Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry
2012; Elsevier BV; Volume: 12; Issue: 2 Linguagem: Inglês
10.1074/mcp.m112.025221
ISSN1535-9484
AutoresMartial Rey, Meng‐Lin Yang, Kyle M. Burns, Yaping Yu, Susan P. Lees‐Miller, David C. Schriemer,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoStudies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5–10 years. The predominant technology requires fast digestion at pH 2–3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners. Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5–10 years. The predominant technology requires fast digestion at pH 2–3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners. Mass spectrometry has served the biochemical and biological communities by providing the capacity for protein identification and characterization, but in the last several years it has also become a powerful tool for interrogating protein structure and dynamics (1Konermann L. Pan J. Liu Y.H. Hydrogen exchange mass spectrometry for studying protein structure and dynamics.Chem. Soc. Rev. 2011; 40: 1224-1234Crossref PubMed Scopus (578) Google Scholar, 2Xu G. Chance M.R. Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.Chem. Rev. 2007; 107: 3514-3543Crossref PubMed Scopus (539) Google Scholar). Solution-phase hydrogen/deuterium exchange (HDX) 1The abbreviations used are:HDX-MShydrogen/deuterium exchange mass spectrometryNHEJnonhomologous end-joiningXRCC4X-ray repair cross-complementing protein 4BRCTBRCA1 (breast cancer suppressor protein) C-terminusXLFXRCC4-like factorPNKpolynucleotide kinaseNMRnuclear magnetic resonancecryoEMcryo electron-microscopySAXSsmall-angle x-ray scatteringCK2casein kinase 2. 1The abbreviations used are:HDX-MShydrogen/deuterium exchange mass spectrometryNHEJnonhomologous end-joiningXRCC4X-ray repair cross-complementing protein 4BRCTBRCA1 (breast cancer suppressor protein) C-terminusXLFXRCC4-like factorPNKpolynucleotide kinaseNMRnuclear magnetic resonancecryoEMcryo electron-microscopySAXSsmall-angle x-ray scatteringCK2casein kinase 2.when coupled with mass spectrometry (MS), provides rich sets of data that can be mined to extract structural and dynamic parameters from proteins (3Soon F.F. Ng L.M. Zhou X.E. West G.M. Kovach A. Tan M.H. Suino-Powell K.M. He Y. Xu Y. Chalmers M.J. Brunzelle J.S. Zhang H. Yang H. Jiang H. Li J. Yong E.L. Cutler S. Zhu J.K. Griffin P.R. Melcher K. Xu H.E. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases.Science. 2012; 335: 85-88Crossref PubMed Scopus (349) Google Scholar, 4Devarakonda S. Gupta K. Chalmers M.J. Hunt J.F. Griffin P.R. Van Duyne G.D. Spiegelman B.M. Disorder-to-order transition underlies the structural basis for the assembly of a transcriptionally active PGC-1 alpha/ERR gamma complex.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 18678-18683Crossref PubMed Scopus (49) Google Scholar, 5Zhang J. Chalmers M.J. Stayrook K.R. Burris L.L. Wang Y. Busby S.A. Pascal B.D. Garcia-Ordonez R.D. Bruning J.B. Istrate M.A. Kojetin D.J. Dodge J.A. Burris T.P. Griffin P.R. DNA binding alters coactivator interaction surfaces of the intact VDR-RXR complex.Nat. Struct. Mol. Biol. 2011; 18: 556-563Crossref PubMed Scopus (156) Google Scholar, 6Roberts V.A. Pique M.E. Hsu S. Li S. Slupphaug G. Rambo R.P. Jamison J.W. Liu T. Lee J.H. Tainer J.A. Ten Eyck L.F. Woods Jr., V.L. Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase.Nucleic Acids Res. 2012; 40: 6070-6081Crossref PubMed Scopus (25) Google Scholar, 7Chung K.Y. Rasmussen S.G. Liu T. Li S. DeVree B.T. Chae P.S. Calinski D. Kobilka B.K. Woods Jr., V.L. Sunahara R.K. Conformational changes in the G protein Gs induced by the beta2 adrenergic receptor.Nature. 2011; 477: 611-615Crossref PubMed Scopus (290) Google Scholar). In many cases, the technique is used in experimental situations in which x-ray diffraction analysis or other biophysical techniques (e.g. NMR, cryoEM) are difficult to apply, which is particularly true in the structure-function analysis of protein interactions (8Iacob R.E. Engen J.R. Hydrogen Exchange Mass Spectrometry: Are We Out of the Quicksand?.J. Am. Soc. Mass Spectrom. 