Article pubs.acs.org/ac
Trypsin Immobilization on Hairy Polymer Chains Hybrid Magnetic Nanoparticles for Ultra Fast, Highly Efficient Proteome Digestion, Facile 18O Labeling and Absolute Protein Quantification Weijie Qin, Zifeng Song, Chao Fan, Wanjun Zhang, Yun Cai, Yangjun Zhang,* and Xiaohong Qian* State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, No. 33 Life Science Park Road, Changping District, Beijing 102206, P. R. China S Supporting Information *
ABSTRACT: In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile 18O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using 18O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.
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labeling16,17 are the most widely adopted ones. Among these methods, 18O labeling, which introduces either +2- or +4-Da mass tags depending on the number of C-terminal oxygen atoms exchanged, is emerging as a powerful labeling strategy for quantitative proteomics applications, since it has several advantages over other methods including high labeling specificity, compatibility with almost any source of proteins and relatively lower price.18,19 In 18O labeling coupled with MRM strategy, protein quantification is achieved by quantifying the corresponding proteolytic peptides of the proteins using 18O labeled standard peptides as the reference, therefore complete protein proteolysis to peptides and 18O labeling are two crucial factors that affect accuracy of the quantification analysis. In the routinely adopted free enzyme digestion and labeling methods, 12−20 h incubation with free protease (typically trypsin) is needed to achieve high efficiency of protein proteolysis or peptide labeling.20−22 However, even with such prolonged
he ability to accurately quantify difference or variation in protein expression under different biological and pathological conditions is one of the most important goals of proteomic research.1−4 The development of methods for accurate proteins quantification in complex samples, such as whole cell lysate, plasma/serum, or tissues, is currently one of the most demanding and challenging tasks, due to the extreme complexity and wide dynamic range of the protein composition in these samples.5,6 Over the past 10 years, mass spectrometry (MS) based shotgun proteomics strategy coupled with stable isotopes labeling has become one of the most popular approaches for quantitative proteomic study, because of its relatively higher accuracy, reliability, and reproducibility.7,8 Among the commonly used MS techniques, multiple reaction monitoring (MRM) which is capable of sensitive and highly specific quantification of multiple targeted proteins in highly complex sample is ideal for quantitative proteomic study and has been proven successful for absolute quantification of proteins in cell lysates and human plasma/serum.9−12 For stable isotopic labeling, methods including metabolic labeling (stable-isotope amino acids in cell culture, SILAC),13,14 chemical labeling (iTRAQ/mTRAQ)15 and enzymatic18O© 2012 American Chemical Society
Received: November 5, 2011 Accepted: March 9, 2012 Published: March 9, 2012 3138
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Scheme 1. Schematic Overview of Preparation Procedures of PHMN−Trypsin (a), Conventional Single Layer of Trypsin Modified Nanoparticle (b), and Aggregated Trypsin Nanoparticles (c)
highly controllable by in situ growth of polymer chains from the initiators immobilized surface via living/controlled polymerization. For hybrid magnetic nanoparticles, three advantages are obtained by SI-ATRP modification. First, SIATRP modification leads to surface confined growth of hairy polymer chains on the surface of the magnetic nanoparticles. These polymer chains and the numerous binding sites on them work as scaffolds to support three-dimensional trypsin immobilization. Second, since no cross-linkage is applied between the surface anchored polymer chains, the internal spaces between these polymer chains allow protein substrates to penetrate into. As a result, both the loading amount and accessibility of the immobilized trypsin is increased and results in remarkably improved digestion efficiency, completeness and reduced digestion time. Finally, the magnetic nanoparticle immobilized trypsin can be easily and completely removed by a magnet after 18O labeling and eliminates the possible back exchange from 18O to 16O. Therefore, improved quantification accuracy and assay throughput can be expected by coupling our immobilized trypsin for sample preparation and MRM for protein quantification.
