WO2020198865A1 - Oligopeptides pour procédés d'analyse protéomique virale quantitative et utilisations - Google Patents

Oligopeptides pour procédés d'analyse protéomique virale quantitative et utilisations Download PDF

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WO2020198865A1
WO2020198865A1 PCT/CA2020/050431 CA2020050431W WO2020198865A1 WO 2020198865 A1 WO2020198865 A1 WO 2020198865A1 CA 2020050431 W CA2020050431 W CA 2020050431W WO 2020198865 A1 WO2020198865 A1 WO 2020198865A1
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protein
amount
peptides
level
biological sample
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François JEAN
Steven J. MCARTHUR
Leonard J. Foster
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The University Of British Columbia
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus

Definitions

  • the present invention relates to a series of oligopeptides, labelled oligopeptides, and stable isotope labelled oligopeptides and methods of using the same.
  • the invention relates to oligopeptides derived from flavivirus and coronavirus amino acid sequences.
  • Flaviviridae family of viruses are positive, single-stranded, enveloped RNA viruses found primarily in ticks and mosquitoes, which can lead to human infections (1).
  • members of the genus Flavivirus include viruses, like dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassan virus (POWV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV), which are important human pathogens (1-3).
  • Most human flavivirus infections are incidental, since the viruses are unable to replicate the virus to high enough titers in their human host.
  • DENV dengue virus
  • WNV West Nile virus
  • ZIKV Zika virus
  • POWV Powassan virus
  • JEV Japanese encephalitis virus
  • YFV yellow fever virus
  • flaviviruses are transmitted by an arthropod vector, and for that reason are also classified as arboviruses (1, 4).
  • arboviruses there are readily available and effective vaccines (i.e. YFV and JEV).
  • YFV and JEV a vaccine for ZIKV and the DENV vaccine
  • Dengvaxia which is a tetravalent chimeric vaccine that splices structural genes of the four dengue viruses onto a 17D yellow fever backbone, has proved to be less than optimal (5, 6).
  • the development of flavivirus vaccines as well as novel therapeutic approaches, including direct- and indirect-acting antivirals, could benefit from a better understanding of viral protein expression and viral proteolytic maturation in human hosts (7).
  • a key determinant of flaviviral infectivity is the proteolytic maturation of the virus-associated structural precursor membrane (prM) glycoprotein, which is cleaved by host protease(s) as nascent virions traffic through the secretory pathway (8-10). While this proteolysis is thought to be primarily mediated by the ubiquitous membrane-anchored cellular proprotein convertase (PC) furin (1, 11, 12), prM proteolysis mediated by other human PCs has not been
  • the currently accepted model for flaviviral prM activation proposes that prM endoproteolysis is mediated in the trans-Golgi network (TGN) by furin, yielding two products: soluble pr, and membrane-anchored M protein (8, 11-15).
  • Furin is predominantly localized to the TGN at steady state.
  • furin is not statically retained in the TGN; it traffics between two local cycling loops, one at the TGN and the other at the cell surface (16).
  • the prM proteolysis event is required for the fusogenicity of the virion, allowing pr to dissociate from its interaction with domain II of the flaviviral E protein and exposing the fusion peptide (8-10, 17-20).
  • Absolute quantification of dengue virus serotype 4 and multiplexed targeted mass spectrometry assay for one-shot flavivirus diagnosis have been previously shown (21, 22).
  • the inventors established a methodology to evaluate, for the first time, the proteolytic cleavage efficiency of DENV-1-4 prM in a human cell culture-based setting. Previous studies have universally relied on immunoblot-based means of detecting prM (11, 23, 24). Unfortunately, the quantitativeness of such approaches is relative at best and relies upon successful interaction between the molecule of interest and an antibody, therefore depending on the specificity and affinity of the antibody for its target (25). Since no antibodies targeting immature prM and mature M of all four DENV serotypes were available at the time, and given the lack of inter-serotype quantitativeness that would undermine conclusions drawn with such a methodology, the inventors chose a targeted quantitative proteomics approach (26, 27). This approach is referred to as multiple reaction monitoring mass spectrometry (MRM-MS); other names for this technique include SRM-MS (selected reaction monitoring) and PRM-MS (parallel reaction monitoring) (22, 26, 27).
