Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide

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Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres to Achieve Automated Proteome Quantification Huiming Yuan, Shen Zhang, Baofeng Zhao, Yejing Weng, Xudong Zhu, Senwu Li, Lihua Zhang, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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Analytical Chemistry

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres to Achieve Automated Proteome Quantification

Huiming Yuan†#, Shen Zhang†‡#, Baofeng Zhao†, Yejing Weng†‡, Xudong Zhu†‡, Shenwu Li†‡, Lihua Zhang*†,Yukui Zhang†

†

CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National

Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; ‡

#

University of Chinese Academy of Sciences, Beijing 100049, China. These authors contributed to this article equally.

* To whom correspondence should be addressed. Prof. Lihua Zhang: phone, +86-411-84379710; fax, +86-411-84379720; E-mail, [email protected].

1

ACS Paragon Plus Environment


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ABSTRACT Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultra-performance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the on-line integration with nanoHPLC-ESI MS/MS. Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility and throughput for proteome quantification.

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INTRODUCTION The accurate quantification of proteins in biological systems is of great significance for discovering proteins with important functions [1]. Among various proteome quantification methods, proteolytic 18O labeling is one of the commonly applied strategies [2], by which the isotopically labeled tag is enzymatically incorporated into the peptide C-terminus, facilitating the quantification of all types of proteomic samples. Furthermore, since protein digestion and peptide 18O labeling could be achieved in H218O without adding extra labeling reagents, the secondary reactions inherent to other chemical labeling strategies, such as dimethyl labeling [3], iTRAQ [4], TMT [5] and ICAT [6] can be avoided. Therefore, in recent years, this strategy has been widely applied into relative proteome quantification [7], de novo sequencing [8], posttranslational modification analysis [9], cross-linking chemistry [10] and multiple reaction monitoring based absolute proteome quantification [11]. The main challenges of the direct 18O labeling methods are the incomplete labeling and back exchange from

18

O to

16

O [12]. Although incomplete labeling has been addressed by

decoupling digestion and labeling [13], in which proteolytic

18

O labeling is performed after

digestion, multi-step sample preparation not only increases the analysis time but also leads to sample loss, making the analysis of trace biological samples difficult. In addition, many efforts have been devoted to solve the back-exchange from 18O to 16O [14-15], and to date, the most effective method is the use of immobilized protease. However, limited by the insufficient amount of immobilized trypsin on matrices [16], the digestion and labeling efficiency still needs to be improved. Furthermore, the nonspecific binding of peptides on matrices might also result in unavoidable sample loss [17]. Therefore, it is imperative to develop a new method to achieve the simultaneous protein digestion and 18O labeling with high efficiency, low non-specific adsorption, and ignorable back-exchange. To achieve this goal, immobilized enzymatic reactors (IMERs) with large specific surface area and good hydrophilicity might be the favorite solution. Graphene oxide (GO), which possesses various polar moieties, such as hydroxy, epoxy, and carboxy groups, and high specific surface area, has exhibited great potential in the fields such as nanocatalysts [18], sensors [19], and sorbents [20-22]. Recently, GO nanomaterials have been widely applied as sorbents for biological [21, 22] and environmental [23] sample treatment. However, IMERs with GO 3



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hybrid microparticles as the matrix to achieve the on-line protein digestion and isotopic labeling simultaneously has not been developed. In our recent study, we developed a novel ultra-performance IMER (upIMER) by immoblizing trypsin on the self-assembled GO nanosheets onto polyetherimide (PEI)-modified acrylic

polymer

microspheres,

and

further

integrated

such

an

IMER

with

nanoLC-ESI-MS/MS to achieve fully automated relative quantification of proteomes based on 18

O labeling strategy. All the results demonstrated that our developed approaches should pro-

vide new opportunities for achieving proteome quantification with high accuracy, reproducibility, coverage and throughput.

EXPERIMENTAL SECTION Sample preparation Protein extracts from Escherichia coli (E. coli), hepatocarcinoma ascites syngeneic cells with high (Hca-F) and low (Hca-P) lymph node metastasis rates were denatured by heating at 90°C for 10 min. After being cooled to room temperature, the denatured proteins were reduced with 10 mM DTT at 56°C for 1.5 h and alkylated in the dark at room temperature for 40 min. For 18O labeling, all buffers and reaction solutions were made with 18O-water instead of 16O-water. Preparation and characterization of GO@PEI@polymer particles (See supporting information) Preparation of the IMER and Trypsin immobilization (See supporting information)