incubation, which limits the sample processing throughput, incomplete protein proteolysis is still present for high complex proteomic samples.23,24 Thought may not be a serious problem for discovery based proteomic study, this incomplete digestion compromises the accuracy of quantification analysis.2 To improve protein proteolysis efficiency and reduce digestion and labeling time required, methods that promote trypsin catalysis are highly demanded. As a promising alternative, the usage of immobilized trypsin drew much attention in the past few years. Compared with free trypsin, trypsin immobilized on different types of supports, such as micro/nanoparticles,25−29 porous reactor,30−32 columns,33−37 MALDI plates,38 as well as microchips39−43 has obvious advantages of high catalysis rate, reusability, and easy for automation. The digestion and labeling time required is largely decreased. However, the digestion efficiency and completeness is still not satisfactory, as revealed by the relatively low sequence coverage of the identified proteins obtained by immobilized trypsin digestion. We think this incomplete digestion can be attributed to the insufficient amount of trypsin that immobilized on the commonly used matrix materials and reduced accessibility of the immobilized trypsin. In commonly used trypsin immobilization strategy, a single layer of trypsin is immobilized on the solid surface of the matrix material. Unfortunately, these solid matrix materials have relatively less surface area for immobilization and large steric hindrance that impedes the interaction between immobilized trypsin and protein substrates. Therefore, the digestion efficiency and completeness is limited. To address these issues, we developed a new type of immobilized trypsin using hairy non-cross-linked polymer chains hybrid magnetic nanoparticles as the matrix material for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification using multiple reaction monitoring (MRM) method. The hybrid nanoparticles are fabricated by in situ growth of linear polymer chains on the surface of magnetic nanoparticle using surface initiated atom transfer radical polymerization (SI-ATRP) technique. SI-ATRP is one of the most successful “grafting from” strategies44,45 and has been widely applied in nanomaterials synthesis and surface modification of various of materials.46−51 Compared with “grafting to” method, in which long polymer chains are directly attached on the substrate, SI-ATRP “grafting from” technique gives significantly increased surface grafting density and well controlled polymer structure and thickness, since the grafting is
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EXPERIMENTAL SECTION Materials and Reagents. Bovine serum albumin (BSA), TPCK-treated trypsin and glycidyl methacrylate (GMA) were obtained from Sigma (St. Louis, MO, U.S.A.). The standard reference peptide (SSIIDIYAR and AGYTAIVSHR) was synthesized by Beijing Scilight Biotechnology Co., Ltd. (Beijing, China). H218O (97%) was obtained from Shanghai Research Institute of Chemical Industry. Deionized water (with resistance >18MΩ/cm) was prepared by using Millipore purification system (Billerica, MA, U.S.A.) and used throughout this work. Preparation of Hairy Non-cross-linked Polymer Chains Hybrid Magnetic Nanoparticles (PHMN) and Monolayer of Aldehyde Functional Groups Modified Magnetic Nanoparticles. Details of synthesis and properties of the silica coated Fe3O4 magnetic nanoparticles have been described in our previous work.52 The procedure of SI-ATRP polymer grafting on the magnetic nanoparticles surface is shown in Scheme 1a. Briefly, the initiator, 3-(2bromoisobutyramido)propyl(triethoxy)-silane (BIBAPTES) carrying a triethoxysilane group was synthesized according to the reported method53,54 and the detailed preparing procedure 3139
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Figure 1. FT-IR spectra of (a) silica coated magnetic nanoparticles and (b) hybrid magnetic nanoparticles with GMA polymer chains.
and incubated at 37 °C for 12−20 h. Finally, 2 μL of formic acid was added to the solution to terminate the reaction. Characterization of PHMN, Mass Spectrometry Analysis of Digestion Products, and Data Processing. The detailed procedures of thermogravimetric analysis (TGA), FTIR analysis of PHMN, dynamic laser scattering (DLS), MALDITOF-MS, LC-MS analysis of digestion products and data processing are shown in the Supporting Information.
