Facile Approach To Observe and Quantify the αIIbβ3 Integrin on a

Letter pubs.acs.org/ac

Facile Approach To Observe and Quantify the αIIbβ3 Integrin on a Single-Cell Jiao Zhai,† Yaling Wang,*,† Chao Xu,†,§ Lingna Zheng,† Meng Wang,† Weiyue Feng,† Liang Gao,† Lina Zhao,† Ru Liu,† Fuping Gao,† Yuliang Zhao,† Zhifang Chai,†,‡ and Xueyun Gao*,† †

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China ‡ School of Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, Suzhou, Jiangsu 215123, China § College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China S Supporting Information *

ABSTRACT: We report for the first time seeing and counting integrin αIIbβ3 on a single-cell level. The proposed method is based on the using of the Au cluster probe. With the fluorescent property of Au24 cluster and the specific targeting ability of peptide, our probe can directly visualize integrin αIIbβ3 on the membrane of human erythroleukemia cells (HEL) via confocal microscopy. On the basis of the accurate formula of our probe (Au24Peptide8), the number of integrin αIIbβ3 can be precisely counted by quantifying the gold content on a single HEL cell via laser ablation inductively coupled plasma mass spectrometry. Our results reveal that the number of integrin αIIbβ3 on a single cell varies from 5.75 × 10−17 to 9.11 × 10−17 mol, because of the heteroexpression levels of αIIbβ3 on individual cells.

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related to blood coagulation and cancer progression such as invasion of human melanoma cells and metastasis in prostate cancer and human erythroleukemia (HEL) cells.11−16 An effective method to realize the precise quantification of αIIbβ3 on single cells is desired. Herein, the human erythroleukemia cell line, which expresses integrin αIIbβ3,14,16 was chosen as a model. We designed a peptide−metal cluster probe emitted red fluorescence (647 nm) and had accurate molecular composition (Au24Peptide8). Utilizing the fluorescence and the specific targeting ability of our probe, we can recognize integrin αIIbβ3 on HEL cell membranes. On the basis of the accurate formula of our probe (Au24Peptide8), we for the first time precisely analyze the number of integrin αIIbβ3 on single HEL cells via LA-ICPMS. The expression amount of integrin αIIbβ3 on single HEL cells range from 5.75 × 10−17 to 9.11 × 10−17 mol. This provides a novel and facile approach for the design of probe for any desired protein in situ observation and quantification at the single cell level. Before synthesis of the Au cluster probe, a peptide, H2NCCYKKKKQAGDV-COOH (abbreviated as CV), with two functional parts was designed. The first part is CCY with the ability to capture Au cluster and the second is KQAGDV, which is specific for integrin αIIbβ3.15 The detailed synthetic process is

he ability to analyze proteins on a single-cell level would promote studies of the role of cellular heterogeneity in disease progression and drug response.1,2 Clinically, accurate proteomic analysis of rare cells, such as circulating tumor cells, could contribute to early disease detection and monitoring the prognosis of patients.3 Therefore, reliable, sensitive, accurate protein quantification methods with single-cell resolution are in great demand. Although a few methods have realized single-cell protein quantification, their quantitative accuracy is limited. For example, one attractive approach is mass cytometry.4 In this method, the abundance of proteins in single cells is assessed by the number of lanthanides conjugated to each antibody. However, because the amount of lanthanide conjugated to an antibody cannot be precisely controlled, the quantification may be poor. Besides, this method is limited to analyzing single cells in suspension. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS), which has the advantage of easy sample preparation and high sensitivity,5−7 has been established very recently for single cell quantification analysis, and analysis of Au nanoparticles endocytosed by single cells has been carried out.8 However, direct analysis of specific protein from single cells using LA-ICPMS has not been performed. This is significant as we believe it facilitates the development of single cell protein in situ quantification. Integrins are cell membrane proteins that mediate cell−cell adhesion events and are essential for the attachment of cells to extracellular matrixes.9,10 The integrin αIIbβ3 expression level is © XXXX American Chemical Society

Received: December 14, 2014 Accepted: February 12, 2015

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DOI: 10.1021/ac504639u Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry described in Supporting Information 2 and Figure S1. After the probe was obtained, we characterized its absorption, fluorescence, and mass spectral properties. Figure 1a shows

Figure 2. Specificity of the Au24Peptide8 probe for integrin. Confocal fluorescence images of HEL cells exposed to (a) 12.5 μM Au24Peptide8 probe only, (b) 12.5 μM Au24Peptide8 probe and antibody (integrin αIIb/β3 antibody, dilution 1:100 in phosphate-buffered saline for 45 min followed by goat antimouse IgG-FITC, 1:100 dilution in phosphate-buffered saline for 25 min, protected from light at room temperature), (c) a blocking dose of 5 mM CV peptide for 1 h at room temperature, followed by 12.5 μM Au24Peptide8 probe. Images are shown for Hoechst 33342 (blue), antibody (green), and Au24Peptide8 probe (red).

