Letter pubs.acs.org/ac
Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples Yuki Ichihashi,† Toru Komatsu,*,† Etsu Kyo,† Hiroyuki Matsuzaki,§ Keisuke Hata,§ Toshiaki Watanabe,§ Tasuku Ueno,† Kenjiro Hanaoka,† and Yasuteru Urano*,†,‡,∥ †
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ∥ Core Research for Evolutional Science and Technology (CREST) Investigator, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan
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‡
S Supporting Information *
ABSTRACT: We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.
C
ellular functions are mediated by a variety of enzymatic functions. Characterizing their alterations is a straightforward means to discover, to understand, and to treat diseases. The importance of globally monitoring enzymatic activities for characterizing disease-related phenotypes can be emphasized by the fact that enzymatic activities, influenced by various factors such as post-translational modifications, protein− protein interactions, and intrinsic inhibitors, should have a direct connection with their phenotypes.1 In this context, we have recently developed a method to globally monitor the enzymatic activities using a library of substrates such as fluorescent probes and peptides.2,3 In the global peptidemetabolizing activity profiling method, assays are performed by mixing a library of peptides with biosamples and reading the substrate consumption with LC-MS-based analysis (Figure 1a).3 While the usability of the assay has been confirmed by characterization of cell-type specific or disease-related enzymes, there was one drawback: the assay only picked up activities with high metabolic rates. It was inevitable as far as the assay was performed in an enzyme mixture, in which multiple enzymes react with multiple substrates and only the fast-acting enzyme activities are detected. To solve the problem is highly desirable to detect more diverse enzymatic activities for discovering ones with interesting features. In this study, we tried to do this by combining the separation of proteome and the subsequent global activity assay in each protein spot (Figure 1b). For protein separation, column chromatography-based or native electrophoresis-based (zymography) approaches have often been applied.4 We © XXXX American Chemical Society
Figure 1. Concept of separation-based enzymomics assay. (a) LCMS-based analysis of global peptide metabolism. Adapted from ref 3. Copyright 2017 American Chemical Society. (b) The concept of the assay where the global peptide-metabolism monitoring is performed after protein separation by native electrophoresis and loading into a multiwell plate.
Received: July 4, 2019 Accepted: August 19, 2019 Published: August 19, 2019 A
DOI: 10.1021/acs.analchem.9b03016 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry employed the latter and developed the assay methodology where the metabolism of 73 peptides is simultaneously monitored after the separation of the protein mixture into 304 fractions based on molecular weights and isoelectric points. By comparing the prepared activity maps between tumor and nontumor lysates of a colorectal tumor specimen, we identified a novel peptide-metabolizing enzymatic activity that was highly elevated in colorectal tumor cells. Native electrophoresis is a useful tool for separating proteins of complex biosamples. It has been traditionally applied in combination with activity assays such as the analysis of subtypes of enzymes in biosamples. However, it is still difficult to apply this process to activity assays read out by methods other than precipitation colorimetric tests and conventional gelatin zymography methods,4 as the reaction products easily diffuse from the gels. To solve this problem, we have recently developed a novel form of zymography in which the enzymatic assay is performed after dicing the electrophoresis gels into small pieces and loading them into a 384-well plate for subsequent activity assays.5,6 We consider that this methodology will offer an ideal platform for monitoring global enzyme activity in combination with a peptide library-based multiplexed assay. In the assay, after loading the gel pieces containing separated proteins, a prepared peptide library is loaded in each well. Afterward, the substrate consumptions during the enzymatic reactions are monitored in each well by LC-MS-based experiments. The challenges in establishing this strategy were (1) shortening the analysis time for the single assay since we want to treat more than 300 protein spots independently and (2) preventing the presence of gel from significantly perturbing the analysis. We solved the first problem by optimizing the separation conditions and using the MS/MS-based targeted analysis. The second problem was managed by selecting the right set of peptides to be used in the assay. First, we tried to make the analysis as a high-throughput 5 min assay. The original assay was designed to monitor the library of peptides prepared from the residue-specific cleavage of purified proteins,3 in which an analysis time of 60 min was used per assay. In this study, we used the peptide library prepared by trypsinization of bovine serum albumin, βgalactosidase, and transferrin and converted the gradient conditions of the original assay to a new one with a tightly packed silica column with a fast gradient. After optimization, the assay was confirmed to work well with almost a similar order of peptide elution, a comparable theoretical plate number, and a shortened analysis time to 5 min (Figures 2 and S1). However, for quantitative analyses by detecting MS signals, the original assay based on XCMS analysis (the analysis of MS scan data to search for the statistically meaningful alternation between different sets of total ion chromatogram files)7 did not have the required peak detection reliability in the high-throughput analysis; namely, peak intensities were not stable. Hence, we switched from the untargeted analysis (with MS scanning) to a targeted one and constructed multiple reaction monitoring (MRM) files of the selected peptides in the library. Then, we tried to construct the peptide library assay in combination with native electrophoresis. In this method, peptides whose solution concentration is highly influenced by the presence of gels, presumably due to the nonspecific binding as result of hydrophobicity or relative instability in the presence of gels, were removed from the list to construct the library of 73 peptides used in the assay
Figure 2. Optimization of peptide analysis. (a) Conditions of the original assay and optimized assay. (b) Total ion chromatograms (TICs) of peptide library analysis in each condition. (c) Comparison of the average number of theoretical plates of the MS peaks of seven randomly selected peptides (YNGVFQECCQAEDK, VPQVSTPTLVEVSR, TVMENFVAFVDK, QTALVELLK, LVVSTQTALA, LVNELTEFAK, and DAFLGSFLYEYSR). (d) Correlation of retention time of each peptide in original and optimized assays.