was stated in the Supporting Information. Next, the magnetic nanoparticles were treated with BIBAPTES for initiator immobilization. After 10 h, excess initiators were removed by repeated washing with methanol and SI-ATRP grafting was carried out by adding nitrogen purged mixture of 2 M GMA, 0.01 M CuCl, 0.001 M CuCl2, and 0.015 M N,N,N′,N″,N″pentamethyldiethylenetriamine to the initiator immobilized magnetic nanoparticles solution, sealed and agitated at 60 °C for 4−6 h. After reaction, excess reagents were removed by repeated washing with methanol. Finally, the epoxy groups on the side chains of the hairy polymer chains on PHMN were converted to aldehyde groups by sequential treatment with 40% ethylenediamine at 60 °C for 4 h and 40% glutaraldehyde at RT overnight for trypsin immobilization. Conventional magnetic nanoparticles modified with monolayer of aldehyde groups was prepared by direct deposition of 3-aminopropyltriethoxysilane (APTES) on the nanoparticle surface, followed by glutaraldehyde treatment to convert the amine groups of APTES to aldehyde groups (Scheme 1b). Trypsin Immobilization on PHMN. The procedure of trypsin immobilization onto PHMN is shown in Scheme 1a and Supporting Information. The amount of trypsin immobilized on PHMN was determined by comparing the UV absorption value (at 280 nm) of the supernatant solution before and after the immobilization reaction. The detailed procedures were shown in the Supporting Information. PHMN−Trypsin or Free Trypsin Digestion of Standard Proteins and Protein Extracts from Thermoanaerobacter tengcongensis (TT) Cultured in 80 °C. BSA, IgG, or protein extracts from TT (100 μg) were dissolved in 25 mM ammonium bicarbonate containing 8 M urea for denaturation, followed by DTT reduction and IAA alkylation. For PHMN− Trypsin digestion, the protein solution was directly mixed with 10 μL of a slurry of PHMN−Trypsin and incubated at 37 °C for 1−2 min depending on the sample complexity. After digestion, 30−50% of ACN was introduced to the system to prevent possible nonspecific adsorption caused sample loss. PHMN−Trypsin was retained by an external magnet and the supernatant was collected for mass spectrometry analysis. Free trypsin digestion was carried out by first diluting the protein solution with 25 mM ammonium bicarbonate to reduce urea concentration below 1M. Next, free trypsin was introduced into the protein solution at substrate to trypsin ratios of 50:1 or 25:1
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RESULTS AND DISCUSSION Preparation and Characterization of Polymer Chains Hybrid Magnetic Nanoparticles (PHMN). Polymer chains hybrid magnetic nanoparticles (PHMN), consisting of Fe3O4 magnetic core, silica shell, and hairy noncross-linked polymer chains have been synthesized via the combination of coprecipitation, sol−gel reaction and SI-ATRP reaction. This combined technique provides a versatile tool for synthesis of multifunctionalized nanoparticles with well-defined surface property and have broad potential application in proteomics studies. The preparation procedure of PHMN is illustrated in Scheme 1a. Briefly, silica coated magnetic nanoparticles are prepared using reported method.52 After immobilizing initiators on the surface of the magnetic nanoparticles, SI-ATRP reaction is carried out using glycidyl methacrylate (GMA) as the monomer and hairy noncross-linked polymer chains are in situ grown from the surface of the nanoparticles. After reaching the preset grafting time, SI-ATRP reaction is stopped and the epoxy groups on the obtained GMA polymer chains are converted to aldehyde group for trypsin immobilization. The prepared PHMN was characterized by FT-IR spectroscopy, thermogravimetric analysis (TGA) and dynamic laser scattering (DLS) to confirm successful growth of GMA polymer chains on the surface of the magnetic nanoparticles as well