  • MRM-MS multiple reaction monitoring mass spectrometry
  • the inventors developed and optimized specific MRM assay methods directed against the tryptic peptide immediately C-terminal to the furin cleavage site in prM (the N-terminal peptide of the proteolysis product M - see FIGURE 1). Since furin cleavage occurs following Arg in the conserved prM consensus sequence (-R-[D/E]-K-R-j-S-V-A-L- (SEQ ID NO: 126)) that will also be cleaved by trypsin, the endogenous furin-generated and in vitro trypsin- generated M peptides would be identical following trypsin digestion.
  • N-terminus of endogenous furin-cleaved M labelling the exposed N-terminus of endogenous furin-cleaved M with an acetyl group, allows differential detection of endogenous furin-cleaved and in vitro trypsin-cleaved peptides by MS (FIGURE 2).
  • proteinase for example, Arg-C proteinase or clostripain
  • trypsin Arg-C proteinase or clostripain
  • a method for determining the maturation status of a flavivirus in a human biological sample including: N- terminal acetyl labelling of the human biological sample; trypsin digestion of the human biological sample; adding stable isotope labelled flavivirus peptides; detecting and quantifying the amount of a flavivirus protein fragments in the human biological sample, using mass spectrometry; and calculating the level of the flavivirus protein in said sample; wherein the flavivirus peptides are one or more of the peptides selected from SEQ ID NOs: 1- 102, and wherein said amount is a relative amount or an absolute amount.
  • a method for measuring the level of the flavivirus protein in a human biological sample including detecting and quantifying the amount of a flavivirus protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of flavivirus protein in said sample; wherein the flavivirus peptide is one or more of the peptides of those presented in TABLES 10A-10D, 11, 12 and 13, and wherein said amount is a relative amount or an absolute amount.
  • a method for measuring the level of the DENV protein in a human biological sample including detecting and quantifying the amount of a DENV protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of DENV protein in said sample; wherein the DENV peptide is one or more of the peptides of those presented in TABLE 10A- 10D, and wherein said amount is a relative amount or an absolute amount.
  • a method for detecting the presence and measuring the level of DENV protein and truncated DENV protein in a human biological sample including detecting and quantifying the amount of a DENV fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry; and calculating the level of full length and truncated DENV protein in said sample wherein said level is an absolute level, wherein the DENV peptide is one or more of the peptides of those presented in TABLE 10A-10D.
  • the flavivirus peptide may be one or more of the peptides of those presented in TABLES 10A.
  • the flavivirus peptide may be one or more of the peptides of those presented in TABLES 10B.
  • the flavivirus peptide may be one or more of the peptides of those presented in
  • the flavivirus peptide may be one or more of the peptides of those presented in TABLES 10D.
  • a method for measuring the level of the ZIKV protein in a human biological sample including detecting and quantifying the amount of a ZIKV protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of ZIKV protein in said sample; wherein the ZIKV peptide is one or more of the peptides of those presented in TABLE 11, and wherein said amount is a relative amount or an absolute amount.
  • a method for detecting the presence and measuring the level of ZIKV protein and truncated ZIKV protein in a human biological sample including detecting and quantifying the amount of a ZIKV fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry; and calculating the level of full length and truncated ZIKV protein in said sample wherein said level is an absolute level, wherein the ZIKV peptide is one or more of the peptides of those presented in TABLE 11.
  • a method for measuring the level of the WNV protein in a human biological sample including detecting and quantifying the amount of a WNV protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of WNV protein in said sample; wherein the WNV peptide is one or more of the peptides of those presented in TABLE 12, and wherein said amount is a relative amount or an absolute amount.
  • a method for detecting the presence and measuring the level of WNV protein and truncated WNV protein in a human biological sample including detecting and quantifying the amount of a WNV fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry; and calculating the level of full length and truncated WNV protein in said sample wherein said level is an absolute level, wherein the WNV peptide is one or more of the peptides of those presented in TABLE 12.
  • a method for measuring the level of the POWV protein in a human biological sample including detecting and quantifying the amount of a POWV protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of POWV protein in said sample; wherein the POWV peptide is one or more of the peptides of those presented in TABLE 13, and wherein said amount is a relative amount or an absolute amount.
  • a method for detecting the presence and measuring the level of POWV protein and truncated POWV protein in a human biological sample including detecting and quantifying the amount of a POWV fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry; and calculating the level of full length and truncated POWV protein in said sample wherein said level is an absolute level, wherein the POWV peptide is one or more of the peptides of those presented in TABLE 13.
