAW264.7 cell. (B) Electropherograms of cell lysates with pretreatments of different concentrations of inhibitor E64: a, 0 μM; b, 10 μM; c, 100 μM. (C) SDS-PAGE result of ABP labeled cell populations lysate. CE conditions: running buffer, 7.5% Dextran, 5 mM HEPES, and 5 mM SDS (pH = 8.0); applied voltage, 15 kV; capillary, 50 cm × 75 μm i.d. × 365 μm o.d., with an effective length, 40 cm.
supported the significance of SCCP. It should be pointed out that the throughput of this SCCP protocol was low. Attempt to increase the throughput to thousands of single cells in several minutes is currently under our considerations.
proteins expression of Pattern I, while another 60% cells exhibited proteins expression of Pattern II. It was the first time to discover the cysteine cathepsins expression pattern at singlecell resolution for RAW264.7. This result, as an important example, demonstrated the significance of SCCP and single cell resolution analysis. Analysis of cell populations undermined this heterogeneity as shown in Figure 4C. There were three bands in SDS-PAGE of cell populations.
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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.analchem.5b04645.
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CONCLUSIONS In this paper, we demonstrated a novel SCCP approach for identify the cysteine cathepsins in lysosome of tumor at single cell resolution. By using membrane-permeable ABP (CpFABPG), the cysteine cathepsin family proteins located in lysosome of the cytosol were specifically labeled after two consecutive transmembrane procedures such as trafficking in plasma membrane and lysosome membrane. The labeled cell was then injected into a capillary and was lysed with SDS solution together with heating. The lysate was subjected to high performance CE separation and high sensitive LIF detection. With this protocol, the expression of cysteine cathepsins in individual HeLa cells was analyzed, revealing the heterogeneity among those cancer cells. Further work was focused on SCCP analysis of mouse leukemia cell of monocyte macrophage. The results at single-cell resolution suggested different two expression modes among RAW264.7 cells. It was for the first time to find out this phenomena, which was undermined easily by the analysis at cell populations’ level. This finding strongly
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Additional supporting figures and tables (PDF).
AUTHOR INFORMATION
Corresponding Authors
*E-mail: bfl
[email protected]. *E-mail:
[email protected]. Tel.: +86-27-87792203. Fax: +86-27-8779217. Author Contributions †
These authors contributed equally to this work (D.C., F.F., and X.Z.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from National Basic Research Program of China (2011CB910403) 2470
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and National Natural Science Foundation of China (21475049, 31471257, 21275060, and 31400732).
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