WO2020198865A1 - Oligopeptides for quantitative viral proteomic analysis methods and uses - Google Patents

Abstract

Provided are purified oligopeptides, labelled oligopeptides, and stable isotope labelled oligopeptides, methods for their use in determining the maturation status of the flaviruses or coronaviruses in a human biological sample. The purified oligopeptides, labelled oligopeptides, and stable isotope labelled oligopeptides may also be used to measure the level of the flavirus protein or coronavirus protein in a human biological sample. Methods are also provided for treating a subject based on the maturation status of the flaviruses or the coronaviruses in the subject's biological sample and/or based on the level of the flavirus or coronavirus protein in the subject's biological sample.

Description

OLIGOPEPTIDES FOR QUANTITATIVE VIRAL PROTEOMIC ANALYSIS

METHODS AND USES

TECHNICAL FIELD

The present invention relates to a series of oligopeptides, labelled oligopeptides, and stable isotope labelled oligopeptides and methods of using the same. In particular, the invention relates to oligopeptides derived from flavivirus and coronavirus amino acid sequences.

BACKGROUND

The Flaviviridae family of viruses are positive, single-stranded, enveloped RNA viruses found primarily in ticks and mosquitoes, which can lead to human infections (1). For example, 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. However, there are exceptions in the case of DENV, WNV, ZIKV, and YFV. Most flaviviruses are transmitted by an arthropod vector, and for that reason are also classified as arboviruses (1, 4). For some flaviviruses, there are readily available and effective vaccines (i.e. YFV and JEV). However, there is no 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

conclusively ruled out. 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. However, 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).

SUMMARY

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).

To allow differential detection and quantification of immature prM and mature M, 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. However, in vitro N- terminal acetyl (NT Ac) labelling of DENV protein extracts before trypsinization, 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). Moreover, by developing MRM assays targeting the NTAc-labelled light peptides with the corresponding NTAc-labelled heavy (SIS) peptides, differential quantification of furin- cleaved M and uncleaved prM peptides was possible, allowing absolute quantification of proteolytic cleavage of DENV prM in biological samples for the first time. The inventors developed, optimized, and applied this NTAc-MRM methodology to the characterization of the prM proteolytic maturation state of DENV-1-4 progeny and further developed the methodology for the analysis of ZIKV, WNV and POWV derived from cultured human cells. This allowed them to test their research hypothesis that DENV-1-4 prM proteolytic maturation was not equivalent across the four serotypes and that it was not universally and exclusively furin-dependent.

Alternatively, another proteinase (for example, Arg-C proteinase or clostripain) may be used instead of trypsin.

In accordance with one embodiment, there is provided a method for determining the maturation status of a flavivirus in a human biological sample, the method 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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

TABLES IOC. The flavivirus peptide may be one or more of the peptides of those presented in TABLES 10D.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided a purified oligopeptide, wherein the oligopeptide is selected from one or more of those presented in TABLES 10A-10D and 11-13

In accordance with a further embodiment, there is provided a labelled oligopeptide, wherein the oligopeptide is selected from one or more of those presented in TABLES 10A-10D and 11-13

In accordance with a further embodiment, there is provided a stable isotope labelled oligopeptide, wherein the oligopeptide is selected from one or more of those presented in

TABLES 10A-10D and 11-13

In accordance with a further embodiment, there is provided a method of diagnosing a flavivirus infection in a subject, the method 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;

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.

In accordance with a further embodiment, there is provided a method for determining the maturation status of a flavivirus in a human biological sample, the method 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. Alternatively, 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.

In accordance with a further embodiment, there is provided a method for testing an attenuated flavivirus vaccine sample, the method including: N-terminal acetyl labelling of the attenuated flavivirus vaccine sample; trypsin digestion of the attenuated flavivirus vaccine sample;

adding stable isotope labelled flavivirus peptides; detecting and quantifying the amount of a flavivirus protein fragments in the attenuated flavivirus vaccine 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 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.

In accordance with a further embodiment, there is provided a method , there is provided a method for determining the maturation status of a flavivirus in a human biological sample, the method 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.

In accordance with a further embodiment, there is provided a method for determining the maturation status of a coronavirus in a human biological sample, the method 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. In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided a method for measuring the level of the SARS-CoV-2 protein in a human biological sample, including detecting and

quantifying the amount of a 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.

In accordance with a further embodiment, there is provided 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.

In accordance with a further embodiment, there is provided a purified oligopeptide, wherein the oligopeptide is selected from one or more of those presented in TABLE 14.

In accordance with a further embodiment, there is provided a labelled oligopeptide, wherein the oligopeptide is selected from one or more of those presented in TABLE 14.

In accordance with a further embodiment, there is provided a stable isotope labelled oligopeptide, wherein the oligopeptide is selected from one or more of those presented in

TABLE 14 In accordance with a further embodiment, there is provided a method of diagnosing a coronavirus infection in a subject, the method 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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).