  • oligopeptide is selected from one or more of those presented in TABLES 10A-10D and 11-13
  • oligopeptide is selected from one or more of those presented in TABLES 10A-10D and 11-13
  • a stable isotope labelled oligopeptide wherein the oligopeptide is selected from one or more of those presented in
  • a method of diagnosing a flavivirus infection in a subject including: measuring the level of the flavivirus protein in a human biological sample, including detecting and quantifying the amount of a flavivirus protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of flavivirus protein in said sample;
  • the flavivirus peptide is one or more of the peptides of those presented in TABLES 10A-10D, 11, 12 and 13, and wherein said amount is a relative amount or an absolute amount.
  • a method for determining the maturation status of a flavivirus in a human biological sample including: N- terminal acetyl labelling of the human biological sample; Trypsin digestion of the human biological sample; adding stable isotope labelled flavivirus prM, pr and M peptides; detecting and quantifying the amount of a flavivirus prM, pr and M protein fragments, using mass spectrometry; and calculating the level of the flavivirus prM, pr and M protein in said sample; wherein the flavivirus prM, pr and M peptides may be one or more of the peptides selected from SEQ ID NOs: l, 2, 18, 19, 29, 30, 45, 46, 61, 62, 74, 75, 90 and 91, and wherein said amount is a relative amount or an absolute amount.
  • the flavivirus prM, pr and M peptides may be one or more of the peptides selected from SEQ ID NOs: 1, 2, 3, 18, 19, 20, 29, 30, 43, 44, 45, 46, 47, 61, 62, 63, 64, 74, 75, 76, 77 90, 91 and 92.
  • a method for testing an attenuated flavivirus vaccine sample including: N-terminal acetyl labelling of the attenuated flavivirus vaccine sample; trypsin digestion of the attenuated flavivirus vaccine sample;
  • flavivirus peptides are one or more of the peptides may be selected from SEQ ID NOs: 1-102, and wherein said amount is a relative amount or an absolute amount is an indication of the maturation status of the attenuated flavivirus vaccine.
  • a method for determining the maturation status of a flavivirus in a human biological sample including: N-terminal acetyl labelling of the human biological sample; Trypsin digestion of the human biological sample; adding stable isotope labelled flavivirus peptides; detecting and quantifying the amount of a flavivirus protein fragments in the human biological sample, using mass spectrometry; and calculating the level of the flavivirus protein in said sample; wherein the flavivirus peptides are one or more of the peptides selected from SEQ ID NOs: 1,2, 18, 19, 29, 30, 45, 46, 61, 62, 74, 75, 90 and 91, wherein said amount is a relative amount or an absolute amount.
  • a method for determining the maturation status of a coronavirus in a human biological sample including: N- terminal acetyl labelling of the human biological sample; Trypsin digestion of the human biological sample; adding stable isotope labelled coronavirus peptides; detecting and quantifying the amount of a coronavirus protein fragments in the human biological sample, using mass spectrometry; and calculating the level of the coronavirus protein in said sample; wherein the coronavirus peptides are one or more of the peptides selected from SEQ ID NOs: 103-120, and wherein said amount is a relative amount or an absolute amount.
  • a method for measuring the level of the coronavirus protein in a human biological sample including detecting and quantifying the amount of a coronavirus protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of coronavirus protein in said sample; wherein the coronavirus peptide is one or more of the peptides of those presented in TABLE 14, and wherein said amount is a relative amount or an absolute amount.
  • a method for measuring the level of the SARS-CoV-2 protein in a human biological sample including detecting and
  • SARS-CoV-2 protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of SARS-CoV-2 protein in said sample; wherein the SARS-CoV-2 peptide is one or more of the peptides of those presented in TABLE 14, and wherein said amount is a relative amount or an absolute amount.
  • a method for detecting the presence and measuring the level of SARS-CoV-2 protein and truncated SARS-CoV-2 protein in a human biological sample including detecting and quantifying the amount of a SARS- CoV-2 fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry; and calculating the level of full length and truncated SARS-CoV-2 protein in said sample wherein said level is an absolute level, wherein the SARS-CoV-2 peptide is one or more of the peptides of those presented in TABLE 14.
  • oligopeptide is selected from one or more of those presented in TABLE 14.
  • oligopeptide is selected from one or more of those presented in TABLE 14.
