Coupling the Immobilized Trypsin Microreactor of Monolithic Capillary

A nanoliter trypsin-based monolithic capillary microreactor coupled with μRPLC−MS/MS system was used for Shotgun proteome analysis, 1578 unique pep...
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Coupling the Immobilized Trypsin Microreactor of Monolithic Capillary with µRPLC-MS/MS for Shotgun Proteome Analysis Shun Feng,†,‡,§ Mingliang Ye,†,§ Xiaogang Jiang,† Wenhai Jin,† and Hanfa Zou*,† National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, China, and College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi, Xinjiang 830046, China Received August 18, 2005

A nanoliter trypsin-based monolithic microreactor coupled with µRPLC-MS/MS was reported for shotgun proteome analysis. The proteins were rapidly digested by the microreactor, and the resulting protein digests were directly loaded onto a µRPLC column for separation followed with detection of the eluted peptides by tandem mass spectrometer. The digestion efficiency and stability of the microreactor was demonstrated by using bovine serum albumin as a model protein. When compared with an incubation time of more than 10 h by free trypsin in the conventional digestion approach, protein mixtures can be digested by the microreactor in several minutes. This system was applied to the analysis of the total cell lysate of Saccharomyces cerevisiae. After a Sequest database search, a total of 1578 unique peptides corresponding to 541 proteins were identified when 590 ng yeast protein was digested by the microreactor with an incubation time of only 1 min. Keywords: immobilized trypsin • microreactor • monolithic matrix • shotgun proteomics • µRPLC-MS/MS

Introduction The need to develop new technologies for shotgun proteome analysis has attracted a lot of attention in recent years. A series of new technologies on sample preparation,1-3 peptide or protein separation,4-8 mass spectrometry analysis, and data processing7-12 have been developed over past few years. A key element for sample preparation is the digestion of protein mixtures by a protease, in most cases, by trypsin. The protein mixture is often digested by free trypsin in solution. However, there are several disadvantages for this approach. To avoid the serious interference of fragments from autodigestion, the free trypsin concentration should be very low, typically 1/50 (w/w) of proteins, which results in long incubation times.13 Also, because of the autodigestion, a free enzyme often quickly loses its catalytic activity and cannot be used repeatedly. Besides, the automation of a digestion procedure using free trypsin is also difficult. Compared with free trypsin, the trypsin immobilized on substrates has the advantages of high digestion efficiency, high stability, and easy to automation. Trypsin has been immobilized on many kinds of substrates such as silica,14,15 polymeric particles,16-18 polymeric,19-23 and silica monolithic materials.24-26 Due to their properties concerning the fast mass transfer between the substance within the eluent and the active sites inside the skeleton, the monolithic materials are ideal supports for the immobilization of enzymes.27,28 Another significant advantage of the monolithic * To whom correspondence should be addressed. E-mail: hanfazou@ dicp.ac.cn. † The Chinese Academy of Sciences. ‡ Xinjiang University. § Authors contributed equally to this work.

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enzyme reactor is that it can be easily prepared with any size, which depends on the container where polymerization takes place. Frechet et al.22 immobilized trypsin on a porous polymer rod that was polymerized in a HPLC column and the system was used successfully for the digestion of proteins. Because convective transport of the liquid within the pores was dominant, the monolithic trypsin reactor provided higher apparent activity than the beads-packed reactor at high flow velocity. Ye et al.26 presented nanoliter enzymatic microreactors prepared in capillaries by polymerization of glycidyl methacrylate and ethylene dimethacrylate. The nanoliter trypsin microreactor was successfully coupled with capillary electrophoresis (CE) for on-line digestion followed by CE peptide mapping. There are a lot of publications on digestion of proteins by immobilized trypsin bioreactor followed with separation of digest by HPLC or CE.17,24,25,29-31 However, the application of these systems mostly limit to peptide mapping of some standard proteins. To the best of our knowledge, the immobilized trypsin reactor was not applied to digest complex protein mixture such as total cell lysate for shotgun proteomics. Recently, Calleri et al.24 reported the applicability of a trypsinbased monolithic reactor coupled on-line with LC-MS/MS system for protein digestion and variant identification in standard protein solutions and serum samples. In their work, a reactor with dimensions of 50 × 4.6 mm I.D. was used, which consumed a large volume of protein samples. Now the dominant platform for high throughput proteome analysis employs nanoscale reversed phase liquid chromatography coupled with nanospray tandem mass spectrometer (µRPLC-MS/MS). Very low volume of the injected sample solution requires the nanoscale of the immobilized enzyme microreactor to be 10.1021/pr0502727 CCC: $33.50

