Article pubs.acs.org/ac
Simultaneous Online Enrichment and Identification of Trace Species Based on Microfluidic Droplets Ji Ji,† Lei Nie,†,‡ Yixin Li,† Pengyuan Yang,*,† and Baohong Liu*,† †
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, 220 HanDan Road, Shanghai 200433, China Shanghai Institute of Quality Inspection and Technical Research, Shanghai 201114, China
‡
S Supporting Information *
ABSTRACT: A facile online enrichment protocol has been proposed based on microfluidic droplets acting as an interface between a liquid chromatography separation system and detection systems of ESI-MS/MS and laser-induced fluorescence. Low-abundance species were successfully concentrated and analyzed in this system via droplet shrinkage. The proposed platform significantly increased the enrichment efficiency and detection sensitivity with reduced sample handling steps, short analysis time, and no cross-contamination. The presented system is universal, shows no discrimination, and is easily coupled with other separation and detection approaches.
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many oil phases, whereas ions, solutes, or biomolecules can remain in the water droplets.26−28 This performance has been applied for fabrication of various particles and protein crystallization.29−31 In recent research, Chiu et al.32 have demonstrated a flexible method to concentrate dissolved solutes and nanoparticles in aqueous microdroplets and investigated the kinetics and factors affecting droplet shrinkage. So far there has been no report to introduce the droplet shrinkage method with ESI-MSn to realize online concentration and simultaneous detection of trace species. In this work, we have developed a facile and versatile protocol capable of online enrichment of separated peptides based on droplet shrinkage in a microfluidic chip coupled with ESI-MS/MS. Herein, the separated sample effluents were compartmentalized into droplets sequentially, where the solvents of each droplet were dissolved in continuous phase in microchannels, and the remaining samples were concentrated in these shrinking droplets and detected by ESI-tandem mass spectrometry (ESI-MSn) with high sensitivity. Such an approach was successfully demonstrated for peptide mixtures of bovine serum albumin (BSA) digestion at low concentrations by using reverse-phase liquid chromatography (RPLC), online concentration in droplet-based microchips, and identification with ESI-MS/MS.
iomarkers are valuable targets for early diagnosis of diseases and for monitoring therapy and postsurgical recurrence.1−5 Electrospray ionization mass spectrometry (ESIMSn) coupled with liquid-based separation, such as reversed phase chromatography,6−8 electrophoresis,9 or electrochromatography,10 has been the foundation of biomarker discovery in large scale.11,12 Medically significant biomarkers are usually lowabundance molecules compared with normal proteins or peptides. 13−15 To effectively detect and predict these biomarkers, the ability to concentrate limited molecules within the analytic vessels is critical. Various sample preprocessing methods have been developed, such as selective enrichment of targeted biomolecules or depletion of abundant species. To some extent, these methods are still challenging for the general applications of online and undiscriminating enrichment of lowabundance molecules where the key markers may be covered.5 To address this challenge, we propose a new concept of online enrichment of low-concentration targets in microfluidic droplets acting as a simple and universal interface between liquid-based separation and mass spectrometry. Droplet-based microfluidic chips, with their flexible manipulation, precise volume control, and ability to integrate multiple analysis steps, offer promise for performing high throughput and automated chemical and biological experiments without cross-contamination.16−24 We have recently developed a droplet-based microfluidic chip used not only as a microreactor for protein digestion but also as an interface between liquid-based separation and ESI-tandem mass spectrometry.25 This concept would be an ideal tool for online enrichment of trace analytes, if the species within individual droplets could be concentrated to a high level. The droplet shrinkage method has been developed based on the phenomenon that water has slight solubility in © 2013 American Chemical Society
Received: June 17, 2013 Accepted: September 15, 2013 Published: September 16, 2013 9617