  • a stable isotope labelled oligopeptide wherein the oligopeptide is selected from one or more of those presented in
  • a method of diagnosing a coronavirus infection in a subject including: measuring the level of the coronavirus protein in a human biological sample, including detecting and quantifying the amount of a coronavirus protein fragment peptide in a protein digest prepared from said biological sample, using mass spectrometry; and calculating the level of coronavirus protein in said sample; wherein the coronavirus peptide is one or more of the peptides of those presented in TABLE 14, and wherein said amount is a relative amount or an absolute amount.
  • FIGURE 1 shows an overview of proteotypic peptides, wherein (A) shows proteotypic peptides corresponding to M and nonstructural protein 1 (NS1) for DENV-1-4 in the context of the DENV proteome, wherein the M peptide is the N-terminal tryptic peptide of M, immediately following the site of host-mediated proteolysis (scissors); and (B) shows proteotypic peptides mapped to 3D structures of prM-E and M-E complexes as well as NS1, wherein the left panel shows the immature DENV-1 cryo-EM structure (PDB 4B03), the centre panel shows the mature DENV-1 cryo-EM structure (PDB 4CCT) and the right panel shows the ZIKV NS1 dimer crystal structure (PDB 5GS6).
  • A shows proteotypic peptides corresponding to M and nonstructural protein 1 (NS1) for DENV-1-4 in the context of the DENV proteome, wherein the M peptide is
  • FIGURE 2 shows an overview of NTAc labelling approach, wherein biological samples contain an amount of cleaved (mature) M as well as uncleaved (immature) prM.
  • N-terminal acetyl (NTAc) labelling covalently adds an acetyl (Ac) moiety to exposed primary amines, including protein N-termini, and subsequent trypsin digestion cleaves all pr-M junctions not already cleaved by host protease(s), yielding the same proteotypic peptide lacking an acetyl label; the acetyl label allows differential quantification by MRM-MS of the mature and immature peptide.
  • FIGURE 3 shows extracted ion chromatograms demonstrating multiplexed detection and quantification of SIS peptides by MRM-MS;
  • A-B DENV-1 peptides.
  • Peptide 1-NSl is proteotypic for both DENV-1 and DENV-2;
  • C-D DENV-2 peptides;
  • E-F DENV-3 peptides;
  • G-H DENV-4 peptides, wherein all chromatograms were obtained in a single run of a single sample comprising 100 fmol/pL of each SIS peptide, with one representative of four replicate injections shown.
  • FIGURE 4 shows NTAc-MRM analysis of DENV-1-4 reveals serotype-specific prM proteolytic maturation rates, where Huh-7.5.1 or LoVo cells were infected with (A) DENV-1, (B) DENV-2, (C) DENV-3, or (D) DENV-4 at MOI 0.1 for 4 days; the media were then collected and analyzed by NTAc-MRM; concentrations of (A) 1-M/l-M-Ac (1D2/1 AcD2), (B) 2-M/2-M-Ac (2D2/2AcD2), (C) 3-M/3-M-Ac (3D2/3AcD2), and (D) 4-M/4-M-Ac (4D2/4AcD2) in media are shown.
  • Error bars represent SD among 2-3 replicate injections. Hatched bars representing LOQ are shown where values below LOQ were obtained. Concentrations in fmol/pL are annotated above bars where applicable.
  • FIGURE 5 shows MRM-MS analysis of DENV-1-4 reveals a lack of NS1 in furin-deficient LoVo cells, wherein the Huh-7.5.1 or LoVo cells were infected with (A) DENV-1, (B)
  • stable isotope-labeled standard (SIS) peptides refers to peptides that have been synthesized using stable isotope-labeled amino acids to produce a peptide that has a greater mass than a corresponding unlabeled target peptide for use as an internal standard to quantify the amount of a target peptide within a sample.
  • Suitable isotopes are non-radioactive chemical isotopes that do not decay spontaneously.
  • stable isotopes that may be used for the study of biological systems include those of 18 0, 13 C, 15 N, 2 H and 32 S.
  • a“subject” refers to an animal, such as a bird or a mammal.
  • Specific animals include rat, mouse, dog, cat, cow, sheep, horse, pig or primate.
  • a subject may further be a human, alternatively referred to as a patient.
  • a subject may further be a transgenic animal.
  • a subject may further be a rodent, such as a mouse or a rat.