 2006 American Chemical Society

Coupling the Monolithic Capillary with mRPLC-MS/MS

coupled with µRPLC-MS/MS system. Because of the high mass transfer process and easy preparation, the monolithic substrate is a good candidate to prepare the immobilized microreactor for coupling on-line with µRPLC-MS/MS. We have developed an integration system by coupling a nanoscale of the immobilized trypsin microreactor with µRPLC-MS/MS for shotgun proteome analysis. This system was first evaluated by bovine serum albumin (BSA) and then applied to analyze yeast cell lysate.

Experimental Section Chemicals and Materials. Fused-silica capillaries were obtained from Yongnian Optic Fiber plant (Hebei, China) and Polymicro Technologies (Phoenix, AZ). Glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA), cyclohexanol, 1-dodecanol and sodiumcyanoboro hydride were obtained from Aldrich (Milwaukee, WI), γ-methacryloxypropyl trimethoxysilane (γ-MAPS), glutaraldehyde solution, sodium azide, iodoacetamide, and dithiothreitol (DTT) were from Sigma (St. Louis, MO). BSA and trypsin (from bovine pancreas, TPCK-treated) were also obtained from Sigma. Azobisisobutyronitrile (AIBN) was purchased from Shanghai Chemical Reagent Co. (Shanghai, China). Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, Milford, MA. USA). All other chemicals and solvents were of analytical or HPLC grade. Preparation of Monolithic Trypsin Microreactor. The preparation of monolithic trypsin microreactor is similar to that reported by Ye et al.,26 which involves two steps. The first step is to prepare a monolithic capillary. The capillary with inner diameter of 100 µm was filled with the polymerization reaction solution containing GMA, EDMA, cyclohexanol, dodecyl alcohol, and AIBN and the polymerization was allowed to proceed at 50 °C in a water bath for 12 h. The second step is to immobilize trypsin onto the surface of monolithic rod. The prepared monolithic column was aminated by 29% ammonium hydroxide solution and activated by 2.5% aqueous solution of glutaraldehyde. Trypsin was then coupled to the support by pumping borate buffer (0.1 M, pH 8.2) containing trypsin at 3 mg/mL through the column for 24 h at 4 °C. The trypsin immobilized monolithic capillary was treated with 25 mM sodium cyanoborohydride overnight. And finally, it was cut into 10 cm length with a volume of about 0.59 µL, and filled with 0.02% NaN3 solution, and stored at 4 °C until use. Protein Digestion. BSA (5 nmol) was dissolved in 0.5 mL 50 mM Tris-HCl (pH 8.1) buffer containing 8 M urea. To above solution, 10 µL of 100 mM dithiothreitol (DTT) was added, and the tube was incubated at 50 °C for 20 min. The solution was allowed to cool to room temperature, and then 10 µL of 100 mM iodoacetamide was added. The solution was incubated at room temperature in the dark for 20 min, and stored in the refrigerator before use. For the digestion with free trypsin, the denatured BSA was first diluted by 50 mM Tris-HCl buffer to reduce the concentration of urea. Then trypsin was added at a protein/enzyme ratio of 50:1 by weight, and the solution was incubated at 37 °C for 16 h. Finally, the solution was acidified by 0.1% formic acid. For the digestion with monolithic trypsin capillary microreactor with dimension of 10 cm × 100 µm I.D., the denatured BSA solution was first diluted to the required concentration with 50 mM Tris buffer (pH 8.1), and then it was delivered into the monolithic capillary microreactor by a syringe. The capillary microreactor was sealed with Tris-HCl buffer at both ends and incubated at room-temperature for 1, 10, 15, or 30 min according to the requirement of experiment.