2012; 23: 1003-1010Crossref PubMed Scopus (95) Google Scholar). Most applications of the method involve deuteration of a protein in two or more states, and differential labeling data is extracted at the highest structural resolution possible. Although there have been some impressive developments in top-down protein analysis using newer ion fragmentation methods for label localization (9Pan J. Han J. Borchers C.H. Konermann L. Hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation for characterizing structural transitions of a 17 kDa protein.J. Am. Chem. Soc. 2009; 131: 12801-12808Crossref PubMed Scopus (155) Google Scholar, 10Rand K.D. Zehl M. Jensen O.N. Jørgensen T.J. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry.Anal. Chem. 2009; 81: 5577-5584Crossref PubMed Scopus (179) Google Scholar), most studies continue to employ a bottom-up strategy, in which the protein is digested with an enzyme, and the label is quantified by mass analysis of the resulting peptides. hydrogen/deuterium exchange mass spectrometry nonhomologous end-joining X-ray repair cross-complementing protein 4 BRCA1 (breast cancer suppressor protein) C-terminus XRCC4-like factor polynucleotide kinase nuclear magnetic resonance cryo electron-microscopy small-angle x-ray scattering casein kinase 2. hydrogen/deuterium exchange mass spectrometry nonhomologous end-joining X-ray repair cross-complementing protein 4 BRCA1 (breast cancer suppressor protein) C-terminus XRCC4-like factor polynucleotide kinase nuclear magnetic resonance cryo electron-microscopy small-angle x-ray scattering casein kinase 2. The reasons for the prominence of the bottom-up approach to HDX-MS are shared with the corresponding proteomics method. Peptides may be detected with more sensitivity than proteins, and samples of considerably higher complexity can be interrogated (11Garcia B.A. What Does the Future Hold for Top Down Mass Spectrometry?.J. Am. Soc. Mass Spectrom. 2010; 21: 193-202Crossref PubMed Scopus (98) Google Scholar). Analytically, this shifts the focus toward optimizing protein digestion, to cover 100% of the sequence and generate a high degree of overlapping fragments to increase opportunities for localizing the deuterium label at high resolution (12Fajer P.G. Bou-Assaf G.M. Marshall A.G. Improved Sequence Resolution by Global Analysis of Overlapped Peptides in Hydrogen/Deuterium Exchange Mass Spectrometry.J. Am. Soc. Mass Spectrom. 2012; 23: 1202-1208Crossref PubMed Scopus (31) Google Scholar, 13Mayne L. Kan Z.Y. Chetty P.S. Ricciuti A. Walters B.T. Englander S.W. Many Overlapping Peptides for Protein Hydrogen Exchange Experiments by the Fragment Separation-Mass Spectrometry Method.J. Am. Soc. Mass Spectrom. 2011; 22: 1898-1905Crossref PubMed Scopus (88) Google Scholar, 14Landgraf R.R. Chalmers M.J. Griffin P.R. Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution.J. Am. Soc. Mass Spectrom. 2012; 23: 301-309Crossref PubMed Scopus (71) Google Scholar). The unique requirements of the HDX-MS workflow unfortunately place restrictions on the digestion enzymes that may be used. To avoid label loss through back-exchange to nondeuterated solvent, the kinetics of exchange must be dramatically slowed by pH and temperature reduction, and even under these conditions the digestion must be done rapidly. Conventional methods employ a pH of ∼2.5 and temperatures of 4–10 °C. The aspartic protease pepsin can function under such conditions, which has led to its prominence for HDX-MS applications. This does not mean the enzyme is ideal. Several laboratories have sought to identify other enzymes or develop analytical solutions that address its shortcomings, which include extensive autolysis, modest efficiency, and nonideal substrate specificity. Currently, most methods involve either enzyme microreactors presenting high concentrations of pepsin in a flow-through system (15Wang L. Pan H. Smith D.L. Hydrogen exchange-mass spectrometry - Optimization of digestion conditions.Mol. Cell. Proteomics. 2002; 1: 132-138Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), or solution-phase digestions using pepsin and protease XIII in separate experiments (16Cravello L. Lascoux D. Forest E. Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteins.Rapid Commun. Mass Spectrom. 2003; 17: 2387-2393Crossref PubMed Scopus (126) Google Scholar, 17Mazon H. Marcillat O. Forest E. Vial C. Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases.Biochimie. 