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; and (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. One representative of two independent experiments is 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)

DENV-2, (C) DENV-3, or (D) DENV-4 at MOI 0.1 for 4 days. Media were then collected and analyzed by MRM-MS. Concentrations of (A) 1-NSl, (B) 1-NSl, (C) 3-NS1, and (D) 4- NS1 in media are shown. One representative of two independent experiments is 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.

DETAILED DESCRIPTION

DEFINITIONS

The term“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. For example, stable isotopes that may be used for the study of biological systems include those of ¹⁸0, ¹³C, ¹⁵N, ²H and ³²S. As used herein 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.

The terms“peptide”,“polypeptide” and“protein” 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.

Nomenclature used to describe the peptide compounds of the present invention follows the conventional practice where the amino group is presented to the left and the carboxyl group to the right of each amino acid residue. In the sequences representing selected specific embodiments of the present invention, the amino- and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue may be generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with TABLE 1.

TABLE 1: Nomenclature and abbreviations of the 20 standard L-amino acids commonly found in naturally occurring peptides.

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. Examples of nonstandard amino acids 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. Examples of such 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

(Shirley, MA). Maturation efficiency is defined as [M]/([prM]+[M]). For example, TABLE 9 shows DENV prM maturation levels as being serotype-specific in Huh-7.5.1 and furin-deficient LoVo cells.

Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

MATERIALS AND METHODS

In silico digest and proteotypic candidate selection

Primary protein sequences for prM, E, and NS1 of DENV-1 strain Hawaiian-3

(USA/Hawaii/1945, accession AF425619, genotype I), DENV-2 strain NGC (Thailand/NGS- C/1944, accession M29095, Asian genotype II), DENV-3 strain H-87 (Philippines/H-87/1956, accession M93130, genotype I), and DENV-4 strain H-241 (Philippines/H-241/1956, accession U18433, genotype I) were subjected to an in silico trypsin digest using the

PeptideMass tool (28, 29). 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.

Peptide synthesis, verification, and preliminary characterization

Solid-phase synthesis of crude, unlabelled peptides for initial MRM development was performed on an Intavis Multipep Peptide Synthesizer™ (Intavis Bioanalytical Instruments AG™, Koln, Germany) using standard Fmoc chemistry with self-purification as previously described (32). Successful peptide synthesis was verified by MALDI-TOF MS analysis. Peptides were then analyzed by LC-MS using the QQQ in MS/MS scan mode to confirm LC- MS compatibility and to identify the dominant precursor charge state (FIGURE 3).

For the peptides that produced the best signal in the QQQ, heavy (SIS) peptides bearing a C- terminal Arg-[¹³C₆ ¹⁵N4] or Lys-[¹³C6¹⁵N2] were custom synthesized by Thermo Fisher Scientific™ and delivered at >98% purity, pre-quantified by amino acid analysis and pre solubilized in 5% acetonitrile (ACN) at 5 pmol/pL.

Cell culture

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/Invitrogen™). All cell lines used in this work were tested for mycoplasma contamination using the MycoAlert Plus™ detection kit (Lonza Group AG™, Basel, Switzerland) according to the manufacturer’s instructions and confirmed to be mycoplasma-free. Cells were grown as monolayers at 37°C with 5% CO2 and allowed to reach 90% confluency before infection.

Virus stock generation

Samples of 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 cm² 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).

Viral infection

Huh-7.5.1 cells or LoVo cells were plated at 5x 10⁴ or 1 ^c 10⁴ 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.

Sample preparation and in-solution trypsin digestion

Samples were exchanged into an ammonium bicarbonate (50 mM, pH 8.0; ABC) buffer by ultrafiltration using 10 kDa molecular weight cutoff (MWCO) centrifugal filter units (Pall Corporation™, Port Washington, NY, USA). Concentrated samples were then reconstituted in ABC buffer supplemented with sodium deoxycholate (1% w/v) to a final volume of 26 pL. From each sample, 1 pL was taken for protein quantification, which was performed using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific™, Waltham, MA, USA) according to the manufacturer’s directions. With the protein concentration determined, 20 pg of sample was taken for subsequent analysis.

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 Corporation™, 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.

SIS peptide spike and LC-MS

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 Empore™ Cl 8 SPE material (3M Company™, 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. 1-5 pL of sample containing 2-10 pg of protein was then injected and analyzed by LC- MS/MS. Mass selection and analysis was performed on an Agilent 6460™ triple quadrupole mass spectrometer, operating with parameters as noted below. Data analysis, including generation of extracted ion chromatograms (EIC) and manual peak integration, was performed with MassHunter™ Qualitative Analysis software (Agilent Technologies™, Santa Clara, CA, USA).

LC-MS operation parameters

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 Zorbax™ 300SB-C18 5 pm material, pore size 300 A) in a Chip Cube™ (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.

In the first stage of MRM method development, 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.