Integrated quantitative analysis On-line protein digestion and 18O labeling

The protein extracts from E. coli and Hca-P prepared with H216O were digested by the IMER at a flow rate of 1 µL/min at 37°C in 50 mM NH4HCO3 buffer (pH 7.8); the resulting peptides were captured by a C18 precolumn (150 µm i.d×3 cm). After the IMER was equilibrated with NH4HCO3 (pH 7.8) prepared with H218O for 10 min, the same aliquots of protein extracts from E. coli and Hca-F dissolved in 50 mM NH4HCO3 (dissolved in 18O-water, pH 4


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7.8) were then digested and 18O labeled by the IMER under the same conditions as described above. The 18O-labeled peptides were captured by the same C18 precolumn and analyzed by either the nano-LC/MS system or 2D-SCX-nano-RPLC/MS system. For comparison, the protein extracts from E. coli were also digested and labeled by the in-solution protocol (See Supporting information). With a ratio (H/L) of 1:1, E. coli was digested with the same amount and analyzed by nano-LC/MS. Peptide separation

For quantitative analysis of H/L-labeled E. coli extracts, the peptides were separated by a C18 capillary column (75 µm i.d. ×150 mm, 5 µm, 100 Å) at a flow rate of 300 nL/min. The mobile phases included H2O containing 2% ACN and 0.1% formic acid (A) and ACN containing 2% H2O and 0.1% formic acid (B). The linear gradients were set as follows: 0% (0 min) → 10% (10 min) →35% (100 min) → 80% (110 min) → 80% (120 min). After each nano-RPLC separation, the column was equilibrated with the initial mobile phase for 20 min. For the quantitative analysis of H/L-labeled Hca-F and Hca-P, the peptides were separated by 2D-SCX-nanoRPLC. A 16 µg sample was digested on-line and labeled by the IMER, and the digests were captured by a RP precolumn (150 µm i.d.× 3 cm). After being desalted by 0.1% FA, the peptides were transferred to an SCX column with elution by 80% ACN containing 0.1% FA for 10 min. The buffers for SCX separation were H2O containing 0.1% formic acid and 1,000 mM ammonium acetate adjusted to pH 3.5 with FA. Eight salt steps—0, 50, 100, 200, 300, 400, 500, and 1,000 mM—were applied, and the peptides were eluted stepwise by 100 µL of each elution solvent. The resulting peptides were then captured and separated by nano-RPLC under the same gradient as described above.

MS detection For LTQ-orbitrap velos-MS analysis, if not stated otherwise, the spray voltage of the MS was set to 2.2 KV, and the temperature of the ion transfer capillary was 200°C. The MS/MS collision energy was set to 35%. During the nano-RPLC-MS/MS analysis, the effluents were sprayed directly into the ESI source using a commercial interface. All MS and MS/MS spectra were acquired in the data-dependent mode, by which the MS acquisition with a mass range of m/z 300-1800 was automatically switched to MS/MS acquisition with the automated control of Xcalibur software. The 15 most intense ions from the full scan were selected for fragmen5



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tation via collision-induced dissociation (CID) in the LTQ. The dynamic exclusion function was set as follows: repeat count: 1, repeat duration: 40 s, and exclusion duration: 40 s.

RESULTS AND DISCUSSION Fully automated proteome quantification is of great significance to improve the accuracy, reproducibility, coverage and throughput. Herein, with a novel kind of upIMER as an interface, an integrated platform was established, by which the protein digestion, 18O labeling, and nanoLC-ESI MS/MS analysis could be online achieved. Preparation and Characterization of upIMER As shown in Figure 1a, acrylic polymer microspheres with amino groups were first modified with PEI by with glutaraldehyde as the space arm. Compared to bare microparticles, the significant increase of zeta potential from 4.8 to 29.2 mV, measured by dynamic light scattering (DLS), and nitrogen atoms percentage increased from 2.89% to 5.37%, obtained by X-ray photoelectron spectroscopy (XPS), demonstrated the successful modification of PEI (Table S-1 and S-2). Subsequently, GO was assembled on polymer@PEI microparticles through electrostatic interaction, confirmed by transmission electron spectroscopy (TEM) images (shown in Fig. S-1). Finally, after polymer@PEI@GO microparticles were packed into a 7 cm×250 µm i.d. capillary, trypsin was immobilized by electrostatic interaction to prepare the upIMER. To determine the enzyme immobilization capacity, a 2 mg/mL trypsin (1 mL) solution was pumped through the capillary (1 cm×250 µm i.d.) packed with polymer@PEI@GO hybrid microparticles. The concentration of residual trypsin in the flow through was analyzed by BCA. The amount of immobilized trypsin on the microreactor was determined to be 19.8 µg/cm microreactor, almost 8 times higher than that obtained by previous reported acrylic polymer particles based IMER [24], attributed to the large surface area of GO. Evaluation on Simultaneous Digestion and 18O Labeling by the upIMER With BSA as the sample, we investigated the capacity of upIMER for the simultaneous digestion and 18O labeling. As shown in Fig. S-2, the obtained sequence coverage (60%) was similar to that obtained with traditional in-solution protocols (59%, decoupling digestion and labeling). However, the labeling efficiency was increased from 93% to 98.5%, and the incu6


