Chinese Chemical Letters  2015, Vol.26 Issue (09): 1068-1072   PDF    
Particulate capillary precolumns with double-end polymer monolithic frits for on-line peptide trapping and preconcentration
Si-Min Xiaa,b, Hui-Ming Yuanb, Zheng Liangb, Li-Hua Zhangb , Yu-Kui Zhangb    
a University of Chinese Academy of Science, Beijing 100039, China;
b National Chromatographic R & A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, China
Abstract: In this work, a novel kind of particulate capillary precolumns with double-end polymer monolithic frits has been developed. Firstly, the polymer monolithic frit at one end was prepared via photo-initiated polymerization of a mixture of lauryl methacrylate and ethyleneglycol dimethacrylate with 1-propanol and 1,4-butanediol as porogens and 2,2-dimethoxy-2-phenylacetophenone as a photo-initiator in UV transparent coating capillary (100 μm i.d.). Subsequently, C18 particles (5 μm, 100Å) were packed into the capillary, and sealed with the polymer monolithic frit at another end. To prevent the reaction of monomers and C18 particles, the packed C18 particles were masked during UV exposure. The loading capacity of such a precolumn was determined to be about 9 μg by frontal analysis with a synthetic peptide APGDRIYVHPF as amodel sample. Furthermore, two parallel precolumns were incorporated into a two-dimensional nano-liquid chromatography (2D nano-LC) system with dual capillary trap columns for peptide trapping and concentration. Compared to 2D nano-LC system with a single trap column, such two dimensional separations could be operated simultaneously to improve the analysis throughput. All these results demonstrated that such capillary precolumns with double frits would be promising for high-throughput proteome analysis.
Key words: Capillary precolumn     Double-end frits     Peptide trapping     2D nano-LC    
1. Introduction

For proteome analysis, multidimensional liquid chromatography- tandem mass spectrometry system (MDLC-MS/MS) has become one of the most popular and widely used approaches, as it provides high resolving power, versatility, sensitivity and automation [1, 2, 3, 4]. In conventional MDLC-MS/MS system, it is indispensable to combine precolumns with separation columns to achieve on-line sample trapping, preconcentration and remove interferents, thus result in the improved detection sensitivity in proteomic analyses [5] and improve the compatibility of different separation modes [6, 7, 8].

Various approaches have been developed to prepare capillary precolumns for nano-flow LC systems, including packed precolumns [9, 10], polymer and silica monolithic precolumns [11] and particle-entrapped precolumns by monoliths [5, 12]. Although monolithic or particle-entrapped monolithic precolumns are fritless, simplifying the column preparation, such columns suffer from the drawbacks, for example, polymer monolith could not endure high organic solvent, while silica monolith is prone to shrinking and cracking during column drying. Moreover, the loading capacity of monolithic precolumns is limited. Thus, particle-packed precolumns are preferable for proteome analysis [13, 14].

To prepare particle-packed precolumns, frits are required to retain the packed stationary phase [15, 16, 17, 18]. To reduce the void volume, most precolumns in nano-HPLC systems used in-capillary frits. To prepare frits with enough strength and high permeability, several methods are used for fabrication of in-capillary frits, such as sintering stationary phase beads inside the capillary [2, 19, 20]. However, by this method, not only the functionality of the stationary phase might be destroyed, leading to the non-specific adsorption of samples at frits, but also precolumns tend to break after the capillary coating is removed by heating [21, 22].

Herein, to solve these problems with photo-initiated polymerization, we successfully prepared C18 particle-packed capillary precolumns with C12 monolithic frits at both ends, and then further incorporated into a nano-RPLC-MS/MS system. With the decreased system dead volume by such precolumns, the separation efficiency of Escherichia coli (E. coli) proteome was improved.

2. Experimental 2.1. Material and reagents

Polyethylene glycol diacrylate (PEGDA), γ-methacryloxypropyltrimethoxysilane (γ-MAPS), lauryl metbacrylate (LMA), ethyleneglycol dimethacrylate (EGDMA), dithiothreitol (DTT), iodoacetamide (IAA), trypsin (bovinepancreas) and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Protease Inhibitor Cocktail Set I and acetonitrile (ACN, HPLC-grade) were bought from Merck (Darmstadt, Germany). 2, 2-Dimethoxy- 2-phenylacetophenone (DMPA) was from Acros Organics (Geel, Belgium). Luna C18 (2) and SCX silica particles (5 μm, 100 Å pore) were bought from Phenomenex (Torrance, CA, USA). Fused silica capillaries were from Ruifeng Chromatographic Device Sino Sumtech (Handan, China). UV transparent coating capillaries (100 μm i.d.) were obtained from Polymicro Technologies (Phoenix, AZ, USA). Water was purified by a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals and solvents were analytical grade.