  • peptide may be used interchangeably, and refer to a compound comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds, for example peptide isosteres (modified peptide bonds) that may provide additional desired properties to the peptide, such as increased half-life.
  • a peptide may comprise at least two amino acids.
  • the amino acids comprising a peptide or protein described herein may also be modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.
  • Nonstandard amino acids may occur in nature, and may or may not be genetically encoded.
  • Examples of genetically encoded nonstandard amino acids include selenocysteine, sometimes incorporated into some proteins at a UGA codon, which may normally be a stop codon, or pyrrolysine, sometimes incorporated into some proteins at a UAG codon, which may normally be a stop codon.
  • Some nonstandard amino acids that are not genetically encoded may result from modification of standard amino acids already incorporated in a peptide, or may be metabolic intermediates or precursors, for example.
  • nonstandard amino acids examples include 4-hydroxyproline, 5-hydroxylysine, 6-N-methyllysine, gamma- carboxy glutamate, desmosine, selenocysteine, ornithine, citrulline, lanthionine, 1- aminocyclopropane-1 -carboxylic acid, gamma-aminobutyric acid, carnitine, sarcosine, or N- formylmethionine.
  • Synthetic variants of standard and non-standard amino acids are also known and may include chemically derivatized amino acids, amino acids labeled for identification or tracking, or amino acids with a variety of side groups on the alpha carbon.
  • side groups are known in the art and may include aliphatic, single aromatic, polycyclic aromatic, heterocyclic, heteronuclear, amino, alkylamino, carboxyl, carboxamide, carboxyl ester, guanidine, amidine, hydroxyl, alkoxy, mercapto-, alkylmercapto-, or other heteroatom-containing side chains.
  • Other synthetic amino acids may include alpha-imino acids, non-alpha amino acids such as beta-amino acids, des-carboxy or des-amino acids. Synthetic variants of amino acids may be synthesized using general methods known in the art, or may be purchased from commercial suppliers, for example RSP Amino Acids LLC
  • Proteotypic peptide candidates were selected that met all of the following criteria: length between 7 and 30 amino acids; not more than one oxidizable residue (Met, Cys, or Trp); no Asp-Pro motif; not more than one Pro-Pro motif; no N-terminal Gin; no putative N-glycosylation sites; and a hydrophobicity score between 15 and 45, as calculated by the SSRCalc algorithm (30).
  • Each proteotypic peptide candidate was then used as the query in a BLASTP® search (31) against the non-redundant (nr) US National Center for Biotechnology Information (NCBI) protein database. Peptides for which the only 100% coverage, 100% identity hits were associated with the appropriate DENV serotype were considered to be proteotypic.
  • heavy (SIS) peptides bearing a C- terminal Arg-[ 13 C 6 15 N4] or Lys-[ 13 C6 15 N2] were custom synthesized by Thermo Fisher ScientificTM and delivered at >98% purity, pre-quantified by amino acid analysis and pre solubilized in 5% acetonitrile (ACN) at 5 pmol/pL.
  • Human hepatoma Huh-7.5.1 cells were kindly provided by Dr. Francis Chisari (Scripps Research Institute, La Jolla, CA, USA) (33); these and African green monkey kidney Vero E6 cells (ATCC #CRL-1586) were maintained as previously described (34).
  • Human colorectal carcinoma LoVo cells that do not produce functional furin ATCC #CCL-229 were maintained in Minimum Essential Medium Alpha (MEM-a) supplemented with 1% each of penicillin, streptomycin, L-glutamine, and 10% FBS (Gibco/InvitrogenTM).
  • DENV-1 strain Hawaiian-3, DENV-2 strain NGC, DENV-3 strain H-87, and DENV-4 strain H-241 were kindly provided by Dr. Mike Drebot (National Microbiology Laboratory, Winnipeg, MB, Canada).
  • Vero E6 cells were cultured in 175 cm 2 flasks to 90% confluence. After the culture medium was removed, the cells were washed with PBS, and an inoculum of 3 mL of culture medium without FBS with 200 pL DENV stock was added. Inoculated cells were incubated at 37°C for 1 h, with the flask gently rocked every 15 min to allow even distribution of the virus. Without removing the inoculum, 30 mL of fresh medium with 2% FBS was then added and the infected cells were cultured for 4 days. The medium was then collected and clarified by centrifuging at 1500 g, 15 min, 4°C before being aliquoted and snap-frozen. Viral stocks were stored at -86°C. Viral titres were determined by plaque assay, performed in Vero E6 cells using the protocol described by Medina et al. (35).