Figure 1. (a) Ultraviolet−visible absorption of the probe. (b) Photoemission (red line, λex = 565 nm) and photoexcitation (black line, λem = 647 nm) spectra of the probe. Inset: Digital photographs of the probe under visible (left) and ultraviolet light (right). (c) MALDITOF-MS of the probe (P, peptide CCYKKKKQAGDV; S, sulfur atom). (d) Enlarged view of peaks (MALDI-TOF-MS) range from 10 to 11k m/z.

CV peptide, the fluorescence from the probe on HEL cells decreased notably because the probe and free CV peptide competed for the same binding site on integrin (Figure 2c). All of these results support excellent specificity of the probe for integrin αIIbβ3. With the purpose of accurately observing and counting integrin αIIbβ3 on a single-cell level, concentration and time parameters were optimized by cell fluorescence labeling experiments and solution-based ICPMS to obtain the average mass of Au on a single cell via observing and digesting a large population of cells. The concentration of Au24Peptide8 probe in cell culture media, which strongly influences cell labeling efficiency, was optimized first. Figure 3a shows that after being incubated for 30 min, the probe fluorescence of cells became basically stable when the probe concentration in PBS was 12.5 μM. This result was consistent with the related fluorescence intensity of cells analyzed by software in Figure 3c. Meanwhile, the results from ICPMS (Figure 3e) showed the average mass of Au on a single cell increased as the probe concentration increased from 2.5 to 12.5 μM and did not increase further for higher probe concentrations. These results imply that the integrin on the cells was saturated completely when the probe concentration was 12.5 μM in PBS. The effect of incubation time on cell labeling was then examined. Confocal microscope observations (Figure 3b) suggested that the optimal incubation time was 30 min. This result almost corresponded to the related fluorescence intensity analyzed by software (Figure 3d), and the average mass of Au on a single cell according to ICPMS (Figure 3f). According to these results, the optimal concentration and time for quantifying integrin αIIbβ3 on HEL cell membranes were 12.5 μM and 30 min, respectively. Under the optimized incubation conditions, integrin αIIbβ3 can be completely saturated by the probe. Therefore, we can quantify the average number of αIIbβ3 on a single HEL cell from ICPMS data, as presented in Figure 3e,f. Because each probe molecule contained 24 Au atoms, the

the absorption peaks of Au cluster at 241 and 295 nm, which were basically consistent with those of a previously reported peptide-Au cluster.17 The probe showed maximum excitation and emission peaks at 565 and 647 nm, respectively (Figure 1b). After excitation at 365 nm using a portable UV light, the probe solution exhibited bright red fluorescence (inset of Figure 1b). To quantify the targeting proteins accurately, we need to determine the composition of our probe. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a popular way to acquire the accurate mass of peptide−Au cluster.18−20 The mass spectrum of the probe (Figure 1c) contained a series of intense peaks, along with weaker peaks, between 5−15k m/z. The spacing between adjacent intense peaks was equal to a peptide of 1370 Da. The m/z of the intense peaks was consistent with the formula Au24PmS16−2m (where P is CCYKKKKQAGDV, m = 0−8; S is sulfur atom). The representative peak at 10−10.8k m/z is shown in Figure 1d. Peaks with m/z 197 and 32 were attributed to the loss of Au and S.21 The mass spectral data revealed that the chemical formula of our probe is Au24Peptide8. To label and quantify integrin αIIbβ3 accurately, the specificity of the probe first needed to be verified. In this study, we chose HEL cells as a model and examined the location of the Au24Peptide8 probe on HEL cells via confocal microscopy. Figure 2a reveals that the Au24Peptide8 probe was located on the HEL cell membrane. We used an immunofluorescence assay to confirm the specificity of the probe toward integrin αIIbβ3. The green emission of the αIIbβ3 antibody and red emission of the probe were strongly colocalized and overlaid on the HEL cell membrane, as depicted in Figure 2b (note that the antibody and our probe target different sites of the integrin). Furthermore, the accurate targeting of αIIbβ3 by the probe was verified with a blocking study. After being blocked with 5 mM B

DOI: 10.1021/ac504639u Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 3. (a,b) Confocal fluorescence images of HEL cells exposed to (a) different concentrations of probe in PBS for 30 min and (b) probe (12.5 μM in PBS) for different times. (c, d) The corresponding fluorescence intensity analyzed by software for HEL cells exposed to Au24Peptide8 probe with different concentrations and times. (e, f) The corresponding amount of Au determined by ICPMS for the incubation conditions in parts a and b.