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Figure 1. Schematic diagram of the integrated system by coupling immobilized enzyme capillary microreactor with µRPLCMS/MS.

Logarithmically growing cells from Saccharomyces cerevisiae were obtained by growing the cells to early log phase in YPD rich medium at 30 °C. The yeast protein extract was prepared by glass bead disruption followed by acetone precipitation. Proteins were then redissolved in 8 M urea with 50 mM TrisHCl buffer (pH 8.1). The amount of protein was determined by BCA assay. The yeast proteins (∼2 mg) were reduced, alkylated as above, and stored in refrigerator for future use. The digestion of yeast proteins by free trypsin and immobilized trypsin microreactor was performed with the same procedure as that of BSA. µRPLC-MS/MS System. A Finnigan surveyor MS pump (ThermoFinnigan, San Jose, CA) was used to deliver mobile phase. The pump flow rate was split by a cross to achieve a column flow rate of about 120 nL/min. For the capillary separation column, one end of the fused-silica capillary (Polymicro Technologies, Phoenix, AZ) was manually pulled to a fine point ∼5 µm with a flame torch. The columns were inhouse packed with C18 AQ particles (5 µm, 120 Å) from Michrom BioResources (Auburn, CA) using a pneumatic pump. The inner diameter for separation column is 75 µm and the packed length is about 12 cm. The mobile phase consisted of A, 0.1% formic acid in H2O, and B, 0.1% formic acid in acetonitrile. The µRPLC column was directly coupled to a LTQ linear ion trap mass spectrometer from ThermoFinnigan (San Jose, CA) with a nanospray source. The LTQ instrument was operated at positive ion mode. A voltage of 1.5 kV was applied to the cross. Coupling of Immobilized Trypsin Capillary Microreactor with µRPLC-MS/MS. The trypsin microreactor was manually coupled with µRPLC-MS/MS. The protein sample was digested in the capillary microreactor for 1, 10, 15, or 30 min, and then it was connected directly to the separation column as shown in Figure 1. The digest was brought to the separation column by pumping mobile phase containing 0.1% formic acid and 5% acetonitrile for 30 min. To avoid the contamination of mass spectrometer by the urea presented in the digest, the separation column was pulled about 2 cm away from the nanospray source during loading the sample. After the digest was loaded, the microreactor was detached from the flow line. Then gradient elution started for the separation. For samples digested in solution by free trypsin, the acidified tryptic digest was pumped into an open capillary (100 µm I.D.) by a syringe, and then the capillary was connected to the capillary separation column. Other procedures were the same as those for the system with coupling of microreactor to µRPLC-MS/MS. A 70 and 100 min gradient elution were applied for the separation of BSA digest and Yeast protein digest, respectively. For the detection of BSA digest, the mass spectrometer was set as 1 full MS scan followed by 4 MS/MS scans on the 4 most intense ions; for the detection of yeast protein digest, the instrument was set as 1 full MS scan followed by 7 MS/MS scans. Journal of Proteome Research • Vol. 5, No. 2, 2006 423

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Database Searching. The acquired MS/MS spectra were searched against protein database using the SEQUEST program. The database for BSA was created in house and the yeast database was downloaded from website [ftp://genome-ftp.stan ford.edu/yeast/data_download/sequence/genomic_sequence/ orf_protein/orf_trans.fasta.gz]. Trypsin was set as enzyme for database search. For the analysis of BSA, the Sequest results were only filtered by the cross-correlation score (Xcorr). The peptides were considered positively identified if the Xcorr were higher than 1.9 for singly charged peptide, 2.2 for doubly charged peptide, and 3.75 for triply charged peptides. For the analysis of yeast sample, two more criteria were used to filter the search results: ∆Cn cutoff values were g 0.1 and the SP rank of the peptides e 4.7,8