2.2. The preparation of particulate capillary precolumn with doubleend polymer monolithic frits

As shown in Fig. 1, UV transparent coating capillaries (100 μm i.d.) were pretreated with γ-MAPS [23], and then filled with 2 cmof the polymerization solution containing 240 μL LMA, 160 μL EGDMA, 400 μL 1-propanol, 200 μL 1, 4-butanediol and 2 mg DMPA, finally sealed at both ends with rubber stoppers and exposed in the ultraviolet light (λ = 360 nm) for 5 min. Subsequently, the C12 monolith frit was washed with methanol to remove unreacted monomers, and C18 silica particles were packed into the capillary till the column length reached 1.5 cm. After being flushed with methanol at 20 MPa for 30 min, the inlet of the capillary was further filled with polymerization solution, followed by exposure in UV for 5 min with covering packed C18 silica particles. Finally, the prepared precolumn was washed with 1-propanol to remove unreacted monomers, and cut into 12 cm long (include 10.5 cm long monolithic frits).

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Fig. 1.Preparation of particulate capillary precolumn with double-end polymer monolithic frits.
2.3. Sample preparation 2.3.1. E. coli whole cell lysate proteins preparation

E. coli (Strain BLT 5403) grown on LB culture medium was cultured at 37 ℃ for 14 h, and then the mixture was centrifuged at 4000 × g for 10 min to precipitate cells. After washed with 1 × PBS for three times, the E. coli cells were lysed with 8 mol/L urea containing 1% (v/v) Protease Inhibitor Cocktail Set I with the ratio of 1:2 (m/v) followed by ultrasonication (Cole-Parmer, IL, USA) for 180 s. The resulting mixture was centrifuged at 20, 000 × g for 20 min, and the supernatant was collected as the soluble fraction of the extracted E. coli lysate proteins. Finally, the protein concentration was determined by Bradford assay.

2.3.2. Proteins digestion

BSA and proteins extracted from E. coli (1 mg/mL, 1 mL) were reduced by DTT and alkylated by N, N-dimethylformamide dimethyl acetal. The denatured proteins (1 mg/mL, 1 mL) were in-solution digested by adding trypsin into pretreated samples with the substrate-to-enzyme ratio of 50:1 (m/m), followed by incubation at 37 ℃ for 20 h. Finally, 2 μL formic acid (FA) was added into the solution to terminate the reaction.

The resulting digested samples were desalted with the following procedures: C18 trap column (4.6 mm i.d × 10 mm, Venusil XBP C18) connected to HPLC pump (Shimadzu Corporation, Japan) was first activated with 80% ACN containing 0.1% TFA, and then equilibrated with 2% ACN containing 0.1% TFA at the flow rate of 1 mL/min. Then samples were loaded on the trap column, and washed with 2% ACN containing 0.1% TFA at 1 mL/min to elute salts. Finally, peptides were eluted with 1 mL of 80% ACN, and then dried at low temperature using a Speed Vac Concentrator (Thermo- Fisher, San Jose, CA). The pellets were collected and stored in the refrigerator under -20 ℃ for further use. Without specific statement, all percentages represented volume percentage.

2.4. Binding capacity measurement of capillary precolumns

The binding capacity of the prepared capillary precolumn was determined by frontal analysis according to previous protocols [24]. Briefly, 1 mg/mL synthetic peptide (APGDRIYVHPF) was dissolved with buffer contained 2% (v/v) ACN 0.1% (v/v) formic acid as a model sample, and introduced into the caillary precolumn at a constant flow rate of 1 μL/min till the precolumn was saturated and detected by UV absorption signal. To estimate the dead time, in our experiment, 250 mmol/L thiourea water solution containing 0.1% FA and 2% ACN (v/v) was pumped through the column at the same flow rate.

2.5. Capillary precolumn with double-end frits for peptide trapping in nano-RPLC-MS/MS system

As shown in Fig. 2a, the capillary precolumn (0.1 mm × 120 mm) was directly connected to two ports of valve 2. The analytical capillary column (Luna C18, 0.075 mm × 150 mm) was also connected to valve 2 and the outlet was connected to MS through electrospray ionization (ESI) source. The syringe pump was used to deliver loading buffer (2% ACN/0.1% FA) at a flow rate of 1 μL/min. Samples were loaded onto the precolumn by using an autosampler and trapped on the head of precolumn. After the precolumn was flushed with the loading buffer for 10 min, the valve 2 was switched to connect the precolumn with capillary RPLC column, and the preconcentrated peptides were backflushed to the analytical column for separation.