Figure 4. (a−e) Transient Au signals of 30 individual cells determined by LA-ICPMS after incubation with different concentrations of probe for 30 min and corresponding statistical distribution of Au signals. (f) The mean mass of Au per cell derived from the statistical distribution of Au signals in parts a−e from the probe concentration labeling experiments with an incubation time of 30 min (left) and the probe incubation time experiments with a probe concentration of 12.5 μM (right, see details in Figure S4 of the Supporting Information). Each point is the average result of 30 individual cells.

amount of probe molecules in one cell was about 6.62 × 10−17 mol, which represented the average amount of integrin αIIbβ3 on a single cell membrane. To better understand the expression of integrin αIIbβ3 on a single HEL cell, we then quantified the abundance of Au, which can be precisely exchanged to the integrin αIIbβ3 number according to the probe formula, per single cell via LA-ICPMS. Figure 4a−e shows Au signal intensities from the ablation of

individual cells on chamber slides. Note that grouped cells in Figure 4a−e were precultured with different concentrations of probe and each group contained 30 cells for LA-ICPMS analysis. Because the signal intensities in control cells were so weak (Figure S3 in the Supporting Information, we can regard the Au signals in our experiments as a response to the probe. The standard deviation in Figure 4a−e reveals the heterogeneity of integrin αIIbβ3 on different single cells. Furthermore, C

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Analytical Chemistry

National Key Basic Research Program of China (Grants 2013CB932703 and 2013CB933704).

we can see the mean intensity of Au signals on a single cell increased as the probe concentration increased from 2.5 to 12.5 μM. At a probe concentration of 12.5 μM, the mean intensity of Au signals on a single cell became basically stable. To obtain the relationship between Au signal intensity and the corresponding Au concentration on a single cell, we used MicroFabJetLab, a commercial inkjet printer, as a calibration standard.8 The intensity of the 197Au signal of the ablation of droplet residues from different concentrations of gold standard solutions on glass slides and the calibration curve were presented in Figure S4 in the Supporting Information. Using the Au standard curve, we can obtain the mass of Au on single cells. After transforming the mean intensity of Au (Figure 4a−e) to Au concentration on a single cell and fitting the Au concentration, we can see that the HEL cells were completely saturated by the probe at a concentration of 12.5 μM (red curve in Figure 4f). The black curve in Figure 4f illustrates the relationship between cell− probe incubation time and the mean mass of Au on a single cell (the detailed signal and statistical distribution of Au on cells can be found in Figure S5 in the Supporting Information). These results suggest that a probe concentration of 12.5 μM and incubation time of 30 min are suitable for quantifying the amount of integrin αIIbβ3 on single HEL cells. Under this optimal labeling condition, the mass of Au on a single cell ranged from 272 to 431 fg (Figure 4c and Figure S4 in the Supporting Information). Because each probe molecule contained 24 Au atoms, the amount of probe (equal to the amount of integrin αIIbβ3) on an individual cell was about 5.75 × 10−17 to 9.11 × 10−17 mol, which was close to the value of 6.62 × 10−17 mol per cell determined from the large-cellpopulation digestion method (Figure 3e,f). In conclusion, we accurately quantified proteins on a singlecell level via peptide-Au cluster and LA-ICPMS. With the help of the peptide-Au cluster probe, integrin αIIbβ3 can be observed spatially by confocal microscopy and quantified accurately on single HEL cells via LA-ICPMS without any enrichment and purification process. Because the variation of the number of membrane proteins on single cells has a significant correlation with the occurrence and progression of disease, our method provides a facile and precise way to quantify any well-identified protein (e.g., biomarker) of interest and helps estimate the progression of the disease. This method will provide new insights into disease diagnosis and therapy on a single-cell level.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Fax: 86-10-88236456. Phone: 86-10-88236709. E-mail: [email protected]. *Fax: 86-10-88236456. Phone: 86-10-88236709. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grants 21390414, 31300827, 31271072, 31200751, 81472851, and 11404333) and the D

DOI: 10.1021/ac504639u Anal. Chem. XXXX, XXX, XXX−XXX