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EXPERIMENTAL SECTION Chemicals and Materials. Bovine serum albumin and trypsin were all obtained from Sigma Chemical Co. Inc. (St. Louis, MO). Fluorescein isothiocyanate (FITC) labeled Pep I (KCDICTDEY-FITC) and Pep II (CYIQNCPRG) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Acetonitrile (HPLC grade) was purchased from Fisher Scientific (Ottawa, ON, Canada) and used without further purification. Formic acid (analytical reagent, 98%) was purchased from BDH Chemicals, (Toronto, ON, Canada). Dimethyl carbonate (DMC) was purchased from Aladdin Regent (Shanghai, China). Aquapel was purchased from PPG Industries. Deionized water (>18 MΩ·cm) was obtained from a Milli-Q Gradient water purification system (Millipore, Bedford, MA). The C18 column (0.2 × 50 μm MagicC18AQ 3 μm, 200 Å) used for reversed-phased chromatography and the trap column (C18, 0.5 × 2 mm) were purchased from Michrom Bioresources Inc. (Auburn, CA). Fabrication of the Microchip. The microfluidic device was made by conventional soft lithographic techniques.33 The detailed design of the PDMS chip is shown in Figure SI-1 of Supporting Information. The height of the channels is 100 μm, and the width and length of the reaction channels are 250 μm and 47 cm, respectively. The microdevice was assembled by attaching the PDMS cast to a glass slide. The bonding strength was provided by pretreating the contact surfaces with oxygen plasma for 60 s in the plasma cleaner (PDC-32G, Harrick Plasma). After plasma bonding, Aquapel, a water repellent agent, was flushed through the microchannel for a few seconds for surface modification. Then the microchannel was flushed with air and baked at 65 °C for 20 min.34 A stainless capillary (250 μm o.d. and 100 μm i.d.) was used as the MS emitter and glued onto the outlet of chip using 502 glue. Protein/Peptide Separation and Enrichment. BSA digests were separated by an HPLC system (Michrom Bioresources Inc. CA) equipped with a C18 column (0.2 × 50 mm Magic C18AQ3 μm, 200 Å). The C18 column was connected to the HPLC system and operated at a flow rate of 2 μL/min. The BSA peptides were loaded and analyzed. Solvent A was 5/94.9/0.1% acetonitrile (ACN)/H2O/formic acid (FA), solvent B was 95/4.9/0.1% ACN/H2O/FA, and solvent C was 99.9/0.1% H2O/FA. First, the sample was loaded by pump C with a flow rate of 30 μL/min for about 5 min to the trap column (C18, 0.5 × 2 mm), and then the valve was switched to begin the gradient. The following separation gradient was set as follows: solvent A 90% (0 min) → 90% (5 min) → 50% (10 min) → 50% (30 min) → 95% (30.1 min) → 95% (35 min). At the end of the HPLC column, a quartz capillary (length 8 cm, 25 μm i.d., and 365 μm o.d.) was used to connect the HPLC and a flexible tube (Pharmed BPT, 250 μm i.d.). The other end of the flexible tube was inserted into the microfluidic chip inlet A (as shown in Figure SI-1, Supporting Information. Dimethyl carbonate (DMC) was employed as a carrying organic phase, which is miscible with acetonitrile and dissolved in water at 2.9% in weight. The oil phase of DMC was injected by multichannel syringe pumps (LONGER Apparatus, Shanghai) from inlet B at a flow rate of 200 μL/h. A photo of the microfluidic-ESI-MS platform is shown in Figure SI-2 of Supporting Information. A self-improved MS ion source was used. The chip was adhered to the three-dimensional platform, and high voltage was applied on the end of the stainless capillary for ESI-MS identification. All standard samples were
dissolved in a solution containing 50% ACN/49.9% water/0.1% FA and injected by syringe pumps at inlet A at a flow rate of 120 μL/h. ESI-MS/MS Identification. LTQ MS (Thermo Corporation, USA) was run under data-dependent mode, the spray voltage was 1.9 kV, the capillary temperature was 220 °C, and the injection time was set to 50 ms. The first mass spectrometry stage was set to full scan, and the mass range was m/z 350− 1800. The ten highest strength ions were selected for the second stage mass spectrometry, and the minimum ion strength for the MS2 was 5000. The energy of CID was adjustable according to the size of peptide, dynamic exclusion was used, and the repeat duration time was 30 s. All the solvent ions were set in the reject list table and would not be selected for the MS2. Data Processing. The data were searched in the Swissport database using Bioworks software. Trypsin was set as the enzyme for database searching, peptides were searched using fully tryptic cleavage constraints, and up to two missed internal sites were allowed for tryptic digestion. The parent ion’s tolerance was 2.0 amu. The fragment ion’s tolerance was set to 1 amu, B and Y ions were used to calculate the series, and the modification was set to methionine (+15.9994 Da). According to the filtrate criterion of SEQUEST,35 the identification of peptides was reliable when its confidence was more than 95%. The Xcorr Vs charges were 3.0 > (+4) > 2.5 (+3), > 2.0 (+2), > 1.50 (+1), ΔCn > 0.1.