Optimal FV and CE settings for each transition were then determined. An MRM method for the precursor ion alone was created (CE = 0 V, selecting for the precursor m/z on Q1 and Q3) with FV varied in 20 V intervals from 60-240 V. Using the optimal FV thus 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)

MS data analysis

All results were processed using MassHunter Qualitative Analysis software (Agilent™). Extracted ion chromatograms (EIC) for each MRM were extracted and visually inspected; 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. In cases where an interfering peak disrupted the shape of the strongest transition peak, 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. Signal-to-noise ratios (SNR) were calculated in peak-to-peak mode, with an interference-free 2 min region from the first 10 min of each EIC defined as noise.

N-terminal acetylation

Sample preparation was performed as described above, with the following exceptions.

Following the initial inactivation step, 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. To the remaining 50 pL, 50 pL of a freshly prepared sulfo-N- hydroxysuccinimide (NHS) acetate (Sigma™) 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 Scientific™) 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.

EXAMPLES

The following working examples are provided for illustrative purposes, and are not intended to be limiting, as such.

EXAMPLE 1: NTAc-MRM Differential Quantification of Cleaved M and Uncleaved prM from DENV-1-4

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. Notably, 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). It is important to note that while our DENV-1- 4 M peptides would be expected to be differentially susceptible to trypsin-mediated cleavage in the immature prM-E and mature M-E conformations (FIGURE IB), all proteins in our samples are denatured by heating in the presence of sodium deoxycholate followed by reduction and alkylation of cysteine thiols, preventing disulfide bond formation so as to allow consistent accessibility to trypsin in vitro.

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. Trypsinization results in the cleavage of all prM junctions that have not already been cleaved endogenously; the purpose of NTAc labelling is therefore to distinguish the trypsin cleavage product (unlabelled N-NEh) from the endogenous cleavage product (N-Ac label) (FIGURE 2). By spiking in correspondingly labelled (N-Ac) or unlabelled (N-NEh) heavy stable isotope labelled standard (SIS) peptides and performing multiplexed MRM-MS analysis, we can differentially quantify mature M and immature prM.

MRM methods were initially developed using the purified, quantified SIS form of each peptide, in which the C-terminal Arg or Lys residue is ¹³C/¹⁵N-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). We next performed a concentration response and linearity analysis; to ensure this was done in a relevant sample matrix, we generated ‘mock serum’ samples composed of 3-4.5 pg/pL FBS with 10³-10⁴ pfu/pL DENV-1-4 derived from cell culture supernatant, intending to mimic approximately 0.2 pL per injection of DENV-infected serum on-column. Sub-aliquots of each sample containing identical endogenous peptide amounts were spiked with dilutions of a SIS peptide cocktail, with concentrations ranging from 0.16- 80 fmol/pL for each peptide. These samples were analyzed by the fully multiplexed pan- serotypic MRM data acquisition method 3-4 times each over the course of a week; the full experiment was repeated four times over the course of five months with varying SIS peptide concentrations.

TABLE 2 Parameters for pan-serotypic MRM and NTAc-MRM assays

MRM acquisition method for DENV-1-4 combined, including NT Ac peptides. Dwell time: 20 ms. Duty cycle: 5217 ms. Cell accelerator voltage: 7 V.

Compound name Precursor ion m/z Product ion m/z Fragmentor ( V) Collision energy (V)

lA13o_hea 379.4 644.2 80 9 lA13o_hea 379.4 497.2 80 5 lA13o_ligh 375.4 636.2 80 9 lA13o_ligh 375.4 489.2 80 5 lA13r_hea 371.4 628.2 80 9 lA13r_hea 371.4 497.2 80 5 lA13r_ligh 367.4 620.2 80 9 lA13r_ligh 367.4 489.2 80 5 lAcD2_hea 786.5 1272.4 60 25 lAcD2_hea 786.5 1088.4 60 23 lAcD2_hea 786.5 854.5 60 27 lAcD2_hea 786.5 544.4 60 21 lAcD2_hea 786.5 413.4 60 21 lAcD2_lig 781.5 1262.4 60 25 lAcD2_lig 781.5 1078.4 60 23 lAcD2_lig 781.5 844.5 60 27 lAcD2_lig 781.5 539.4 60 21 lAcD2_lig 781.5 413.4 60 21 lD2_heavy 510.9 755.3 180 20 lD2_heavy 510.9 672.4 180 8 lD2_heavy 510.9 636.8 180 8 lD2_light 507.6 745.3 180 20 lD2_light 507.6 667.4 180 8 lD2_light 507.6 631.8 180 8 lEl heavy 555.5 879.4 220 14 lEl heavy 555.5 780.4 220 15 lEl heavy 555.5 565.2 220 1^~5 lEl light 550.5 869.4 220 14 lEl light 550.5 770.4 220 15 lEl light 550.5 555.2 220 15