Results and Discussion Digestion Efficiency of the Microreactor. To avoid the serious interference of fragments originated from autodigestion of trypsin, the concentration of trypsin should be much lower than that of proteins in free enzyme digestion, which results in long incubation time. Typically, the ratio of protein to trypsin is 50:1. If the protein concentration is 1 mg/mL, then the concentration of trypsin should be at 0.02 mg/mL. It was reported that the concentration of trypsin in the immobilized enzyme reactor is as high as 7.75 mg/mL. Due to the high concentration of trypsin inside the reactor, the obtained activity of the immobilized trypsin reactor was 86 times higher than that of the 0.02 mg/mL free trypsin.25 For some simple proteins such as β-casein could be completely digested in less than 10 s.25 For some complex proteins, the digestion could also be completed in a few minutes if they were denatured prior to digestion.25 The high digestion efficiency of this kind of enzyme reactor may also attribute to the monolithic material, where convective mass transfer was dominant. Bovine serum albumin (BSA) was selected as a model protein to evaluate the performance of the enzyme reactor in this study. The denatured BSA was filled into a monolithic trypsin microreactor and incubated for 10 min. The concentration of BSA was 2 nmol/mL and the volume of the microreactor was about 0.59 µL, which was calculated according to the porosity of the polymer. The porosity was assumed to be 75% based on the percentage of porogenic solvent used in the reaction. Thus about 1.2 pmol of BSA was digested in the microreactor. The generated tryptic peptides were analyzed by µRPLC-MS/MS. Figure 2 shows the base peak chromatogram for separation of BSA tryptic digest with incubation time of 10 min. Many peaks were observed in the chromatogram, which further indicated that many peptide fragments were generated during digestion of BSA by the microreactor. Manually connecting the microreactor to µRPLC system represent a simple and convenient way to perform fast digest of proteins followed with shotgun proteome analysis. One significant advantage of this approach is that the entire digestion products in the reactor was directly loaded onto the separation column, and so no sample was lost during the procedure of sample transferring and injection. This system is very suitable for the analysis of minute amount of protein sample. Protein sequence coverage was typically used as an index to evaluate the performance of trypsin reactor.18,24,25,32 The sequence coverage of BSA was also used to characterize the performance of monolithic microreactor in this study. To investigate the repeatability of the digestion, 5 consecutive operations for BSA with incubation of 10 min and the analysis of resulted products by µRPLC-MS/MS were 424

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Figure 2. Base peak chromatogram for separation of BSA digest resulted from the immobilized trypsin capillary microreactor. Sample: 0.59 µL of 2 nmol/mL BSA in 50mM Tris-HCl buffer (pH 8.1); immobilized trypsin capillary microreactor, 10 cm × 100 µm I.D. with monolithic matrix; incubation time, 10 min. See experiment section for detail separation conditions. Table 1. Effect of Incubation Time on Sequence Coverage of BSA for the Digestion Conducted by Immobilized Trypsin Capillary Microreactor and Free Trypsina enzyme

incubation time

sequence coverage (%)

unique peptides

trypsin reactor trypsin reactor trypsin reactor free trypsin

1 min 10 min 30 min 16 h

74.96 80.89 78.58 79.41

54 57 56 61

a

See Figure 2 for detail conditions.

conducted. The average protein coverage of 77.6% with relative standard deviation (RSD) of 2.9% was obtained. This means the repeatability of this system is relatively good, and thus the most following measurements in this study were performed only with single operation on a newly prepared enzyme microreactor. As a comparison, the denatured BSA was also digested by free trypsin with incubation time of 16 h. Table 1 lists the µRPLC-MS/MS analysis results for digestion of BSA by microreactor and free enzyme with different incubation time. For the digestion conducted by immobilized trypsin microreactor, there is no significant change on protein coverage when the incubation time decreased from 30 to 10 min. The protein coverage of about 80% was obtained, which is very similar to that obtained in the digestion using free trypsin. Further decrease the incubation time to 1 min, the protein coverage only decreased to 75%. This means the digestion of BSA is still relatively efficient with digestion time of only 1 min, and is almost 3 orders of magnitude faster than that of free trypsin. Trypsin-based monolithic reactor with size of conventional HPLC column (50 × 4.6 mm I.D.) coupled on-line with a regularly sized RPLC column (100 × 2.1 mm I.D.) followed with an electrospray mass spectrometer (ESI-MS) detection was reported to analyze human serum albumin (HSA), and the obtained detection limit was about 151 pmol.24 Miniaturizing the separation column inner diameter significantly improves LC-ESI-MS sensitivity.33 To improve the detection sensitivity, the separation column with 75 µm I.D. was typically used in proteome analysis. Considering the small sample size, our fabricated nanoliter trypsin reactor is well suited with the