2005; 87: 1101-1110Crossref PubMed Scopus (13) Google Scholar). The latter enzyme is also an aspartic protease with a sequence specificity that partially overlaps pepsin, thus the two maps together tend to somewhat improve sequence coverage (18Zhang H.M. Kazazic S. Schaub T.M. Tipton J.D. Emmett M.R. Marshall A.G. Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.Anal. Chem. 2008; 80: 9034-9041Crossref PubMed Scopus (80) Google Scholar). A method that fuses the two strategies has recently been described, involving tandem pepsin and protease XIII microreactors (13Mayne L. Kan Z.Y. Chetty P.S. Ricciuti A. Walters B.T. Englander S.W. Many Overlapping Peptides for Protein Hydrogen Exchange Experiments by the Fragment Separation-Mass Spectrometry Method.J. Am. Soc. Mass Spectrom. 2011; 22: 1898-1905Crossref PubMed Scopus (88) Google Scholar). Neither strategy is likely to be ideal when applied to protein complexes of far greater complexity, to support of structure-building activities or dynamics analysis of multiprotein "machines." The microreactor approach complicates the front-end fluidic system, and can lead to sample loss and carryover (19Wu Y. Kaveti S. Engen J.R. Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry.Anal. Chem. 2006; 78: 1719-1723Crossref PubMed Scopus (37) Google Scholar). The solution-phase method requires large amounts of enzyme resulting in contamination because of enzyme autolysis (18Zhang H.M. Kazazic S. Schaub T.M. Tipton J.D. Emmett M.R. Marshall A.G. Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.Anal. Chem. 2008; 80: 9034-9041Crossref PubMed Scopus (80) Google Scholar). More importantly, neither fully overcomes the low efficiency of these enzymes. In many cases, changing the presentation of substrate can change the sequence map considerably. For example, the sequence map of a protein can be distorted when bound to a second protein (20Zhang Q. Willison L.N. Tripathi P. Sathe S.K. Roux K.H. Emmett M.R. Blakney G.T. Zhang H.M. Marshall A.G. Epitope Mapping of a 95 kDa Antigen in Complex with Antibody by Solution-Phase Amide Backbone Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.Anal. Chem. 2011; 83: 7129-7136Crossref PubMed Scopus (103) Google Scholar). A map dependent on the protein load complicates the comparison of deuteration levels for the protein in different states. This alteration suggests a level of substrate inhibition (21Ruan C.Q. Chi Y.J. Zhang R.D. Kinetics of hydrolysis of egg white protein by pepsin.Czech J. Food Sci. 2010; 28: 355-363Crossref Google Scholar), and probably reflects a wide range of specificity constants (kcat/Km) across the many individual cleavage sites presented in a protein substrate (22Sachdev G.P. Fruton J.S. Kinetics of Action of Pepsin on Fluorescent Peptide Substrates.Proc. Natl. Acad. Sci. U.S.A. 1975; 72: 3424-3427Crossref PubMed Scopus (33) Google Scholar). Similar issues have been noted with trypsin (23Slysz G.W. Schriemer D.C. Blending protein separation and peptide analysis through real-time proteolytic digestion.Anal. Chem. 2005; 77: 1572-1579Crossref PubMed Scopus (81) Google Scholar). Overall, the sequence maps generated using these methods are serviceable for samples of lower complexity, but low enzymatic efficiency of the available proteases remains an important limitation and a key driver in the search for novel proteases (24Marcoux J. Thierry E. Vivès C. Signor L. Fieschi F. Forest E. Investigating Alternative Acidic Proteases for H/D Exchange Coupled to Mass Spectrometry: Plasmepsin 2 but not Plasmepsin 4 Is Active Under Quenching Conditions.J. Am. Soc. Mass Spectrom. 2010; 21: 76-79Crossref PubMed Scopus (25) Google Scholar). In this study, we characterize the proteolytic activity of secretions from the Nepenthes genus (25Vines S.H. On the digestive ferment of nepenthes.J. Anat. Physiol. 1876; 11: 124-127PubMed Google Scholar), arising from the aspartic protease nepenthesin (26Amagase S. Nakayama S. Tsugita A. Acid protease in nepenthes . 2. Study on specificity of nepenthesin.J. Biochem. 