lE2_heavy 597.1 966.4 240 15 lE2_heavy 597.1 794.4 240 15 lE2_heavy 597.1 680.4 240 17 lE2_light 593.1 958.4 240 15 lE2_light 593.1 786.4 240 15 lE2_light 593.1 672.4 240 17 A10_heavy 607 692.8 200 11 A10_heavy 607 675 200 13 A10_heavy 607 660.6 200 15 A10_light 605.4 690.8 200 11 A10_light 605.4 673 200 13 A10_light 605.4 658.6 200 15 A12a_heav 575 820.4 220 19 A12a_heav 575 595.2 220 23 A12a_heav 575 441 220 27 A12a_light 571 812.4 220 19 A12a_light 571 587.2 220 23 A12a_light 571 437 220 27 A13o_heav 508.5 801.2 160 13 A13o_heav 508.5 688.4 160 15 A13o_heav 508.5 541.2 160 15 A13o_light 504.5 793.2 160 13 A13o_light 504.5 680.2 160 15 A13o_light 504.5 533.2 160 15 A13r_heav 500.5 785.2 160 13 A13r_heav 500.5 672.2 160 15 A13r_heav 500.5 541.2 160 15 A13r_light 496.5 777.2 160 13 A13r_light 496.5 664.2 160 15 A13r_light 496.5 533.2 160 15

AcD2o_hea 817.7 1122.4 160 25AcD2o_hea 817.7 725.4 160 31AcD2o_hea 817.7 561.6 160 27AcD2o_hea 817.7 413 160 29AcD2o_lig 812.7 1112.4 160 25AcD2o_lig 812.7 720.4 160 31AcD2o_lig 812.7 556.6 160 27AcD2o_lig 812.7 403 160 29AcD2r_hea 809.7 1106.4 60 27AcD2r_hea 809.7 872.2 60 33AcD2r_hea 809.7 773.2 60 31AcD2r_hea 809.7 553.8 60 23AcD2r_hea 809.7 413 60 29AcD2r_ligh 804.7 1096.4 60 27AcD2r_ligh 804.7 862.2 60 33AcD2r_ligh 804.7 763.2 60 31AcD2r_ligh 804.7 548.8 60 23AcD2r_ligh 804.7 403 60 29D2o_heavy 531.6 703.6 60 9D2o_heavy 531.6 611.6 60 9D2o_heavy 531.6 562.2 60 15D2o_light 528.3 698.6 60 9D2o_light 528.3 606.6 60 9D2o_light 528.3 557.2 60 15D2r_heavy 526.3 695.6 60 9D2r_heavy 526.3 660 60 9D2r_heavy 526.3 554.2 60 15D2r_light 523 690.6 60 9D2r_light 523 655 60 9D2r_light 523 549.2 60 15

E13_heavy 493.2 668.2 60 19E13_heavy 493.2 402.8 60 19E13_light 489.9 658.2 60 19E13_light 489.9 397.8 60 19E2_heavy 618 1008.4 220 17E2_heavy 618 808.2 220 17E2_heavy 618 694.2 220 17E2_light 614 1000.4 220 17E2_light 614 800.2 220 17E2_light 614 686.2 220 17A13o_heavy 591.7 867.2 60 19A13o_heavy 591.7 641 60 19A13o_heavy 591.7 433.8 60 19A13o_light 587.7 859.2 60 19A13o_light 587.7 633 60 19A13o_light 587.7 429.8 60 19A13r_heavy 583.7 951 60 ^~Ϊ9A13r_heavy 583.7 851.2 60 19A13r_heavy 583.7 625.2 60 23A13r_light 579.7 943 60 19A13r_light 579.7 843.2 60 19A13r_light 579.7 617.2 60 23A14_heavy 384.4 696.2 80 9A14_heavy 384.4 609.2 80 7A14_heavy 384.4 496.2 80 7A14_light 380.4 688.2 80 9A14_light 380.4 601.2 80 7A14_light 380.4 488.2 80 7AcD2o_heav 796.6 1108.4 180 26AcD2o_heav 796.6 775.4 180 27

AcD2o_he 796.6 554.6 180 26AcD2o_he 796.6 413 180 27AcD2o_li 791.6 1098.4 180 26AcD2o_li 791.6 765.4 180 27AcD2o_li 791.6 549.6 180 26AcD2o_li 791.6 403 180 27AcD2r_he 788.7 1092.4 60 27AcD2r_he 788.7 858.4 60 29AcD2r_he 788.7 547 60 25AcD2r_he 788.7 413.2 60 25AcD2r_lig 783.7 1082.4 60 27AcD2r_lig 783.7 848.4 60 29AcD2r_lig 783.7 542 60 25AcD2r_lig 783.7 403.2 60 25D2a_heav 512.3 858.2 80 21D2a_heav 512.3 674.4 80 8D2a_heav 512.3 547 80 13D2a_light 509 848.2 80 21D2a_light 509 669.4 80 8D2a_light 509 542 80 13D2b_heav 517.6 682.6 200 8D2b_heav 517.6 647 200 8D2b_heav 517.6 590.4 200 9D2b_light 514.3 677.6 200 8D2b_light 514.3 642 200 8D2b_light 514.3 585.4 200 9El_heavy 557.5 883.4 240 1^~5El_heavy 557.5 812.4 240 17El_heavy 557.5 711.4 240 16El_light 552.5 873.4 240 15