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RESULTS AND DISSCUSION The microfluidic device was fabricated by a soft-lithography technique using polydimethylsiloxane (PDMS) as the base material to form microchannels with a height of 100 μm. The general design of the device and schematic illustration of the droplet-based protocol are shown in Figure SI-1 (Supporting Information) and Scheme 1. A covalent surface modification Scheme 1. General Design of the Device and Schematic Illustration of the Droplet-Based Protocol
(Aquapel modification) was used to obtain stable hydrophobic coatings on PDMS microchannel surfaces,34 which tends to form water-in-oil droplets and can effectively avoid sample adsorption. By introducing DMC and sample solution into the microchannels, uniform water-in-oil (w/o) droplets were generated at the confluence. The volumes of produced droplets decreased when droplets traveled through the microchannel because water and ACN dissolved into DMC. In this way, samples were in situ concentrated within the shrunken droplets and consequently detected by mass spectrometry. 9618
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Figure 1. (a) The fluorescence signals corresponding to different concentration times within the microchip. The fluorescence signals of FITClabeled peptides (Pep I) in droplets after (b) 0.14 s, (c) 20 s, and (d) 2 min concentration. The inserts are the corresponding images of a droplet taken by a microscope coupled with a digital camera (scale bar 100 μm).
Figure 2. The base peak chromatography of peptides (Pep II, 20 nM) by the integration of the microfluidic chip and ESI-MS/MS (a) with online concentration and (c) without concentration. (b) The mass spectrum of peptides at RT = 1.25 min from spectrum a. (d) The mass spectrum of peptides at RT = 0.48 min from spectrum c.
induced fluorescence (LIF) detector. Figure 1a displays the fluorescence signals detected at different locations in the microfluidic channels corresponding to different concentration times. The fluorescence signal of the initial droplet (10 μg/mL) was 0.0065 V, while after a 20 s and 2 min concentration in the microchannel, the signals increased to 0.035 and 0.08 V,
To explore the possibility of this platform for concentrating analytes, a peptide solution (containing 50% acetonitrile and 50% water) of Lys-Cys-Asp-Ile-Cys-Thr-Asp-Glu-Tyr (KCDICTDEY, PepI) labeled with fluorescein isothiocyanate (FITC) was used as analyte for the experiment. The fluorescence signals of droplets were measured by the laser9619
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respectively. The fluorescence signal was enhanced more than 10 times. The inserted images in Figure 1b−d show the expected droplet shrinkage during this process. The significant enhancement of fluorescence signals can be ascribed to two factors. First, the decrease in droplet volume during shrinkage led to the concentration increase. Second, the fluorescence signals were affected by the solvents. The fluorescence signals of the FITC-labeled peptide in different solvents (acetonitrile concentration from 0% to 80%) were measured and are shown in Figure SI-3 of Supporting Information. There are enhanced fluorescence signals for the lower acetonitrile concentration, and the signals were maximally enhanced 4−5 times when the acetonitrile concentration was decreased (from 50% to 0%). Overall, this may not be a good method for quantification. On the other hand, no obvious fluorescence signal was observed in the DMC phase at any position of the microchannel, indicating that only the solvents diffused into the organic phase while the peptides stayed in the droplets for enrichment. Given that peptides contain charges and polar groups, their partition coefficients in most organic phases are negligible. Thus, these peptide molecules cannot dissolve into the oil phase as the solvent molecules exit the droplet.32 The above results show that this method can effectively concentrate the analytes. ESI-MS has been developed as an attractive technique for the analysis of segmented flows.36−40 In proteomics research, complex proteins are enzymatically digested, separated by RPLC, and identified by ESI-MS/MS, which is one of the most important protocols for biomarker discovery. Although many peptides at very low concentrations can be separated, without enrichment it is difficult to detect them successfully with satisfactory signal-to-noise ratios. To evaluate the concentration efficiency of this approach for ESI-MS analysis of lowconcentration analytes, a peptide (CYIQNCPRG, MW 1050.22, PI 8.06, Pep II) was used as a sample model. The electrospray voltage was optimized with a series of Pep II samples. At a voltage of 1.9 kV, the total ion current (TIC) graph (Figure SI-4 of Supporting Information) revealed a higher signal of peptide while no interferential signal from DMC was observed in this system. Thus, 1.9 kV was applied for the following experiments. A 20 nM Pep II solution and DMC with flow rates of 120 and 200 μL/h, respectively, were pumped into the microchannels to form droplets, where the peptides were online concentrated and subsequently identified by ESI-MS at the end of the channel. Two well-resolved peaks of Pep II corresponding to the single charged (m/z 1051.55) and double charged (m/z 526.36) signals were clearly observed (Figure 2b) with a high intensity of 105, which was theoretically enough for the identification with ESI-MSn. For comparison, only a 20 nM Pep II solution was pumped into the microchannel without enrichment for ESI-MS detection, and no obvious signal could be observed in the mass spectrum (as shown in Figure 2d). The significant improvement obtained in the mass spectrometry may be induced by two factors. First, an enhancement of concentration results from the droplet volume decrease. Second, the water−oil interface also plays an important role in the signal enhancement. Previous reports have investigated this issue. According to Chiu’s research,32 the solute molecules accumulate along the W/O interface, which can hinder further water diffusion. In Kennedy’s work, the interface phenomenon was also referenced.40 At present, a clear mechanism has not yet been reported to explain the interface effect on the mass spectrometry. The results show that this droplet-based protocol has a high efficiency and good