1969; 66: 431-439Crossref PubMed Scopus (24) Google Scholar), and evaluate the enzyme for use within HDX applications. Nepenthesin displays remarkably high cleavage efficiency for a broad range of substrates at low pH and temperature, which promotes high sequence coverage for a collection of proteins selected from ongoing HDX projects in our laboratories. Globally, we demonstrate that it outperforms pepsin in sequence coverage and can be used in a simple workflow for broad sequence coverage, or targeted toward a desired area of protein sequence. This new tool was applied to an HDX-MS characterization of a protein complex involved in the nonhomologous end-joining (NHEJ) pathway of DNA damage repair, and the results support a model of the complex proposed from SAXS data (27Hammel M. Yu Y. Fang S.J. Lees-Miller S.P. Tainer J.A. XLF Regulates Filament Architecture of the XRCC4.Ligase IV Complex.Structure. 2010; 18: 1431-1442Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Transplants of several Nepenthes varieties were acclimated in a small terrarium. On pitcher maturity, the plants were fed with one or two Drosophila per pitcher and the pitcher fluid harvested 1 week later. Pitchers and their secretions were left to recover for 1 week before a second round of feeding and extraction. All pitcher fluid was combined and clarified through a 0.22 μm filter, and then concentrated 80- to 100-fold using an Amicon Ultra 10 kDa molecular weight cutoff centrifugal filter (both from Millipore). Before use in digestions, the concentrate was acid-activated with 100 mm glycine HCl (pH 2.5) for 3 h, then washed 3 × with 100 mm glycine-HCl (pH 2.5) in the filtration device, using 10 × fluid volume for each wash. The final isolate was then rediluted to an 11 × concentration based on the original sampling of pitcher fluid. Additional details on plant horticulture, feeding and fluid extraction can be found in Supplementary Methods. Digestions were carried out in solution using a HTX-PAL autosampler and dispensing system designed for HDX applications (Leap Technologies, Carrboro, NC), and data were collected using an AB Sciex Triple-TOF 5600 QqTOF mass spectrometer. Peptides were identified with Mascot (v2.3) from MS/MS data, from .mgf files created in Analyst TF v1.51. Data were mapped to sequence using the following search terms: a mass tolerance of 10 ppm on precursor ions and 0.6 Da on fragment ions, no modifications, and no enzyme specificity. A standard probability cutoff of p = 0.05 was implemented and matches near the cutoff manually verified. For nepenthesin-based digestion, 8 μm protein solutions (XRCC4, XLF, Ligase IV-tandem BRCT domains, PNK, myoglobin, or cytochrome C) were mixed with 11 × concentrated fluid for 2 min at 10 °C, in various ratios (see text). Information on the production of XRCC4, XLF, BRCT domains, and PNK can be found in Supplementary Methods. Myoglobin and cytochrome C were purchased from Sigma. After dilution to 1 μm substrate concentration, 15 μl were injected into the chilled reversed-phase LC system (4 °C) connected to the mass spectrometer. The peptides were trapped on a 5 cm, 200 μm i.d. Onyx C18 monolithic column (Phenomenex Inc., Torrance, CA) and eluted with an acetonitrile gradient from 3% to 40% in 10 min. Peptides detected in these analyses were selected for CID fragmentation in multiple information-dependent acquisitions of MS/MS spectra, akin to the gas-phase fractionation strategy (28Blonder J. Rodriguez-Galan M.C. Lucas D.A. Young H.A. Issaq H.J. Veenstra T.D. Conrads T.P. Proteomic investigation of natural killer cell microsomes using gas-phase fractionation by mass spectrometry.Biochim. Biophys. Acta. 2004; 1698: 87-95Crossref PubMed Scopus (25) Google Scholar). Spectra were searched against a miniature database containing the sequences for all six proteins. Sequencing results were manually verified. Stock solutions of XRCC4(1–200) with BRCT, and XRCC4(full length) with BRCT were diluted in buffer (10 mm Tris-HCl, pH 7.5) to equimolar concentrations (10 μm), and incubated at 4 °C for a minimum of 30 min to promote complexation. The samples were held at 4 °C until HDX analysis. Aliquots were deuterated for 2 min at 20 °C with the addition of D2O (25% v/v). Aliquots were then digested in two ways. In the first digestion strategy, protein deuteration was quenched by adding the sample to chilled 100 mm glycine-HCl (pH 2.5), and the quenched protein solution was injected into a pepsin microreactor. This microreactor was installed in the HTX-PAL system between the injector valve and the C18 column. Protein digest was captured