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bation time was shortened from 38 h to 2.5 min (Table S-3). Moreover, the comparison with other microparticle-based IMERs [25, 26] demonstrated that the labeling efficiency of 18O by upIMER could be improved from 80% to over 98% within the same incubation time (Fig. S-3), which should be attributed to the large amount of immobilized trypsin on polymer@PEI@GO. Besides labeling efficiency, another important issue for IMERs based proteome quantification is the incomplete digestion [27, 28]. Herein, to study the effect of missing cleavage by upIMER on proteome quantification, two identical aliquots of Escherichia coli (E. coli) extracts were digested in 50 mM NH4HCO3 (pH 7.8), respectively prepared with H218O and H216O. With the heavy/light (H/L) ratio as 1:1, the mixed digests were analyzed by nano-RPLC-MS/MS. Through database searching, 1,141 peptides, corresoponding to 425 proteins were quantified. Of all quantified peptides, only 8% are of one or more missed cleavage sites, considerably less than those obtained by in-solution overnight digestion protocols (15- 20%) and other previously reported IMERs (20-30%) [29, 30], which should be contributed to the high digestion efficiency of our prepared upIMER. As shown in Fig. S-4a, the peptides with missed cleavage sites had ignorable effects on quantitative accuracy and precision. Furthermore, as shown in Fig. S-4b, almost all proteins (98%) were quantified with log2 ratios (H/L) ranging from -1 to 1, with average CV as 17.8% (n=3), approximately half of that achieved by the decoupling ultrasound-immobilized trypsin digestion and 18O labeling method [31]. Furthermore, we investigated the carryover of peptides on the upIMER. It could be observed from Fig. S-5 that the ignorable light labeled peptides were found in the heavy labeled fraction, and the average ratio of L/H labeled peptides was 3.97%±0.26% (n=3), demonstrating the low peptide carryover of upIMER, which should be contributed by the good hydrophilicity of the matrix with substantial GO and PEI modification on polymer particles. For free enzyme catalyzed 18O labeling, the back exchange from 18O to 16O is inevitable [32], which would result in the decreased quantitative accuracy. However, it could be most readily avoided by immobilized protease, because after digestion, the labeled peptide could be easily separated from protease, avoiding the enzyme catalyzed back exchange. In our experiments, the average 16O/18O abundance ratios of 6 most intense peptides from BSA were cal7



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culated. The results showed that compared to those in 18O-water, there was no obvious difference in the

16

O/18O abundance ratios of these peptides redissolved in

16

O-water (Fig.S-6),

demonstrating that the back-exchange occurred by upIMER could be ignored. The lifetime of the upIMER was also studied by the digestion and O18 labeling of 0.1 mg/mL BSA at the flow rate of 1 µL/min in consecutive 10 days (100 runs), as shown in Fig S-7, BSA could be still digested and labeled sufficiently by the IMER even after 10 d run, with the obtained sequence coverage (61.6%±2.3% vs. 60.6%±2.5%, n=10) and labeling efficiency (98%±0.3% vs.97%±0.5%, n=10), comparable to the freshly prepared one. These results demonstrated that such an IMER could endure long time usage. Furthermore, compared to off-line protocols, such an IMER have several advantages in reducing the cost of analysis (1) Due to high enzymatic activity of the IMER, the digestion and

18

O labeling time was greatly reduced, which significantly improved the analysis

throughput, thus reduced the time cost per analysis. (2) The IMER could endure long-time usage, which could reduce the experimental cost. (3) The IMER could be online integrated with nanoHPLC-ESI-MS/MS system, which could be operated automatically, which is beneficial to save the labor cost. Integrated platform for automated proteome quantification Based on such an upIMER, an integrated platform, combining on-line protein digestion, 18

O labeling and peptide analysis by nano-RPLC-MS/MS was established. To evaluate the

performance for automated proteome quantification, E. coli extracts with 500 ng, 250 ng and 125 ng starting materials were respectively quantified with the H/L isotopically labeled ratios as 1. Compared to traditional off-line protocols, not only the number of commonly quantified proteins among three replicate runs was increased 1.7, 2.3 and 4.3-fold (Fig. 2a), but also the total analysis time, was shortened from 40 h to 2 h. With the decreasing of starting sample amount, the superiority on the quantification accuracy and precision by the integrated platform was more obvious (Fig.2b), contributed by automated operation, with the improved sample preparation reproducibility and decreased risk for sample loss.