  • Huh-7.5.1 cells or LoVo cells were plated at 5x 10 4 or 1 c 10 4 cells/well in 12- or 24-well plates, respectively. After the culture medium was removed, the cells were washed with PBS, and 2 mL of fresh culture medium (including 10% FBS) containing the appropriate amount of DENV stock was added. Infected cells were maintained for 4 days at 37°C with 5% CO2, after which the medium was collected and clarified by centrifuging at 1500 g for 15 min at 4°C. Samples were aliquoted; portions destined for LC-MS analysis were rendered non- infectious by heat inactivation (99°C for 10 min) (35) before being processed immediately as described below.
  • the sample was then denatured by heating to 99°C for 5 min.
  • Thiol groups were reduced by adding dithiothreitol (0.5 pg) followed by incubating (37°C, 30 min in an air incubator to prevent condensation on the lid).
  • Reduced thiols were then alkylated by adding
  • iodoacetamide (2.5 pg) followed by incubating (37°C, 30 min). Trypsin digestion was performed by adding sequencing-grade modified porcine trypsin (Promega CorporationTM, Madison, WI, USA) (minimum 0.5 pg; final proteimtrypsin ratio at least 1 :50 w/w) and incubating for 18 h at 37°C. Tryptic digests were then acidified to pH ⁇ 2.5 with 0.5% formic acid (FA)/3% acetonitrile (ACN) and centrifuged (16000 g, 10 min) to stop the trypsin digestion and precipitate out the deoxycholic acid.
  • FA formic acid
  • ACN acetonitrile
  • a SIS peptide cocktail of 100 fmol/pL of each SIS peptide was prepared fresh in 0.5% FA, and the appropriate amount was added to each sample.
  • Solid-phase extraction and desalting using self-made C18 stop-and-go extraction (STAGE) tips containing EmporeTM Cl 8 SPE material (3M CompanyTM, Maplewood, MN, USA) was performed as described elsewhere (36, 37), eluting each sample with 2x 10 pL of 70% ACN. Samples were then dried by vacuum evaporation without heating for 1 h. Dried samples were reconstituted in 20 pL LC Buffer A (0.1% FA/3% ACN) and sonicated for 90 s to ensure thorough reconstitution.
  • Peptides were separated by nano-HPLC on a water/ ACN/0.1% FA mobile phase using an HPLC Chip II (G4240-62010, Agilent; 160 nL enrichment column, 75 pm x 150 mm analytical column packed with ZorbaxTM 300SB-C18 5 pm material, pore size 300 A) in a Chip CubeTM (G4240A) ESI ion source.
  • Peptides were enriched at 2 pL/min in 3% buffer B before being analyzed at 300 nL/min using a 55 min gradient of 3-80% B, followed by a 10- min wash and re-equilibration of the trap and analytical columns before injecting the next sample.
  • the dominant precursor charge state for each peptide was determined by analyzing 500 fmol of SIS peptide alone in MS/MS scan mode. Fragmentation patterns were obtained in product ion scan mode, using arbitrary fragmentor voltage (FV) and collision energy (CE) settings of 175 V and 10, 20, and 30 V, respectively. Peak identities were assigned manually; of these, the strongest 3-5 assignable peaks were selected for MRM.
  • FV fragmentor voltage
  • CE collision energy
  • Optimal FV and CE settings for each transition were then determined.
  • a series of MRMs for each product ion were created with CE varied in 2 V intervals from 5-35 V. This information was used to construct the fully multiplexed MRM methods, including heavy and light peptides: one MRM method targeting all four DENV serotypes (TABLE 2), and one for each individual DENV serotype (TABLES 3-6)
  • EIC Extracted ion chromatograms
  • peptide elution was verified by the co-elution of at least 3 transitions.
  • EIC were smoothed (quartic/quintic Savitsky-Golay algorithm over 15 points) and then manually integrated on the strongest transition.
  • a secondary transition was consistently used for integration (e.g. peptides 2-M and 4-M).
  • Light peptide concentration was calculated by determining the lightheavy peak area ratio and dividing this value by the known concentration of the spiked-in heavy peptide.