on the monolithic C18 capillary column and eluted into the mass spectrometer. All fluidic elements, including the microreactor, were chilled at 4 °C to minimize deuterium back-exchange during the analysis time (<15 min). In the second digestion strategy, an equivalent amount of deuterated protein was simultaneously quenched and digested with 3 or 5 μl of 11 × nepenthes fluid for 3 or 5 min, respectively, at 10 °C. The samples were then injected into the chilled LC-system connected to the mass spectrometer. Replicate mass shift measurements were made (four or more) and referenced to control protein states—free XRCC4(1–200), free XRCC4(full length), and free LigIV-BRCT. The average deuterium level for each peptide was determined using Mass Spec Studio (manuscript in preparation), which is a rebuild of Hydra v1.5 (29Slysz G.W. Baker C.A.H. Bozsa B.M. Dang A. Percy A.J. Bennett M. Schriemer D.C. Hydra: software for tailored processing of H/D exchange data from MS or tandem MS analyses.BMC Bioinf. 2009; 10Crossref PubMed Scopus (67) Google Scholar). Perturbed mass shifts were considered significant if they (a) passed a two-tailed t test (p < 0.05) using pooled standard deviations from the analyses of each state, (b) passed a distribution analysis to guard against spectral overlap, and (c) exceeded a threshold shift value (±2 s.d.) based on a measurement of the shift noise and assuming its normal distribution (30Bennett M.J. Barakat K. Huzil J.T. Tuszynski J. Schriemer D.C. Discovery and Characterization of the Laulimalide-Microtubule Binding Mode by Mass Shift Perturbation Mapping.Chem. Biol. 2010; 17: 725-734Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Water and acetonitrile, HPLC grade from Burdick and Jackson, were purchased from VWR. Formic acid, Tris, and glycine were purchased from Sigma Aldrich. The fluidic secretions of the pitcher plant were filtered, concentrated and the nepenthesin activated by pH reduction (pH 2.5), approximately the same pH as found in the wild. A low complexity and protein content of the activated fluid was observed by SDS-PAGE, with the presence of nepenthesin confirmed by LC-MS/MS (see Supplementary Methods). The total protein concentration of the activated and 80 × enriched fluid was measured by a BCA assay to be 22 ng/μl. The simplicity of the fluid proteome is expected based on earlier studies (31Hatano N. Hamada T. Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata.J. Proteome Res. 2008; 7: 809-816Crossref PubMed Scopus (90) Google Scholar, 32Hatano N. Hamada T. Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata.J. Proteom. 2012; (In press)Crossref PubMed Scopus (47) Google Scholar), in which nepenthesin was found to be a major component. For the purposes of our study, we assume 22 ng/μl represents the nepenthesin concentration in the 80 × enriched fluid, recognizing that it is likely an overestimate. Yields of this magnitude are consistent with earlier reports (33Tokes Z.A. Woon W.C. Chambers S.M. Digestive enzymes secreted by carnivorous plant Nepenthes-Macferlanei-L.Planta. 1974; 119: 39-46Crossref PubMed Scopus (69) Google Scholar). We then digested a series of proteins with the enriched fluid under conditions suitable for HDX-MS experiments, and characterized the digestion specificity of the concentrate at the P1 and P1′ positions (Fig. 1), to support a comparison with similar studies applied to pepsin (34Hamuro Y. Coales S.J. Molnar K.S. Tuske S.J. Morrow J.A. Specificity of immobilized porcine pepsin in H/D exchange compatible conditions.Rapid Commun. Mass Spectrom. 2008; 22: 1041-1046Crossref PubMed Scopus (112) Google Scholar). In our experiments, the enzyme-to-substrate ratio was 1:85 based on the above assumption that all the measured protein in the enriched fluid is nepenthesin. The nepenthesin data represents an assessment of 1612 residues and although not as extensive as the corresponding pepsin data (13,766 residues), the sequence diversity is sufficiently high in the protein set to warrant a comparison at the level of P1 and P1′ positions at least. The greatest specificity for pepsin is clearly in the P1 position. It presents high-efficiency cleavage for the hydrophobic residues F, L, and M but cleavage after P, H, K, and R is essentially forbidden. Nepenthesin cleaves after most