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Fig. 2.Schematic diagram of HPLC–ESI–MS/MS system, (a) 1D LC–MS/MS system, (b) 2D HPLC–MS/MS system.

Binary solvents of A (2% ACN/0.1% FA) and B (98% ACN/0.1% FA) were used for peptide separation. Gradient elution for BSA digests was as follows: 0% B (0 min) - 5% B (5 min) - 40% B (35 min) - 80% B (40 min) - 80% B (50 min). Gradient elution for E. coli digests was as follows: 0% B (0 min) - 5% B (10 min) - 40% B (95 min) - 80% B (105 min) - 80% B (120 min). After each nano-RPLC separation, the column was equilibrated with the initial mobile phase.

For comparison, a commercial precolumn (C18, 0.3 mm × 5 mm, Dionex, Sunnyvale, CA) and homemade one-end frit capillary precolumn (0.1 mm × 120 mm, with polymer monolithic frit 2 cm and C18 particles 1.5 cm) were also used to trap samples in nano-RPLC-MS/MS system. To use the commercial precolumn, two 50 mm i.d. fused-silica capillaries of 7 cm length were used to connect the precolumn to the valve 2, to reach the minimum length required by the system. When single frit precolumn was used, the flow path of the system adjusted slightly: the connection line to port 1 in valve 2 was changed to port 6, to make sure that the buffer passed the precolumn only along one direction. Besides, the other conditions were as the same as those for double-end frits capillary precolumn system.

2.6. 2D-nano-SCX-dual capillary trap columns-RPLC-MS/MS for E. coli digests

As shown in Fig. 2b, the 2D-nano-SCX-dual capillary trap columns-RPLC-MS/MS System was constructed with double capillary precolumns (0.10 mm × 120 mm).

For analysis of E. coli digests, 10 μg sample was injected, and the buffers for SCX separation were 0.1% formic acid solution and 1000 mmol/L ammonium acetate adjusted to pH 3.0 with formic acid. Five salt steps, including 40 mmol/L, 80 mmol/L, 120 mmol/L, 250 mmol/L and 1000 mmol/L ammonium acetate were applied, and peptides were eluted stepwise by 20 μL of each elution solvent. The resulting peptides were in turn captured by precolumns with double-end polymer monolithic frits, and finally analyzed by nano-RPLC-ESI-MS/MS system, which was as same as that described above. Binary solvents of A (2% ACN/0.1% FA) and B (98% ACN/0.1% FA) were used for sample separation. The gradient was set as follows: 0% B (0 min) - 5% B (5 min) - 40% B (95 min) - 80% B (105 min) - 80% B (115 min). After each nano-RPLC separation, the column was equilibrated with the initial mobile phase for 10 min.

2.7. MS analysis

An LTQ XL ion trap mass spectrometer was used for ESI-MS/MS detection. The temperature of the ion transfer capillary was 200 ℃, and the spray voltage was 2.5 kV. The normalized collision energy was set at 35.0%. During nano-RPLC-MS/MS analysis, the effluents were sprayed directly into ESI source using a commercial interface. All MS and MS/MS spectra were acquired in the data-dependent mode, by which MS acquisition with the mass range of m/z 400- 1800 was automatically switched to MS/MS acquisition with the automated control of Xcalibur software. The 9 most intense ions from the full scan were selected to fragmentation via collisioninduced dissociation (CID) in the LTQ. The dynamic exclusion function was set as follows: repeat count 1, repeat duration 30 s, and exclusion duration 60 s.

2.8. Data analysis

All MS/MS spectra by nano-RPLC-ESI-MS/MS analysis in raw file were converted to mgf file by pXtract (version 1.0) [25], and searched against ecoli_REVERSE database using MASCOT (version 2.3.2). Mass tolerances for LTQ were set as 2 Da for parent ions and 1 Da for fragments. Peptides were searched using fully tryptic cleavages constraint, up to 2 missed cleavages. Cysteine residues were searched as a static modification of +57.02 Da, and methionine residues were searched as a variable modification of +15.99 Da. The MASCOT results were filtered by pBuild [25]. The filter parameters were: FDR ≤ 1%, distinct peptides ≥ 1.

3. Results and discussion 3.1. Evaluation of the capillary precolumns with double-end monolithic frits 3.1.1. Binding capacity of double-frit capillary precolumns

To avoid different retention behaviors of samples on frits of precolumns, herein, C12 polymer monolith, with reversed phase mechanism, similar to C18 particles [18], was prepared by photoinitiated polymerization as double-end frits of RP capillary precolumns.