E 1 light 552.5 802.4 240 17 E 1 light 552.5 701.4 240 16E12_heav 594 829.2 60 21E12_heav 594 537 60 19E12_heav 594 396.8 60 27E12_light 590 821.2 60 21E12_light 590 533 60 19E12_light 590 388.8 60 27A14_heav 523.5 790.2 100 11A14_heav 523.5 431.2 100 8A14_heav 523.5 395.6 100 9A14_light 519.5 782.2 100 11A14_light 519.5 427.2 100 8A14_light 519.5 391.6 100 9A15_heav 407.4 700.2 120 7A15_heav 407.4 553.2 120 7A15_heav 407.4 398.2 120 5A15_light 403.4 692.2 120 7A15_light 403.4 545.2 120 7A15_light 403.4 394.2 120 5AcD2o_he 812.6 1110.4 180 27AcD2o_he 812.6 555.6 180 27AcD2o_he 812.6 514 180 24AcD2o_he 812.6 413 180 29AcD2o_li 807.6 1100.4 180 27AcD2o_li 807.6 550.6 180 27AcD2o_li 807.6 504 180 24AcD2o_li 807.6 403 180 29AcD2r_he 804.6 1094.4 60 27AcD2r_he 804.6 860.2 60 31

AcD2r_heav 804.6 547.8 60 27AcD2r_heav 804.6 413.2 60 23AcD2r_light 799.6 1084.4 60 27AcD2r_light 799.6 850.2 60 31AcD2r_light 799.6 542.8 60 27AcD2r_light 799.6 403.2 60 23D2o_heavy 528.2 698.6 60 9D2o_heavy 528.2 663.2 60 9D2o_heavy 528.2 555.8 60 15D2o_light 524.9 693.6 60 9D2o_light 524.9 658.2 60 9D2o_light 524.9 550.8 60 15D2r_heavy 522.9 690.6 100 9D2r_heavy 522.9 598.04 100 9D2r_heavy 522.9 548 100 15D2r_light 519.6 685.6 100 9D2r_light 519.6 593.04 100 9D2r_light 519.6 543 100 15E12a_heavy 690 877.4 60 21E12a_heavy 690 458 60 19E12a_heavy 690 344.8 60 19E12a_light 685 867.4 60 21E12a_light 685 448 60 19E12a_light 685 334.8 60 19E14o_heavy 463.4 536 60 19E14o_heavy 463.4 449 60 19E14o_heavy 463.4 347.8 60 19E14o_light 458.4 526 60 19E14o_light 458.4 439 60 19E14o_light 458.4 337.8 60 19

4E14r_heavy 455 778.4 60 19

4E14r_heavy 455 664.6 60 19

4E14r_heavy 455 536.2 60 19

4E14r_light 450 768.4 60 19

4E14r_light 450 654.6 60 19

4E14r_light 450 526.2 60 19

TABLE 3 Parameters for DENV-1 MRM and NTAc-MRM assays.

MRM acquisition method for DENV-1 only, including NTAc peptides. Dwell time: 20 ms. Duty cycle: 987 ms. Cell accelerator voltage: 7 V.

Compound name Precursor ion m/z Product ion m/z Fragmentor (V) Collision energy (V) lAcD2_heavy 786.5 1272.4 60 25