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Coupling the Monolithic Capillary with mRPLC-MS/MS Table 2. Detection Sensitivity of the Integrated System by Coupling Immobilized Trypsin Capillary Microreactor with µRPLC-MS/MS for the Identification of BSAa BSA concentration (nmol/mL)

injected amount

peptide count

unique peptides

sequence coverage (%)

2 0.1 0.05

1.2 pmol 60 fmol 30 fmol

330 212 68

59 51 24

77.43 71.33 35.75

a

The incubation time was 15 min. See Figure 2 for other conditions.

capillary separation column. Different amount of BSA was digested by a same coupling microreactor followed by the analysis of the digest by µRPLC-MS/MS. The obtained results were summarized in Table 2. When the amount of BSA decreased from 2 pmol to 60 fmol, the peptide count decreased from 339 to 212 and the number of unique peptides decreased from 59 to 51. The protein sequence coverage slightly decreased from 77.42% to 71.33%. When the amount of BSA further decreased to 30 fmol, the peptide count decreased dramatically from 212 to 68, and the protein sequence coverage decreased to 36%. If 36% is considered as lowest acceptable sequence coverage, then the detection limit of this system is about 30 fmol. By comparing above microreactor system with that by using bigger size of trypsin reactor coupling with bigger size of RPLC column, then the former system by coupling nanoliter trypsin microreactor with µRPLC-MS/MS is several orders of magnitude more sensitive than the latter system. Stability of the Microreactor. Because of the inaccessibility of internal peptide bonds, proteolytic digestion of globular proteins in their native form is a very slow process. To accelerate the proteolytic digestion process, these proteins need to be denatured before digestion. Typically, high concentrations of denaturing agents such as 8 M urea or 6 M guanidine hydrochloride were used to denature proteins. However, free trypsin can only tolerant low concentration of denaturing agents. Therefore, severalfold dilution of the sample is necessary to bring down the concentration of denaturing agents, which results in the dilution of the proteins and trypsin simultaneously and thereby decrease the digestion efficiency. However, immobilized trypsin may have higher tolerance on denaturing agent. Urea was typically used as denaturant to denature proteins. To test if the immobilized trypsin has higher tolerance on denaturing agent, the influence of urea concentration on the enzyme activities was investigated. Denatured BSA solutions with different urea concentration were pumped into capillary microreactor and digested for 15 min, then the digested product was analyzed by µRPLC-MS/MS. In comparison, same samples were also digested by free trypsin and analyzed by µRPLC-MS/MS. The obtained results were summarized in Table 3. For the sample digested by free trypsin, the sequence coverage of BSA decreased with the increase of urea concentration. This is consistent with the fact that the activity of free trypsin decreased with the increase of urea concentration. However, for the sample digested by the immobilized trypsin, the sequence coverage increased with the increase of the urea concentration. And the highest sequence coverage of 84.18% was observed at 8 M urea. This means that the immobilized trypsin still preserve high activity in high urea concentration. In other words, the immobilized trypsin have higher tolerance on urea. Because the concentration of urea has no significant influence on activity of the immobilized trypsin with relatively short incubation time, but the internal

Table 3. Effect of Urea Concentration in Sample Solution on Sequence Coverage of BSA for the Digestion Conducted by Immobilized Trypsin Capillary Microreactor and Free Trypsina peptide count

unique peptides

sequence coverage

urea concentration (M)

free trypsin

trypsin reactor

free trypsin

trypsin reactor

free trypsin (%)

trypsin reactor (%)