(Figures 3, S2, and S3). Before going into the separation-based assay, we confirmed that the assay conditions developed here
Figure 3. Construction of MRM files for peptide library. m/z values of parent ions and daughter ions and assignment of daughter ions are shown.
can be applied for monitoring the peptide metabolism in complex biosamples containing a mixture of proteins. We studied the peptide-metabolizing activities of tumor sites of a colorectal tumor specimen derived from a surgical specimen, in which we confirmed that the LVVSTQTALA peptide, which has been characterized to be a substrate of neurolysin, was preferably metabolized in tumor tissue-derived lysates (Figure S4).3 Then, this condition was applied to the separation-based assay, where the analysis was conducted in each well containing diced gels after performing a native electrophoresis. One set of the colorectal tumor specimen (lysates of tumor and nontumor tissues) was included in the analysis with B
DOI: 10.1021/acs.analchem.9b03016 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 4. Global peptide enzymomics assay. An averaged activity map of three repeated assays with a single set of tumor and nontumor tissuederived lysates of a colorectal cancer surgical specimen. Blue areas represent the activity spots where the consumption of peptides was higher than the average + 3 × S.D. of the background.
we acquired the list of proteins contained in the activity spot (Figure S5). Then, we did the database and literature search and picked up enzymes in the list that are known to act on peptide substrates (Figure 5d). Among those candidate enzymes, we considered that thimet oligopeptidase (THOP1) would be a reasonable target, as the enzyme is known to exhibit the endopeptidase activity against diverse peptides of approximately 10 amino acids long.8 In a gratifying way, QNNFNAVR was indeed confirmed to act as the substrate of recombinant THOP1 being cleaved at the C-terminus of QNNF (Figure S6), namely, the same position observed in our separation-based assays. Although the role of this enzyme is relatively less characterized compared to other enzymes,9 neurotensin is known as one of its substrates (Figure 6a). From this perspective, we studied if the metabolism of neurotensin could be affected by the elevated activity of THOP1. In metabolism of neurotensin NT (1−13) in tumor tissue-derived lysate, four major fragments were observed, namely, NT (1− 12), NT (1−10), NT (1−8), and NT (1−7) (the number in parentheses meaning the number of amino acids from the Nterminus, Figures 6a and S7). NT (1−8) is known to be generated by the activity of THOP1,10 and its elevation was indeed confirmed in the assay (Figure 6b). The elevated activity of neurolysin, another neurotensin-metabolizing
triplicates. In each well, activity was determined by the decrease of LC-MS peak areas of the peptides compared to the corresponding peak areas in the empty wells. Then, the results of the triplicates were averaged to construct the overall peptide-metabolizing activity map for each sample in which spots with peptide consumption rates higher than the average + 3 × S.D. of the backgrounds were assigned as the activity spots for each peptide. The results summarized in Figure 4 showed many possible alterations between tumor and nontumor tissue-derived lysates. This result could be considered as an initial screening process since it was performed with only a single set of samples. Also, at this stage, there is still the possibility that the decrease of peaks might reflect other reasons than the metabolic consumptions of the substrates. Hence, peptides with conspicuous activities were chosen, and confirmation was attempted. In this context, we synthesized and purified the hit peptides, and the separation-based assay was performed by monitoring both the substrate consumption and the product formation with three different sets of tumor and nontumor samples. Among those, we observed that the enzyme spot with the ability to generate the QNNF peptide fragment from QNNFNAVR was significantly altered in tumor tissue-derived lysates compared to nontumor ones in three sample sets (Figure 5a−c). Consequently, we tried to identify the target protein by performing the peptide mass fingerprinting analysis. As a result, C
DOI: 10.1021/acs.analchem.9b03016 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 5. (a) Structure of the QNNFNAVR peptide. (b) Analysis by the diced electrophoresis gel assay of the enzymes able to metabolize QNNFNAVR (10 μM) into QNNF within a colorectal cancer specimen (30 μg). The results of three subjects (tumor and nontumor samples) are shown. (c) The average MS peak areas of the QNNF peptide fragment regarding the activity spot of (b) (n = 3). Error bars represent S.D. (d) List of possible targets of QNNFNAVRmetabolizing enzymes detected by peptide mass fingerprinting (PMF) analysis regarding the activity of (b). The whole list of detected proteins is shown in Figure S5.