lAcD2_heavy 786.5 1088.4 60 23

lAcD2_heavy 786.5 854.5 60 27

lAcD2_heavy 786.5 544.4 60 21

lAcD2_heavy 786.5 413.4 60 21

lAcD2_light 781.5 1262.4 60 25

lAcD2_light 781.5 1078.4 60 23

lAcD2_light 781.5 844.5 60 27

lAcD2_light 781.5 539.4 60 21

lAcD2_light 781.5 413.4 60 21

lE2_heavy 597.1 966.4 240 15

lE2_heavy 597.1 794.4 240 15

lE2_heavy 597.1 680.4 240 17

lE2_light 593.1 958.4 240 15

lE2_light 593.1 786.4 240 15

lE2_light 593.1 672.4 240 17

lEl heavy 555.5 879.4 220 14

lEl heavy 555.5 780.4 220 15

lEl heavy 555.5 565.2 220 15

lEl light 550.5 869.4 220 14

lEl light 550.5 770.4 220 15

lEl light 550.5 555.2 220 15

lD2_heavy 510.9 755.3 180 20

lD2_heavy 510.9 672.4 180 8

lD2_heavy 510.9 636.8 180 8

lD2_light 507.6 745.3 180 20

lD2_light 507.6 667.4 180 8

lD2_light 507.6 631.8 180 8

lA12 heavy 401.4 730.2 100 9

lA12_heavy 401.4 643.2 100 7

lA12_heavy 401.4 496.2 100 10

lA12_light 397.4 722.2 100 9

lA12_light 397.4 635.2 100 7

lA12_light 397.4 488.2 100 9

lA13o_heavy 379.4 644.2 80 9

lA13o heavy 379.4 497.2 80 5

lA13o_light 375.4 636.2 8⁽G 9

lA13o_light 375.4 489.2 80 5

lA13r_heavy 371.4 628.2 80 9

lA13r_heavy 371.4 497.2 80 5

lA13r_light 367.4 620.2 80 9

lA13r light 367.4 489.2 80 5

TABLE 4 Parameters for DENV-2 MRM and NTAc-MRM assays.

MRM acquisition method for DENV-2 only, including NTAc peptides. Dwell time: 20 ms. Duty cycle: 1504 ms. Cell accelerator voltage: 7 V.

Compound name Precursor ion m/z Product ion m/z Fragmentor (V) Collision energy (V)

2AcD2o_he 817.7 1122.4 Too 25

2AcD2o_he 817.7 725.4 160 31

2AcD2o_he 817.7 561.6 160 27

2AcD2o_he 817.7 413 160 29

2AcD2o_li 812.7 1112.4 160 25

2AcD2o_li 812.7 720.4 160 31

2AcD2o_li 812.7 556.6 160 27

2AcD2o_li 812.7 403 160 29

2AcD2r_he 809.7 1106.4 60 27

2AcD2r_he 809.7 872.2 60 33

2AcD2r_he 809.7 773.2 60 31

2AcD2r_he 809.7 553.8 60 23

2AcD2r_he 809.7 413 60 29

2AcD2r_lig 804.7 1096.4 60 27

2AcD2r_lig 804.7 862.2 60 33

2AcD2r_lig 804.7 763.2 60 31

2AcD2r_lig 804.7 548.8 60 23

2AcD2r_lig 804.7 403 60 29

2E2_heavy 618 1008.4 220 17

2E2_heavy 618 808.2 220 17

2E2_heavy 618 694.2 220 17

2E2_light 614 1000.4 220 17

2E2 light 614 800.2 220 17

E2_light 614 686.2 220 17A10_heavy 607 692.8 200 11A10_heavy 607 675 200 13A10_heavy 607 660.6 200 15A10_light 605.4 690.8 200 11A10_light 605.4 673 200 13A10_light 605.4 658.6 200 15A12a_heavy 575 820.4 220 19A12a_heavy 575 595.2 220 23A12a_heavy 575 441 220 27A12a_light 571 812.4 220 19A12a_light 571 587.2 220 23A12a light 571 437 220 27D2o_heavy 531.6 703.6 6(G 9D2o_heavy 531.6 611.6 60 9D2o_heavy 531.6 562.2 60 15D2o_light 528.3 698.6 60 9D2o_light 528.3 606.6 60 9D2o_light 528.3 557.2 60 15D2r_heavy 526.3 695.6 60 9D2r_heavy 526.3 660 60 9D2r_heavy 526.3 554.2 60 15D2r_light 523 690.6 60 9D2r_light 523 655 60 9D2r_light 523 549.2 60 15A13o_heavy 508.5 801.2 160 13A13o_heavy 508.5 688.4 160 15A13o_heavy 508.5 541.2 160 15A13o_light 504.5 793.2 160 13A13o_light 504.5 680.2 160 15A13o_light 504.5 533.2 160 15A13r_heavy 500.5 785.2 160 13A13r_heavy 500.5 672.2 160 15A13r_heavy 500.5 541.2 160 15A13r_light 496.5 777.2 160 13A13r_light 496.5 664.2 160 15A13r_light 496.5 533.2 160 15E13_heavy 493.2 668.2 60 19E13_heavy 493.2 402.8 60 19E13_light 489.9 658.2 60 19E13 light 489.9 397.8 60 19 TABLE 5 Parameters for DENV-3 MRM and NTAc-MRM assays.

MRM acquisition method for DENV-3 only, including NTAc peptides. Dwell time: 20 ms.

Duty cycle: 1363 ms. Cell accelerator voltage: 7 V.

Compound name Precursor ion m/z Product ion m/z Fragmentor (V) Collision energy (V)