compatibility for the enrichment and identification of peptides at low concentrations with ESI-MSn. In general proteomics research, the maximum concentration of acetonitrile was 80% for elution buffer.41 To test its universal feasibility, the acetonitrile concentration of the sample solution was altered accordingly. Uniform droplets could be successfully formed when the acetonitrile concentration ranged from 0% to 80% (see Figure SI-5 of Supporting Information), respectively. No obvious changes were observed in the generation frequency of droplets; however, the size of droplet decreased with a higher acetonitrile concentration, which can be attributed to two factors. First, the fluidic dynamics varied with the changed acetonitrile concentrations. Second, with higher concentration, acetonitrile diffused into DMC more quickly and the partition equilibrium state was achieved faster. Furthermore, the droplet-based approach was applied for analyzing the peptide mixture to demonstrate its general enrichment capability without cross-contamination and discrimination. Herein, BSA tryptic digests (20 fM) were separated by RPLC and the effluents were compartmentalized to droplets with DMC. Then the separated peptides were concentrated when proceeding through the microchannel and analyzed by ESI-MS/MS (shown in Figure 3 and Figures SI-6 and SI-7 of Supporting Information). In Figure 3a,b, the graphs have a small difference in peak position and relative abundance. Although the droplet roughly retained the separation ability,42 the effluent flow through the microchip could lead to some peak banding in the chromatogram. Moreover, the diverse enhancement factor of different peptides may induce relative signal intensity variation. There are differences among the peptides to accept the influence of the electrospray. The absolute signal intensity (5.85 × 107) in Figure 3b was far above the intensity (5.59 × 105) in Figure 3a. Although the relative abundances of the peaks (from t = 11 min to t = 17 min) became low in Figure 3b, their absolute intensities were greatly enhanced. For example, the signal intensity in Figure 3a at t = 14.71 min was about 2.8 × 105; however, the signal intensity in Figure 3b at t = 13.77 and 17.78 min was 2.52 × 106 and 7.24 × 106, respectively. After the online enrichment, a total of 22 peptides were successfully identified corresponding to an amino acid coverage of 38.2% (Table SI-1, Supporting Information). Nevertheless, only seven peptides were identified, and the coverage was 10.2% without enrichment (Table SI-2, Supporting Information). When the sample concentration was down to 10 fM, six peptides were still identified after the online enrichment (Table SI-3, Supporting Information). However, only one peptide could be observed without enrichment, leading to a failed identification (Table SI-4, Supporting Information). All compared results without enrichment were obtained by sequential HPLC separation, effluent flowing through the microfluidic channel, and ESI-MSn detection. Figure SI-8 (Supporting Information) showed the blank sample (no sample; just the HPLC mobile phase was compartmentalized into droplets with DMC and analyzed by ESI-MS/MS), and there was little interference with the sample signals. These results indicate that such a microfluidic dropletbased device can be a general protocol for efficient enrichment of peptide mixtures in low abundance.
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CONCLUSION In summary, a facile online enrichment protocol has been proposed based on microfluidic droplets acting as an interface between the separation system (such as HPLC) and the 9620
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universal, shows no discrimination, and is easily coupled with other separation and detection approaches. This protocol could open a new window for the enrichment of low-abundance molecules in a large scale and further application in the field of biomarker discovery.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Fax: (+86) 21-6564-1740. *E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. J. Ji and L. Nie contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by NSFC (20925517, 21175028, 21105014, 21375022), 863 (2012AA020202) and MARC2012D002.
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REFERENCES
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Figure 3. (a) The base peak chromatography of HPLC-separated BSA digests (20 fM) identified with ESI-MS/MS directly. (b) The base peak chromatography of HPLC-separated BSA digests (20 fM) compartmentalized with DMC for concentration and identified with ESI-MS/MS. (c) The mass spectrum of peptides from a fraction of BSA digests eluted at RT = 21.50 min. The insert is the MS/MS spectrum of a parent peak at m/z 653.0 with a sequence of HLVDEPQNLIK.
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