enzyme producing NT (1−10),10 was also observed, as previously reported.3 In most of the samples, the consumption of neurotensin was faster in tumor-derived lysate than in nontumor ones (Figure 6b, left), which seemed to be associated with the increase of the activity of THOP1 and neurolysin. For further characterization, we studied the correlations among consumption of neurotensin and formation of each fragment (Figure 6b). If two reactions share the same enzyme or substrates/products, the reaction rates are expected to show good correlation among different samples. NT (1−12) is a peptide fragment generated by the cleavage of a single amino acid at the C-terminus. We consider that the reaction was catalyzed by the activity of certain types of carboxypeptidases,11,12 but this activity did not correlate well with the consumption of NT (1−13) as underlined by the correlation constants R2 = 0.02. On the contrary, the formation of NT (1− 10), from neurolysin, and NT (1−8), from THOP1, both correlated well with the consumption of NT (1−13) with correlation constants R2 = 0.58 and 0.43, respectively. These features would indicate that THOP1 and neurolysin are the major contributors of the consumption of NT (1−13) in the lysate. Meanwhile, the shorter fragment NT (1−7), whose formation also correlated with the consumption of NT (1−13) (R2 = 0.51), might be generated from NT (1−10), since the formation of two peptides had a good correlation (R2 = 0.60). The metabolism of neurotensin was also evaluated with the separation-based assay (Figure S8). As a result, we were able to
Figure 6. Metabolism of neurotensin monitored by LC-MS/MS. (a) Summary of the observed and reported cleavage sites of neurotensin. (b) The consumption rate of neurotensin (left) and the formation of each product (right) were monitored after incubating neurotensin (10 μM) in the lysate of the colorectal tumor specimen (normal and tumor areas, 15 samples each, 0.2 mg/mL) for 1 h. The P-value was calculated by the Mann−Whitney’s U-test. (c) Correlation between consumption of neurotensin and formation of each fragment in different samples. The correlation constants (R2) in each graph are shown, with the red color as an indication of strong correlation. (d) Plausible metabolic routes of neurotensin in colorectal tumor lysates. Red arrows indicate enzymatic activities elevated in tumor sites compared to nontumor sites.
clearly detect the activity spot of neurolysin, which is able to produce NT (1−10), and THOP1, which is able to produce NT (1−8). Several protein spots were detected for NT (1−12) as well. On the contrary, there were not specific activity spots D
DOI: 10.1021/acs.analchem.9b03016 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Science. We would like to thank Gilles-Olivier Gratien for constructive comments on the manuscript.
detected for NT (1−7), indicating that this peptide fragment was not directly generated from neurotensin. The overall analysis confirmed that the independent activities of both neurolysin and THOP1 were highly elevated in colorectal tumor specimens and were the major cause of the consumption of neurotensin in the lysates. The contribution of those peptidases in tumor pathogenesis is still unknown.13 However, neurotensin is known to function in potentiating immune responses,14,15 and we consider that it is interesting to study how alteration of those enzymes affects the residence time of the peptide hormones for modulating immunologic activities around tumor areas. In addition, we consider that these novel biochemical biomarker candidates could be potentially useful for diagnostic purposes since it was able to completely discriminate tumor and nontumor sites, and studies along these lines are currently underway. In conclusion, we have developed a methodology for characterizing the altered enzymatic activities in biosamples by the combination of proteome separation and activity monitoring on each protein spot. Using the methodology of separating the electrophoresis gels into multiwell plates, we successfully monitored the peptide-metabolizing activities in more than 20 000 data spots. Results were exploited to characterize novel enzymatic activities that were elevated in a colorectal tumor. Rewardingly, it led to the discovery that the characterized enzyme, thimet oligopeptidase, co-operates with neurolysin, another tumor-related endopeptidase, to metabolize immune-response promoting peptide neurotensin toward its elimination. Finally, we consider that our process is widely applicable to globally profile altered enzymatic activities in biosamples of interest.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b03016. Experimental details and photophysical properties of the compounds (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Toru Komatsu: 0000-0002-9268-6964 Hiroyuki Matsuzaki: 0000-0001-8521-3029 Kenjiro Hanaoka: 0000-0003-0797-4038 Yasuteru Urano: 0000-0002-1220-6327 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by MEXT (24655147, 15H05371, 15K14937, 17K19477, 18H04538, and 19H02846 to T.K. and 16H05099 and 18H04609 to K.Hanaoka.), JST (T.K. and K.Hanaoka.), and AMED (Y.U.). T.K. was supported by The Naito Foundation and The Mochida Memorial Foundation for Medical and Pharmaceutical Research and The Tokyo Biochemical Research Foundation. PMF analysis was performed as a contract service by Apro E
DOI: 10.1021/acs.analchem.9b03016 Anal. Chem. XXXX, XXX, XXX−XXX