as determine the content of the surface grafted polymer chains. Typical FT-IR spectra of silica coated magnetic nanoparticles (MN) and PHMN are shown in Figure 1. In Figure 1a, the peak around 3400 cm−1 is attributed to the OH bonds of hydroxyl groups in MN. After SI-ATRP of GMA on the MN surface, strong absorption peaks at 1730 cm−1 and 1150 cm−1 corresponding to CO and C−O bonds in the ester group and peaks characteristic of epoxy ring (906 and 845 cm−1) are observed in Figure 1b demonstrating successful growth of GMA polymer chains on the surface of MN. Next, the content of the surface grafted polymer chains was 3140
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determined by TGA via plotting the weight loss versus temperature as shown in Figure 2. PHMN consists of both
Information). The amount of trypsin immobilized on PHMN was determined by measuring the difference of UV absorbance at 280 nm of the trypsin solution before and after immobilization and was found to be 247 μg/mg, which is obviously higher than that of conventional magnetic nanoparticles (62 μg/mg)55 or microspheres (18.1 μg/mg).56 We attribute this increased trypsin loading amount to the particular structure of PHMN, in which large amount of noncross-linked polymer chains attached on the surface of PHMN provide numerous of binding sites and also work as scaffolds to support 3D trypsin immobilization. In contrast, only a single layer of trypsin can be immobilized on the surface of conventional nanoparticles or microspheres and therefore the trypsin loading amount is limited by the surface area of the nanoparticles or microspheres (Scheme 1b). Furthermore, since no crosslinkage is applied between the surface anchored polymer chains, the internal spaces between these polymer chains allow protein substrates to penetrate into and leads to improved accessibility of the immobilized trypsin. Though even higher trypsin loading amount can be obtained using aggregation based immobilization strategy,37 in which large amount of trypsin aggregates and coats on the matrix surface, the digestion efficiency is not improved much compared with that obtained using single layer of trypsin immobilization, since the trypsin below the surface layer of the aggregates is inaccessible to protein substrates (Scheme 1c). Digestion Performance Evaluation of PHMN−Trypsin Using Standard Protein. Bovine serum albumin (BSA) (66.4 kDa) is the most widely used substrate protein for digestion performance evaluation of immobilized trypsin. BSA (100 μg) was mixed with 10 μL PHMN−-Trypsin slurry to evaluate digestion efficiency and completeness. After incubated at 37 °C for 1 min, PHMN−Trypsin was retained by an external magnet and the supernatant was collected for mass spectrometry analysis. BSA digested in solution with free trypsin using standard protocol was also carried out as a control. The resulting tryptic digests were analyzed by MALDI-TOF-MS. A typical MALDI-TOF-MS mass spectrum of PHMN−Trypsin digestion of BSA is shown in Figure 3 and the protein was profiled using peptide mass fingerprint (PMF). Remarkably, the identified peptides cover 93% amino acid sequence of BSA by allowing one miss cleavage as shown in Figure S-2a (Supporting Information). In contrast, free trypsin digestion of BSA gives obviously lower sequence coverage (69%), though much longer incubation time was used. Lowering the amount of BSA does not reduce the obtained sequence coverage much. As shown in Figure S-2b (Supporting Information), 77% sequence coverage can still be reached using only 100 ng BSA for digestion. To further demonstrate the high digestion efficiency of PHMN− Trypsin, we summarized the literature reported immobilized
Figure 2. TGA curves of silica coated magnetic nanoparticles (a), polymer chains hybrid magnetic nanoparticles with 4 h polymerization (b), and 6 h polymerization (c).