  • SNR Signal-to-noise ratios
  • concentration/buffer exchange into a sodium carbonate buffer (20 mM, pH 8.4) was performed on 10 kDa MWCO centrifugal filter units to a final volume of 51 pL, of which 1 pL was taken for protein quantification by BCA assay as described above.
  • 50 pL of a freshly prepared sulfo-N- hydroxysuccinimide (NHS) acetate (SigmaTM) solution was added to a final concentration of 0.1 mg/mL and incubated at room temperature for 2 h.
  • Samples were then exchanged into ABC buffer, quenching any unreacted sulfo-NHS acetate, and concentrated to 25 pL on 10 kDa MWCO centrifugal filter units. Subsequent sample preparation (denaturation, reduction, alkylation, trypsinization, SIS spike-in, desalting, and LC-MS analysis) were performed as noted above, with the inclusion of N-acetylated forms of each SIS peptide (Thermo Fisher ScientificTM) in the spike-in peptide cocktail. Although certain peptides are indicated as having an N-terminal acetylation (i.e.“Ac-”), other peptides may be similarly acetylated on their N-terminus, but are not exemplified herein.
  • the MRM-MS assay was designed to target the N-terminal tryptic peptide of M, immediately C-terminal to the prM proteolytic cleavage site (FIGURE 1A). Furthermore, the MRM-MS assays were designed to target proteotypic peptides derived from NS1, identified as proteotypic peptide candidates by an in silico digest and manual curation (FIGURE 1A). Peptides were confirmed to be proteotypic by performing a BLASTP search against the nr database; each peptide met the standard for uniqueness and was considered proteotypic if the only 100% coverage, 100% identity hits occurred against the correct DENV protein.
  • the proteotypic peptide we selected for detecting DENV-1 NS1 is precisely conserved in DENV-2; the same peptide (1-NSl) was therefore used to detect and quantify both DENV-1 and DENV-2 NS1 (FIGURE 1A).
  • the mapping of these peptides on the 3D structures of flaviviral prM and NS1 is shown in FIGURE IB.
  • the DENV-1-4 M peptides are located immediately following the host proteolytic cleavage site.
  • the DENV-1-3 NS1 peptides are derived from the wing domain while the DENV-4 NS1 peptide is located within the intertwined loop within the wing domain (38).
  • NTAc N-terminal acetyl
  • FIGURE 2 To allow differential quantification of mature and immature M, we adapted a protocol for the in vitro N-terminal acetyl (NTAc) labelling of peptide substrates, based on a methodology commonly used in positional proteomics (39, 40). This methodology is summarized in FIGURE 2. Briefly, primary amines including all protein N-termini within each sample were covalently modified with acetyl groups through the addition of sulfo-N-hydroxysuccinimide (sulfo-NHS) acetate. This includes the N-terminus of endogenously cleaved M.
  • sulfo-NHS sulfo-N-hydroxysuccinimide
  • NTAc labelling is therefore to distinguish the trypsin cleavage product (unlabelled N-NEh) from the endogenous cleavage product (N-Ac label) (FIGURE 2).
  • MRM methods were initially developed using the purified, quantified SIS form of each peptide, in which the C-terminal Arg or Lys residue is 13 C/ 15 N-labelled. Selecting for the dominant precursor charge state observable in MS/MS scan mode, product ion scans were performed to determine the top three strongest transitions for each peptide. These were then individually optimized dissociation and fragmentation voltage parameters for each of these (see TABLES 2-6).
  • MRM acquisition method for DENV-3 only including NTAc peptides.
  • Dwell time 20 ms.
  • MRM acquisition method for DENV-4 only including NTAc peptides.
  • Dwell time 20 ms.
  • Signal-to-noise ratios were calculated in peak-to-peak mode using an interference-free 2-min region from the first 5 minutes of each run as a noise baseline.
  • Heavy (SIS) to light (endogenous) peak area ratios were calculated and averaged among experimental replicates, then averaged and plotted against SIS concentration as response curves (data not shown).
  • Response factor (RF) plots were also generated to determine the range of linearity in each response curve (data not shown); RF values within 20% of the target concentration response were considered to be linear.
  • LOD Lower limit of detection
  • SNR signal-to-noise ratio
  • LOQ lower limit of quantification
  • human hepatoma Huh-7.5.1 cells were infected with DENV-1, -2, -3, or -4 at MOI 0.1 for 96 h before cell culture supernatant was harvested.