0.8 2 4 8

308 305 209 28

330 339 272 316

71 61 57 15

51 59 60 70

82.21 79.41 74.14 38.22

70.68 77.43 78.09 84.18

a The incubation time for free trypsin was 16 h, and that for trypsin microreactor was 15 min. See Figure 2 for other conditions.

peptide bonds are more accessible to the immobilized trypsin at high urea concentration which resulted in the higher digestion efficiency. The optimal pH for trypsin digestion is around 8.1. With the decrease of pH value the trypsin will gradually lose its activity. The optimal pH for reversed phase separation of peptides is less than pH 3.0 where trypsin will completely lose its activity. In this study, the microreactor was first filled with digestion buffer, 50 mM Tris-HCl (pH 8.1), to perform in column digestion. After digestion, the microreactor was connected to a µRPLC column, and the digest was flushed onto the separation column by the mobile phase containing 5% ACN, 0.1% formic acid (pH < 3.0). Therefore, the solvents for immobilized trypsin were switched from digestion buffer to loading mobile phase repeatedly if the microreactor was used multiple times. No significant change of BSA sequence coverage was observed when the microreactor was used more than 20 times. This means that the stability of the microreactor is quite good when the microreactor was subject to both acidic and basic conditions. This is consistent with the fact that trypsin can reversibly restore its activity when the pH value changes back to 8.1. Digestion of Yeast Protein Extract. Yeast protein extracts were often used as a complex protein mixture to evaluate the performance of proteome analysis technologies.34-38 To further demonstrate the applicability of the trypsin immobilized monolithic capillary microreactor in shotgun proteome analysis, it was applied to digest yeast protein extract followed by analysis of the resulted digest by µRPLC-MS/MS. About 590 nL yeast extract (1 µg/µL) was pumped into the capillary microreactor for digestion for 15 min, then the digest was directly loaded onto the separation column by elution mobile phase and separated by µRPLC with a 100 min gradient elution. Figure 3 (a) shows the base peak chromatogram acquired by LTQ linear ion-trap mass spectrometer. Numerous peaks were observed, which indicates that the protein mixture was efficiently digested in the capillary microreactor. The acquired MS/MS spectra were searched against a Saccharomyces cerevisiae sequence database using the SEQUEST program. The search results were filtered using the accepted reliable criteria of ∆Cn g 0.1, SP e 4, and Xcorr g 1.9, 2.2, 3.75 for singly, doubly, and triply charged peptides, respectively. A total of 1523 unique peptides were identified, corresponding to 550 distinct yeast proteins. Fifty percent of them (278 proteins) were identified with 2 or more unique tryptic peptides. To further demonstrate the high digestion efficiency of the capillary microreactor, the incubation time was further decreased to 1 min. Figure 3b shows base peak chromatogram for the separation of the resulted digest. After database search, a total of 1578 Journal of Proteome Research • Vol. 5, No. 2, 2006 425

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Figure 3. Base peak chromatogram for separation of yeast protein digest by immobilized trypsin capillary microreactor for incubation of (a) 15 min (b) 1 min and (c) free trypsin for incubation of 16 h. The digest of about 0.59 µg protein was analyzed by µRPLC-MS/MS. See Figure 2 for other conditions.

unique peptides from 541 proteins were identified. These results are almost identical to that with incubation time of 15 min. This means that the digestion of complex protein mixture in the capillary microreactor was very efficient. Considering 426

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only 0.59 µg yeast proteins was digested and analyzed, the detection sensitivity of this system was also very impressive. As a comparison, the same sample was also digested by free trypsin with incubation time of 16 h, and same amount of

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Coupling the Monolithic Capillary with mRPLC-MS/MS

Proteins of large size are more difficult to access the catalytic site of immobilized trypsin, because of the steric hindrance. It is of interest to see if there is any difference in digestion efficiency of free and immobilized trypsin on proteins with different sizes. Figure 4 (a) shows the molecular weight (MW) distribution of the identified proteins from the samples digested by free trypsin and immobilized trypsin. Of the 541 proteins identified from the sample digested by immobilized trypsin, 8.7% (47) of them have MW greater than 100 kDa. A slightly higher percentage of 11.1% (69 from 624) was observed for that of free trypsin. This indicates that digestion of proteins with high molecule weight by immobilized trypsin is slightly less efficient than that of free trypsin. The pI distributions of the proteins identified from both cases are also shown in Figure 4b. Same profiles were obtained which means that free and immobilized trypsin are equally efficient for digestion of proteins with different pI values.