thermally stable Fe3O4 core and silica shell that remains in the residue and decomposable polymer chains that contribute to the weight loss after thermo treatment. Curves a, b and c in Figure 2 are the TGA curves of MN, PHMN with of 4 and 6 h polymerization, respectively. The weight loss is 34.07% for PHMN with 4 h polymerization and increases to 42.93% after 6 h polymerization indicating efficient surface confined growth of polymer chains is achieved. Such high content of polymer grafting is hard to be obtained by conventional “graft to” strategy, because of the large steric hindrance induced by direct attachment of large polymer chains to MN surface. Furthermore, the roughly proportional increase of the content of the polymer chains of PHMN with increasing polymerization time exhibits the characteristic of controlled/ “living” polymerization, in which side reactions such as chain transfer and termination is minimized and therefore results in better controlled polymer structure. In contrast, conventional magnetic nanoparticles modified with monolayer of aldehyde groups on the surface (Figure 2, curve a) show only ∼5% of total weight loss which may be attributed to the loss of possible water residue and surface bound aldehyde groups. The size and size distribution of silica coated nanoparticle core, PHMN (6 h SI-ATRP grafting) and PHMN−Trypsin were characterized by DLS and their radii were found to be 28.7 nm, 57.6 nm and 61.0 nm, respectively. All of the samples showed well dispersity and narrow size distribution (Figure S-1, Supporting
Figure 3. MALDI-TOF-MS spectrum of BSA digests obtained by PHMN−Trypsin digestion for 1 min. 3141
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unique proteins and 661 unique peptides identified by free trypsin digestion (trypsin/protein = 1:50, 12 h at 37 °C), 316 unique proteins and 1116 unique peptides were identified by PHMN−Trypsin (Table S-2, Supporting Information) which corresponds to 27.2% and 40.8% increase in the number of identified proteins and peptides, respectively. The increased number of identified unique proteins and peptides clearly demonstrates higher digestion efficiency is achieved using PHMN−Trypsin. Furthermore, obviously increased digestion completeness is also obtained using PHMN−Trypsin as demonstrated by the SDS-PAGE analysis with silver staining (Figure 4). In Figure 4, smear and quite a few fainted bands
trypsin attached on different types of matrix materials, such as nanoparticles, microchips, microspheres, porous materials and monolithic reactors in Table S-1 (Supporting Information). The sequence coverage of BSA reported in the literature was obtained by allowing one miss cleavage and was generally lower than 60%. Comparison between the digestion efficiency of different kinds of immobilized trypsin is complicated by various of sample handling procedure and conditions. Even though, the exceptionally high digestion efficiency of PHMN−Trypsin suggests more complete protein digestion which may lead to improved accuracy in bottom up based proteome quantification. Since the completeness of protein digestion is a key issue for accurate protein quantification, we evaluated the digestion completeness of PHMN−Trypsin by analyzing the tryptic digests of BSA using MALDI-TOF MS in linear model. As shown in Figure S-3 (Supporting Information), no peaks corresponding to intact protein can be found. For MALDITOF-MS analysis of protein, usually 20 ng of BSA is needed to obtained an identifiable peak with S/N > 3 in the MS spectrum. Considering the large amount of BSA (100 μg) added to the digestion system, highly efficient digestion is achieved by PHMN−Trypsin for only 1 min digestion. We attribute this unusually high digestion efficiency and largely accelerated digestion rate of PHMN−Trypsin to the particular structure of PHMN. First, a high localized trypsin concentration is obtained by 3D trypsin immobilization on the hairy noncross-linked polymer chains scaffolds and leads to higher enzyme-to-protein substrate ratio. Second, compared with trypsin immobilized on solid surface, trypsin attached on the flexible noncross-linked polymer chains has more freedom and therefore better interaction with protein substrate. The stability of PHMN− Trypsin is another important issue that needs to be evaluated before it can be applied to complex proteomic samples. For the stability test, we used the same batch of PHMN−Trypsin to digest BSA 10 times in 30 days (once every three days). The sequence coverage of the identified peptides obtained from these tests is shown in Figure S-4 (Supporting Information). No obvious digestion efficiency drop is found and the sequence coverage is all above 90% indicating very good stability of PHMN−Trypsin. Digestion of Complex Protein Sample Extracted from Thermoanaerobacter tengcongensis (TT) using PHMN− Trypsin. In bottom up based proteome quantification strategies, complex protein samples are digested to peptides before subjected to quantitative mass spectrometry analysis. Therefore, highly efficient and complete digestion of proteins is a prerequest for accurate quantification. To evaluate the digestion efficiency and completeness of PHMN−Trypsin toward complex protein sample, we applied it for proteolysis of protein extracts from Thermoanaerobacter tengcongensis (TT). With no prefractionation treatment, the denatured protein extracts of TT were incubated with PHMN−Trypsin at 37 °C for 2 min and went through RPLC-ESI-MS/MS with 90 min gradient elution. Solution digestion with free trypsin using standard protocols and the same LC-MS condition was carried out as the control. The base peak chromatogram of tryptic digests of TT obtained by PHMN−Trypsin is displayed in Figure S-5 (Supporting Information). Numerous of peaks are observed in Figure S-5, which indicates that the protein mixture is efficiently digested by PHMN−Trypsin. The acquired MS/ MS spectra were searched against a TT sequence database using Mascot program. Impressively, compared with 230
Figure 4. SDS-PAGE gel picture of TT protein extracts before and after different trypsin digestion strategies. Lane 1: Marker. Lane 2: TT protein extracts digested by free trypsin (trypsin to substrate ratio 1:25, 20 h incubation in 37 °C). Lane 3: TT protein extracts digested by PHMN−Trypsin (2 min in 37 °C). Lane 4: TT intact protein without digestion. Samples were separated on 12% SDS-PAGE gel and silver stained. 2 μg loading amount of digested or intact protein was used for lanes 2−4.