  • Samples were prepared by denaturation, reduction and alkylation of cysteine thiol groups, and trypsinization for 18 h to produce tryptic peptides. Tryptic digests were then spiked with a cocktail of heavy (SIS) peptides bearing a C-terminal Arg-[ 13 C 6 15 N4] or Lys-[ 13 C6 15 N2] such that the final on-column amount would be 50 fmol per injection for each peptide. Samples were then desalted by solid-phase extraction and analyzed by a pan-serotypic MRM-MS assay targeting all 21 DENV-1-4 peptides.
  • SIS cocktail of heavy
  • TABLE 8 shows the partial primary amino acid sequence of the prM proteolytic cleavage site in the four DENV serotypes and WNV, wherein the furin cleavage site is indicated by the downwards arrow and our proteotypic peptide for NTAc-MRM is underlined.
  • the NTAc-MRM assays were used to elucidate the putative role of furin in the proteolytic maturation of the four DENV serotypes. To do so, the absolute level of prM maturation in viral progeny derived from DENV-l-4-infected human hepatoma Huh-7.5.1 cells was determined, and this was compared with the maturation of DENV-1-4 derived from furin- deficient human colorectal adenocarcinoma LoVo cells. Following infection at an MOI of 0.1, cell culture supernatant was collected 96 h post-infection and prepared for LC-MS analysis, including NTAc labelling of endogenously cleaved M prior to trypsin digestion.
  • DENV-3 prM is moderately cleaved during infection of Huh-7.5.1 cells, with a rate of maturation around 60% (FIGURE 4C and TABLE 9).
  • a similarly dramatic reduction in maturation efficiency was also observed, with mature M (3-M-Ac) peptide levels again below the assay LOQ, indicating that maturation efficiency was no more than 35%.
  • the results of the in silico trypsin digest and manual curation are shown in TABLE 14.
  • the criteria used to select peptides for the MRM- MS assay for SARS-CoV-2 were length in the range 6-35 residues; not more than one total instance of any of M, C, W, DP, PP; no N-terminal Q; hydrophobicity score (SSRCalc) in the range 15-45; and uniqueness (i.e. only 100% coverage, 100% identity hits in a BLASTP search of the nr database are on 2019-nCoV or related coronaviruses (CoV only)).
  • DENV-1 prM maturation was unaffected by the absence of furin in LoVo cells, whereas DENV-2 and DENV-3 were confirmed as undergoing furin-dependent maturation. It was also found that the extracellular abundance of mature and immature M as well as NS1 was significantly reduced in a furin- deficient cell line, suggesting that furin plays a broader role in the DENV lifecycle than simply cleaving prM, seemingly impacting protein biosynthesis or secretion by an unknown mechanism.
  • cysteine (C) residues are constitutively oxidized to carbamidomethylcysteine (C-CAM).
  • Methionine residues (M) can be in any oxidation state; that is, methionine, methionine sulfoxide, or methionine sulfone. N-terminal acetylation is denoted by“Ac”. No other modifications are present. Peptide positions within the context of the full viral proteome are shown.
  • NTAc-MRM i.e. SEQ ID NO: 119
  • flavivirus precursor membrane-envelope protein complex structure and maturation. Science (80- ) 319(5871): 1830-4.
  • Subtilisin Kexin Isozyme-1 SKI-1)/Site-1 Protease (SIP) regulates cytoplasmic lipid droplet abundance: A potential target for indirect-acting anti-dengue virus agents.

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Abstract

L'invention concerne des oligopeptides purifiés, des oligopeptides marqués et des oligopeptides marqués par un isotope stable, des procédés pour leur utilisation dans la détermination de l'état de maturation de flavirus ou de coronavirus dans un échantillon biologique humain. Les oligopeptides purifiés, les oligopeptides marqués et les oligopeptides marqués par un isotope stable peuvent également être utilisés pour mesurer le niveau de la protéine de flavirus ou de la protéine de coronavirus dans un échantillon biologique humain. L'invention concerne également des méthodes de traitement d'un sujet sur la base de l'état de maturation des flavirus ou des coronavirus dans l'échantillon biologique du sujet et/ou sur la base du niveau de la protéine de flavirus ou de coronavirus dans l'échantillon biologique du sujet.
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US11866485B2 (en) 2021-10-01 2024-01-09 Academia Sinica Antibody specific to spike protein of SARS-CoV-2 and uses thereof

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