3AcD2o_he 796.6 1108.4 180 26

3AcD2o_he 796.6 775.4 180 27

3AcD2o_he 796.6 554.6 180 26

3AcD2o_he 796.6 413 180 27

3AcD2o_li 791.6 1098.4 180 26

3AcD2o_li 791.6 765.4 180 27

3AcD2o_li 791.6 549.6 180 26

3AcD2o_li 791.6 403 180 27

3AcD2r_he 788.7 1092.4 60 27

3AcD2r_he 788.7 858.4 60 29

3AcD2r_he 788.7 547 60 25

3AcD2r_he 788.7 413.2 60 25

3AcD2r_lig 783.7 1082.4 60 27

3AcD2r_lig 783.7 848.4 60 29

3AcD2r_lig 783.7 542 60 25

3AcD2r_lig 783.7 403.2 60 25

3E12_heav 594 829.2 60 21

3E12_heav 594 537 60 19

3E12_heav 594 396.8 60 27

3A13o_hea 591.7 867.2 60 19

3A13o_hea 591.7 641 60 19

3A13o_hea 591.7 433.8 60 19

3E12_light 590 821.2 60 21

3E12_light 590 533 60 19

3E12_light 590 388.8 60 27

3A13o_ligh 587.7 859.2 60 19

3A13o_ligh 587.7 633 60 19

3A13o_ligh 587.7 429.8 60 19

3A13r_hea 583.7 951 60 19

3A13r_hea 583.7 851.2 60 19

3A13r_hea 583.7 625.2 60 23

3A13r_ligh 579.7 943 60 19

3A13r_ligh 579.7 843.2 60 19

3A13r_ligh 579.7 617.2 60 23

3El_heavy 557.5 883.4 240 15

3El_heavy 557.5 812.4 240 17

3El_heavy 557.5 711.4 240 16

3El light 552.5 873.4 240 15

3El_light 552.5 802.4 240 17

3El_light 552.5 701.4 240 16

3D2b_heavy 517.6 682.6 200 8

3D2b_heavy 517.6 647 200 8

3D2b_heavy 517.6 590.4 200 9

3D2b_light 514.3 677.6 200 8

3D2b_light 514.3 642 200 8

3D2b_light 514.3 585.4 200 9

3D2a_heavy 512.3 858.2 80 21

3D2a_heavy 512.3 674.4 80 8

3D2a_heavy 512.3 547 80 13

3D2a_light 509 848.2 80 21

3D2a_light 509 669.4 80 8

3D2a_light 509 542 80 13

3A14_heavy 384.4 696.2 80 9

3A14_heavy 384.4 609.2 80 7

3A14_heavy 384.4 496.2 80 7

3A14_light 380.4 688.2 80 9

3A14_light 380.4 601.2 80 7

3A14 light 380.4 488.2 80 7

TABLE 6 Parameters for DENV-4 MRM and NTAc-MRM assays.

MRM acquisition method for DENV-4 only, including NTAc peptides. Dwell time: 20 ms.

Duty cycle: 1363 ms. Cell accelerator voltage: 7 V.

Compound name Precursor ion m/z Product ion m/z Fragmentor (V) Collision energy (V)

4AcD2o_heavy 812.6 1110.4 180 27

4AcD2o_heavy 812.6 555.6 180 27

4AcD2o_heavy 812.6 514 180 24

4AcD2o_heavy 812.6 413 180 29

4AcD2o_light 807.6 1100.4 180 27

4AcD2o_light 807.6 550.6 180 27

4AcD2o_light 807.6 504 180 24

4AcD2o_light 807.6 403 180 29

4AcD2r_heavy 804.6 1094.4 60 27

4AcD2r_heavy 804.6 860.2 60 31

4AcD2r_heavy 804.6 547.8 60 27

4AcD2r_heavy 804.6 413.2 60 23

4AcD2r_light 799.6 1084.4 60 27

4AcD2r_light 799.6 850.2 60 31

4AcD2r_light 799.6 542.8 60 27

4AcD2r_light 799.6 403.2 60 23

4E12a heavy 690 877.4 60 21

E12a_heav 690 458 60 19E12a_heav 690 344.8 60 19E12a_light 685 867.4 60 21E12a_light 685 448 60 19E12a_light 685 334.8 60 19D2o_heavy 528.2 698.6 60 9D2o_heavy 528.2 663.2 60 9D2o_heavy 528.2 555.8 60 15D2o_light 524.9 693.6 60 9D2o_light 524.9 658.2 60 9D2o_light 524.9 550.8 60 15A14_heavy 523.5 790.2 100 11A14_heavy 523.5 431.2 100 8A14_heavy 523.5 395.6 100 9D2r_heavy 522.9 690.6 100 9D2r_heavy 522.9 598.04 100 9D2r_heavy 522.9 548 100 15D2r_light 519.6 685.6 100 9D2r light