corresponding to the undigested protein residues are shown in lane 2 indicating incomplete digestion by free trypsin, even though relatively high trypsin to protein ratio (1:25) and long incubation time (20 h) was used. In contrast, much more complete digestion is obtained by PHMN−Trypsin and almost no protein residue bands can be identified in lane 3. Since the detection sensitivity of silver staining is very high (∼1 ng/ band), the possible amount of undigested protein residues is fairly low after digestion by PHMN−Trypsin. Therefore, it is clear that PHMN−Trypsin has obvious advantages in digestion throughput, efficiency and completeness over conventional free trypsin. This feature is particularly beneficial for bottom up based protein quantification. Absolute Quantification of Protein from Thermoanaerobacter tengcongensis (TT) Using PHMN−Trypsin Digestion, 18O Labeling and MRM Based Quantification. To evaluate the reliability and robustness of sample preparation using PHMN−Trypsin for absolute protein quantification in complex sample, enolase from TT was chosen as the targeted protein. For bottom up based protein quantification coupled with isotopic labeling, protein sample needs to be digested to peptides and isotopic labeled standard reference peptides need to be prepared. The digestion efficiency and completeness of PHMN−Trypsin have been demonstrated in the previous 3142
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Information), good linear relationship is achieved over 2 orders of magnitude concentration range (0.005−2.5 pmol/μL) with correlation coefficients (R2) of 0.9989−0.9848. For reproducibility evaluation, six batches of equivalent amounts of TT protein extracts and standard reference peptides were processed using the same workflow (PHMN−Trypsin digestion, 18O labeling and LC-MRM analysis). The concentrations of enolase obtained from each transition of each targeted peptide were summarized in Table S-4 (Supporting Information). The mean absolute concentration and relative standard deviations (RSDs) of enolase were calculated by treating the three transitions of each target peptide and the six experimental replicates all as independent analysis. The mean absolute quantity of enolase is 25.67 ± 2.29 ng/μg (ENO-1) and 26.89 ± 4.77 ng/μg (ENO2) of total protein and CVs range from 4.51% to 19.97%. These results support that a satisfied quantification result was obtained using our strategy.