Large Scale Proteome quantification To achieve large-scale proteome quantification, the upIMER was online coupled with 2D-SCX-nanoRPLC-MS/MS (Figure 1b), and applied in the relative quantitative analysis of 8


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hepatocarcinoma ascites syngeneic cells with high (Hca-F) and low (Hca-P) lymph node metastasis rates. A total of 2956±42 (n=3) non-redundant proteins were successfully quantified. Proteins quantified in all three replicates were processed using Benjamini-Hochberg (BH) FDR estimations, and those passed the 1% BH-FDR threshold were retained in the volcano plot (Fig. S-8a). Using two-fold differences as the cutoffs, in total, 69 proteins were considered to be differentially expressed, of which 46 were up-regulated and 23 were down-regulated in Hca-F (Figure S-8a and Table S-5). As high as 46.4% of the 69 differentially expressed proteins were reported as cancer metastasis associated proteins (Table S-4). For example, Histone H3.3 (H3f3a), involved in the structure of chromatin in eukaryotic cells, important for development, transcription and chromosome segregation, showed the largest increase (9.16-fold) in Hca-F; which was proved to promote lung cancer cell migration by activating metastasis-related genes and related with lung cancer progression recently [35]. The adaptor protein SORBS1 also known as CAP/ponsin, showing largely decrease in Hca-F, was also proved to suppress tumor metastasis by inhibiting the epithelial- to- mesenchymal (EMT) process recently[36]. Gene ontology (GO) analysis (Figure S-8b and Table S-5) suggested that many proteins involving in the important biological processes, including RNA processing, protein folding and chromatin remolding, were significantly changed, among which RNA processing has been regarded as a crucial step controlling gene expression, and its alteration is related to the acquisition of metastatic potential [37]. Furthermore, the development and progression of cancer have been attributed to independent or combined genetic and epigenetic events [38], both of which increased in our results. In contrast, protein folding and nuclear transport processes decreased dramatically in our results. The shuttling of proteins between nuclear and cytoplasm is essential for regulating the cell cycle and proliferation. Dysfunction of nuclear transport processes may affect many important cellular processes, including cancer cell metastasis [39].

CONCLUSIONS

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In summary, a novel kind of ultra-performance IMER was developed with polymer@PEI@GO hybrid microparticles as the trypsin-immobilized matrix, and successfully online coupled with nano-LC-MS/MS, to establish a fully automated platform for large-scale relative proteome quantification. Compared to off-line protocols, the integrated platform demonstrated the superiority of improved accuracy, precision, coverage and throughput, especially in the quantitative analysis of trace proteomic samples. Moreover, such a platform can be also extended to achieve other chemical labeling based proteome quantification such as dimethyl labeling, demonstrating the wide applicability in proteome analysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Detailed information on materials and reagents, protein extraction, preparation and characterization of GO@PEI@polymer particles, trypsin immobilization, evaluation of the peptide carryover on the IMER, trypsin digestion and

18

O labeling, MALDI-TOF MS

detection, data analysis, zeta potentials, element analysis, comparison of the labeling efficiencies obtained by the IMER and a traditional off-line protocol, TEM, MALDI-TOF MS spectra of BSA digests by the IMER in H218O and H216O, comparison of peptide labeling efficiencies obtained by different IMERs, evaluation of protein digestion by upIMER in proteome quantification, carryover of peptides on the IMER, comparison of the labeling efficiencies for 16

18

O-peptides produced by the IMER in

18

O-water

and re-dissolved in

O-water for 7 days, evaluation of durability of the IMER and differential proteomics

analysis of Hca-F and Hca-P.

AUTHOR INFORMATION Corresponding Author *Phone: +86-411-84379720. Fax: +86-411-84379720. E-mail: [email protected] Author Contributions 10


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#

HM.Y. and S.Z. contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2016YFA0501401), National Basic Research Program of China (2013CB911202), National Natural Science Foundation (91543201 and 21235005), Innovational Program from DICP,

CAS

(DICP

TMSR201601),

(QYZDY-SSW-SLH017)

and

National

CAS Key

Key

Project

Scientific

in

Instrument

Frontier and

Science

Equipment

Development Projects (2012YQ120044 -2).

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(39) Gravina, G. L.; Senapedis, W.; McCauley, D.; Baloglu, E; Shacham, E; Festuccia, C. J Hematol Oncol. 2014, 7, 85.

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Figure legends Figure 1. a) Preparation of polymer@PEI@GO composites based upIMER. b) Integrated platform for proteome quantification achieved by on-line protein digestion, 18O labeling, and 2D nano-LC-MS/MS analysis.

Figure 2. Comparison of a) the number and b) accuracy and precision of the quantified proteins (H/L=1) achieved by integrated platform and traditional off-line protocol.

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Figure 1.

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Figure 2.

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Graphic for Abstract:

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Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide

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