Conclusion

Figure 4. Physicochemical characteristics of the identified yeast proteins by (a) molecular weight distribution and (b) pI value distribution.

digest was analyzed by µRPLC-MS/MS. The obtained base peak chromatogram is shown in Figure 3c. More peaks were observed by digestion of yeast protein extract with free enzyme than by the immobilized trypsin. After database search, 2099 unique peptides corresponding to 624 distinct proteins were identified. Fifty-five percent of the proteins were identified with 2 or more unique tryptic peptides. Compared with results obtained by capillary microreactor digestion with 1 min, 24.5% of more unique peptides were identified with sample digested by free trypsin. The lower number of peptides identified in the sample digested by enzyme microreactor may be caused an absence of retention of some hydrophilic peptides on the µRPLC column during the loading procedure. As we know for the separation of peptides by reversed-phase liquid chromatography, the peptide samples were typically acidified to neutralize the peptide’s carboxylic acid groups of peptides prior to injection to increase their hydrophobicity. However, the pH of the digest in the capillary microreactor was 8.1 that is the optimal pH for the digestion. When this digest was loaded directly onto the separation column, some hydrophilic peptides cannot be retained on the stationary phase, thereby passing through the separation column and being lost. Another possible reason is that the digestion of some proteins is not so complete because of the short contact time. Thus, only a few peptides were cleaved from the proteins. In theory, one unique peptide can unambiguously identify a protein. Therefore, the loss of some peptides may not seriously impact the number of identified proteins. Although the number of identified peptides increased by 24.5%, the number of identified proteins only increased by 13.3% for the sample digested by free trypsin. Considering the digestion time of only 1 min, the digestion efficiency of capillary microreactor with immobilized trypsin on monolithic matrix is much higher.

A capillary microreactor with immobilized trypsin monolithic matrix was prepared and used for rapid digestion of complex protein mixture for shotgun proteome analysis. It was found that the digestion of proteins by the capillary microreactor is very efficient due to the presence of high concentration of trypsin in the prepared microreactor, and the immobilized trypsin has higher tolerance with denaturing agent like urea. The efficiency of the digestion was further demonstrated by digestion of a real proteome sample, the yeast cell lysate. Compared to the digestion with free trypsin, the digestion time with the capillary microreactor decreased by 960 times from 16 h to 1 min, while the number of identified protein only decreased by 13.3% from 624 to 541 when 590 ng yeast proteins were digested and analyzed. The immobilized trypsin capillary microreactor was manually coupled with RPLC-MS/MS for proteome analysis in this work. The automation of this system is necessary in the future. Usually, the on-line protein digestion was conducted in flowthrough mode.17,18,24,25 Protein solution was pumped through the enzyme reactor at certain flow rate, and the generated proteolytic fragments were retained on a trap or separation column. The disadvantage of this mode is that the incubation time is controlled by the flow rate. If long incubation time is required to increase the digestion efficiency, then the flow rate should be set very low, which is time-consuming and not convenient. One advantage of monolithic column is its low back pressure. The monolithic capillary microreactor may be installed as the sample loop in the 6-port-injector. The protein solution is injected into sample loop, and kept for a certain time in static state to generate proteolytic fragments, and then they are eluted into separation column by switching the valve. This system may be easily automated by an autosampler. This is an optional scheme which we are developing for the automation of a proteome analysis platform based on microtrypsin-reactor.

Acknowledgment. Financial supports from the National Natural Sciences Foundation of China (No. 20327002), the China State Key Basic Research Program Grants (001CB510202, 2004CB520804 and 2005CB522701) and Knowledge Innovation program of DICP to H.Z. is gratefully acknowledged. Journal of Proteome Research • Vol. 5, No. 2, 2006 427

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