sections. The labeling efficiency of PHMN−Trypsin catalyzed 18 O exchange at the C-terminal of peptides and facile removal of PHMN−Trypsin to prevent 18O to 16O back-exchange is evaluated in the following section. (a). Targeted Peptides, Transitions Selection, and 18O Labeling. For MRM based absolute protein quantification, targeted peptides and transitions were selected using MRMpilot 1.0 software (AB SCIEX) and fragmentation information obtained at the discovery step. Common criteria for selecting targeted peptide candidates for MRM analysis were considered including peptide uniqueness, length, charge states, miss cleavage site and modification of peptides. The selected targeted peptides, optimum transitions and collision energies (CE) are listed in Table S-3 (Supporting Information). Two targeted peptides (ENO-1: SSIIDIYAR and ENO-2: AGYTAIVSHR) were chosen for the quantification of enolase and three transitions were chosen for each targeted peptide. The actual purity of the standard reference peptides was determined by amino acids analysis57 using 13C labeled Arginine and was found to be 62.35% (ENO-1) and 68.57% (ENO-2). For traditional 18O labeling using free trypsin, prolonged incubation (10−12 h) in H218O and postlabeling trypsin deactivation is needed to achieve high labeling efficiency. Even though, incomplete exchange of two 18O atoms at peptides C-terminal and back exchange from 18O to 16 O caused by the trypsin residues is sometimes inevitable. In addition, it takes extra steps to deactivate trypsin after labeling and may lead to sample loss. However, in our labeling strategy, free trypsin is replaced by PHMN−Trypsin which can be easily separated from the labeled peptides by a magnet after labeling for only 1 min. Three replicated labeling experiment were carried out to check the labeling efficiency and reproducibility. Typical MALDI-TOF-MS spectra of the labeled and unlabeled peptides are shown in Figure S-6 (Supporting Information). After labeling, peptides with +4 Da dominate the spectra for both ENO-1 and ENO-2. Peptides of other 18O labeled variants (+0 or +2 Da) can hardly be observed indicating complete labeling with two 18O atoms is achieved for the two standard reference peptides. The average labeling efficiency of ENO-1 and ENO-2 in three tests are 96.7% and 96.5%, respectively. No obvious back exchange from 18O to 16O was observed even 7 days after labeling (data not shown) demonstrating that facile removal of PHMN−Trypsin by a magnet is complete. (b). MRM Quantification Method Validation and Absolution Quantification of Enolase from TT Protein Extracts. The identity of the chosen targeted peptides and transitions were verified by MS/MS data triggered by MRM signals using TT protein digests (matrix) and 18O labeled standard reference peptides obtained by PHMN−Trypsin digestion or labeling. Using MRM initiated detection and sequencing (MIDAS) workflow,51 the chromatogram peaks of ENO-1 and ENO-2 (18O-labeled and unlabeled) with retention times of 19.75 and 34.41 min were confirmed by MS/MS analysis (Figure S-7, Supporting Information). The MS/MS spectra of the corresponding peptides shown in Figure S-8 (Supporting Information) provided reliable identifications of the 18O-labeled and unlabeled targeted peptides. Calibration curves of ENO-1and ENO-2 were prepared by spiking TT protein digests with varying amounts of 18O labeled standard reference peptides. The peak areas of variable concentrations of the standard reference peptides (Y) were plotted against the corresponding concentration shown as pmol/μL of peptides (X). As shown in Figure S-9 (Supporting
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CONCLUSION In summary, a new type of immobilized trypsin using hairy polymer chains hybrid magnetic nanoparticles as matrix is developed in this work. The hybrid nanoparticle prepared by SI-ATRP technique leads to increased trypsin loading amount and accessibility. Therefore, obviously enhanced digestion efficiency, completeness as well as facile 18O labeling is achieved with largely reduced sample processing time and steps, which is a key advantage for improving accuracy of protein quantification. Successful application of PHMN− Trypsin in absolution protein quantification is demonstrated by determining the concentration of enolase in Thermoanaerobacter tengcongensis protein extracts using MRM method.
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ASSOCIATED CONTENT
S Supporting Information *
Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (Zhang, Y.J.);
[email protected] (Qian, X.H.). Fax: (+) 86 80705155. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS W.J. Qin and Z.F. Song contributed equally to this work. This work was supported by National Key Program for Basic Research of China (No. 2012CB910603, 2010CB912701), National Key Scientific Instrument Development Program of China (2011YQ09000504, 2011YQ030139, 2011YQ06008408), International Scientific Cooperation Project of China (No. 2011DFB30370) and National Natural Science Foundation of China (No. 20905077, 30900258, 31100591).
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Analytical Chemistry
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dx.doi.org/10.1021/ac2029216 | Anal. Chem. 2012, 84, 3138−3144