In our experiment, a synthetic peptide, APGDRIYVHPF (MW: 1281.2), was used to evaluate the binding capacity of the capillary precolumn by the frontal analysis. As shown in Fig. 3, the void time, was estimated to be about 6.5 min with 250 mmol/L thiourea, and the elution time for the peptide with a concentration of 1 mg/mL was 15.5 min with the flow rate of 1 μL/min, the binding capacity of this precolumn was calculated to be 9 μg, much more than that for the uncovered one, which was attributed to the polymerized monomers in the uncovered precolumn covered the surface of the C18 particles, and hindered peptides adsorbing on the C18 resins.

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Fig. 3.Binding capacity of double-frit capillary precolumns by frontal analysis of synthetic peptide.
3.1.2. Permeability and stability of double-frit capillary precolumns for LC application

An ideal precolumn used for proteome analysis should not only offer enough binding capacity for peptides, but also appropriate permeability to decrease the backpressure of nano flow LC system. Therefore, we packed 1.5 cm long C18 particles in the precolumn to meet these requirements. However, to connect these parallel precolumns onto the 10-port valve, a reversed phase capillary precolumn with two 5 cm-length frits on both ends is necessary. Thus, the permeability of the C12 monolithic frit was crucial.

For capillary precolumn with double-end frits, the evaluation of column permeability was performed by pumping the solvent (2% ACN/0.1% FA) through, and then calculated by the following Eq. (1) [5].

where B0 is the column permeability; μ, the linear velocity; η, the viscosity; L, the column length; and ΔP is the back pressure.

The B0 value of such a precolumn was determined to be 8.94 × 10-14 m2, much lower than that of single-end precolumn with the same dimension. Benefited from the high mechanical strength of C12 polymer monolith, a 12 cm long precolumn, composed of 1.5 cm long packed C18 particle bed, and 10.5 long monolithic frits, could withstand up to 30 MPa pressure, higher than the commonly applied backpressure of nano-LC (20 MPa).

Furthermore, the reproducibility of capillary precolumns with double-end monolithic frits for nano-LC analysis was also evaluated by analyzing BSA digests (200 ng) for three times. The obtained sequence coverage of BSA was 87.3% ± 2.38% (n = 3). Furthermore, precolumns prepared in three batches were integrated with the nano-LC-MS/MS system to analyze the BSA digests respectively and the obtained average sequence 88.3% ± 1.31% (n = 3). These results indicate that the preparation of such precolumns with double-end monolithic frits is reproducible, which ensured robust operation in LC applications.

3.2. Application of the capillary precolumns with double-end monolithic frits

To evaluate the performance of such a precolumn, 300 ng E. coli digests was captured, and then analyzed by nano-LC-MS/MS systems. The obtained results were compared with those by singlefrit precolumn and commercial packed precolumn (Fig. S1 in Supporting information). As shown in Table 1, it could be seen that with the double-frit precolumn for peptide trapping, higher separation efficiency and peak capacity could be achieved due to lower extra-column effects, resulting in better protein identification. Furthermore, such a precolumn was also applied in the development of 2D-nano-SCX-dual capillary trap columns- RPLC-MS/MS system. To evaluate the performance of the 2D-LC- MS/MS system, 10 μg tryptic digests of E. coil extracts was analyzed. By database searching, a total of 1763 protein groups and 6286 unique peptides could be identified in three consecutive analyses, as shown in Fig. 4, among which the percentages of proteins and peptides identified at least 2 or 3 replicates were 58.87%, and 61.06%, which demonstrated that the 2D nano-LC-MS/MS platform with dual capillary precolumns had good performance of peptides separation for proteome study.

Table 1
Comparison of peak intensity, retention time (RT), and peak width at 1/2 height (W1/2) for five peptides in E. coli digests obtained with different precolumns.

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Fig. 4.Overlap of identified protein groups (a) and peptides (b) in three consecutive analyses.
4. Conclusion

The C18 packed capillary precolumns with monolithic C12 frits at both ends were successfully prepared by photo-initiated polymerization. Compared with commercial precolumn and single frit precolumn, the nano-LC system incorporating with the doublefrits precolumn had an obvious improvement on detection sensitivity and peak capacity by decreasing the system dead volume. Furthermore, such precolumns were also applied in multidimensional LC-MS/MS platform for E. coli proteome analysis, and all these results demonstrated that our developed precolumns would provide a promising tool for proteome analysis.

Acknowledgments

The authors are grateful for the financial support from National Basic Research Program of China (No. 2012CB910604), National Natural Science Foundation of China (No. 20935004), the Creative Research Group Project by NSFC (No. 21321064), and National High Technology Research and Development Program of China (No. 2012AA020202).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2015.05.042.

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