residues with the exception of G, S, T, V, I, and W. It supports a high rate of cleavage after the expected pepsin P1 residues but also at the residues forbidden in pepsin digestion, notably K, R, and P. In the P1′ position, pepsin shows a preference for hydrophobic residues in general, including any residue with aromaticity. Conversely, nepenthesin demonstrates little in the way of selectivity at the P1′ position, except perhaps against G, P, and H. The significantly relaxed specificity relative to pepsin is remarkable for an aspartic protease, and the selectivity data provides an early indication of very high enzymatic efficiency. To determine if this relaxed specificity translates into improved sequence mapping for HDX-MS applications, we profiled full-length XRCC4, a protein that contains a globular domain, an extended helical stalk, and a long disordered C-terminal (27Hammel M. Yu Y. Fang S.J. Lees-Miller S.P. Tainer J.A. XLF Regulates Filament Architecture of the XRCC4.Ligase IV Complex.Structure. 2010; 18: 1431-1442Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 35Junop M.S. Modesti M. Guarne A. Ghirlando R. Gellert M. Yang W. Crystal structure of the Xrcc4 DNA repair protein and implications for end joining.EMBO J. 2000; 19: 5962-5970Crossref PubMed Scopus (149) Google Scholar). Such multidomain proteins are challenging to encompass in a single digestion protocol, and in particular, intrinsically disordered regions tend to digest poorly with pepsin as they are relatively depleted in hydrophobic residues and enriched in proline, and charged residues (36Dunker A.K. Lawson J.D. Brown C.J. Williams R.M. Romero P. Oh J.S. Oldfield C.J. Campen A.M. Ratliff C.R. Hipps K.W. Ausio J. Nissen M.S. Reeves R. Kang C.H. Kissinger C.R. Bailey R.W. Griswold M.D. Chiu M. Garner E.C. Obradovic Z. Intrinsically disordered protein.J. Mol. Graphics & Modelling. 2001; 19: 26-59Crossref PubMed Scopus (1836) Google Scholar). The pepsin and nepenthesin maps for this protein are displayed in Fig. 2. In this comparison, an exhaustive mapping was pursued for both enzymes, using a range of different protease/substrate ratios, and recursive MS/MS experiments. Nepenthesin provides superior coverage of the full length protein: 357 peptides for nepenthesin compared with 187 for pepsin, but with the same average peptide length (11 residues). Both enzymes represent the globular and stalk regions with a large number of overlapping peptides but the complementarity provided by nepenthesin is evident. For example, nepenthesin offers considerably deeper coverage of a β-sheet region in the globular domain (residues 1–30). The disordered C-terminal region is covered to a much greater extent as well, and to a considerably higher level of redundancy. On average, each residue in this disordered tail region receives 16X coverage using nepenthesin and only 4.7 × coverage with pepsin. The high cleavage efficiency C-terminal to basic residues prompted us to explore if there exists any bias in peptide detection. This could be tested in several ways, but we chose to select average search score as the metric (Fig. 3). The approach emphasizes confidence in sequence identification as the principal means by which sequence maps are defined. There is only one outlier, R. The higher scores for peptides terminating in R likely reflect a combination of higher average peptide intensity and better fragmentation, which is consistent with what we know from trypsin-based bottom-up proteomics (37Warwood S. Mohammed S. Cristea I.M. Evans C. Whetton A.D. Gaskell S.J. Guanidination chemistry for qualitative and quantitative proteomics.Rapid Commun. Mass Spectrom. 2006; 20: 3245-3256Crossref PubMed Scopus (34) Google Scholar). We then examined enzyme efficiency in greater detail and the degree to which the peptide mass map could be varied, or tuned, simply by altering the enzyme-to-substrate ratio (Fig. 4). Nepenthesin load was varied over a 50-fold range for in-solution digestions. For the pepsin experiment, immobilized pepsin in a slurry format was used rather than free pepsin so that we avoided extensive pepsin autolysis. The enzyme load was varied over an eightfold range; lower amounts led to poor peptide intensities and higher amounts had no ef
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