519.6 593.04 100 9D2r_light 519.6 543 100 1^~5A14_light 519.5 782.2 100 11A14_light 519.5 427.2 100 8A14_light 519.5 391.6 100 9E14o_heavy 463.4 536 60 19E14o_heavy 463.4 449 60 19E14o_heavy 463.4 347.8 60 19E14o_light 458.4 526 60 19E14o_light 458.4 439 60 19E14o_light 458.4 337.8 60 19E14r_heavy 455 778.4 60 19E14r_heavy 455 664.6 60 19E14r_heavy 455 536.2 60 19E14r_light 450 768.4 60 19E14r_light 450 654.6 60 19E14r_light 450 526.2 60 19A15_heavy 407.4 700.2 120 7A15_heavy 407.4 553.2 120 7A15_heavy 407.4 398.2 120 5A15_light 403.4 692.2 120 7A15_light 403.4 545.2 120 7A15 light 403.4 394.2 120 5 Resulting extracted ion chromatograms (EICs) were smoothed and manually integrated on the strongest transition, with individual peak identities confirmed by the co-elution of two additional transitions. Signal-to-noise ratios (SNR) 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. Lower limit of detection (LOD) is defined as the lowest concentration point with a signal-to-noise ratio (SNR) greater than 3.0; lower limit of quantification (LOQ) was defined as the lowest concentration point within the linear response range with an SNR greater than 10.0 (38, 39). LOD and LOQ are summarized in TABLE 7.

TABLE 7: Shows the proteotypic peptide sequences and limits of detection (LOD) and quantification (LOQ). On-column estimated LOD and LOQ values for each N-NLh peptide were determined by response curves and RF plots using a pan-serotypic method to analyze mock serum samples spiked with SIS peptides. In the case of N-Ac peptides, single-serotype methods were used rather than the pan-serotypic method.

*This peptide is proteotypic for both DENV-1 and DENV-2 NS1

Extracted ion chromatograms confirm the favourable elution profile of the N-NEh and N-Ac peptides, showing co-elution of three transitions with good peak shape (FIGURE 3). All transitions monitored for these seven peptides are y ions, ensuring the C-terminal labelled Arg-[¹³C₆ ¹⁵N4] or Lys-[¹³C6¹⁵N2] that distinguishes the SIS peptides is always present and appropriately selected in Q1 and Q3. Importantly, while the N-terminal Ac label is only selected in Ql, the different biophysical properties of the N-NH2 and N-Ac peptides (i.e. charge, hydrophobicity) ensure they will not co-elute (FIGURE 3), reducing the possibility of interference between the two forms.

To confirm the detectability of our seven peptides in biological samples, 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-[¹³C₆ ¹⁵N4] or Lys-[¹³C6¹⁵N2] 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.

Using this methodology, we were able to successfully differentiate and quantify N-NEb (immature) and N-Ac (mature) forms of prM from DENV-1-4 and WNV (TABLE 3A). However, highly conserved Met residues in the DENV-2, DENV-3, and DENV-4 peptides complicated MRM development and analysis. To tackle this, separate MRMs targeting methionine (Met-S, reduced‘r’ form) and methionine sulfoxide (Met-SO, oxidized‘o’ form) forms for all light/heavy N-NEh/N-Ac peptides were optimized. In all results presented here, no quantifiable levels of the Met-SO form of any peptide were consistently observed; all quantification and interpretation is therefore based on the Met-S forms only.

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.

TABLE 8: Sequences of DENV-1-4 and WNV prM.

DENV- 1 NATETWVTYGTCSQTGEHRRDKR j SVALAPHVGLGLETRTETW ( SEQI D NO : 121 ) DENV-2 NST STWVTYGTCTTMGEHRREKR j. SVALVPHVGMGLETRTETW ( SEQI D NO : 122 )

DENV-3 NLT STWVTYGTCNQAGEHRRDKR j. SVALAPHVGMGLDTRTQTW ( SEQI D NO : 123 ) DENV-4 NLTSTWVMYGTCTQSGERRREKRj SVALTPHSGMGLETRAETW ( SEQID NO: 124)

WNV TKSAVYVRYGRCTKTRHSRRSRRj SLTVQTHGESTLANKKGAW (SEQID NO:125)

70. 80. 90. 100. 110

EXAMPLE 2: DENV prM Maturation Rate and Furin Dependence based on Serotype

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. Importantly, since our proteotypic peptides targeting NS1 contain Lys which would be covalently modified by NTAc labelling in addition to the N-terminal amine, all NS1 data was obtained by MRM-MS performed on samples that were not NTAc labelled. The analysis revealed some disparities in the absolute levels of prM and M as well as NS1 found between each serotype. Furthermore, serotype-specific patterns of maturation and dependency on human furin were observed.

For DENV-1, it was found that extracellular prM derived from Huh-7.5.1 cells were mostly mature, to the extent that the unlabelled immature prM (1-M) peptide was below LOQ for the assay, representing an efficiency of maturation greater than 95% (FIGURE 4A and TABLE 9). Unexpectedly, the maturation of DENV-1 derived from furin-deficient LoVo cells remained efficient, again with no quantifiable amount of immature prM peptide detected (FIGURE 4A and TABLE 9). This evidence strongly suggests that DENV-1 prM maturation takes place in a furin-independent manner in LoVo cells. Despite the apparent lack of a furin requirement for prM maturation, a robust effect on the total extracellular abundance of viral proteins was observed in LoVo cell culture supernatant. It was found that levels of DENV-1 structural proteins (total immature + mature M) as well as non- structural proteins (NS1) were reduced by about 70% in LoVo media compared to Huh-7.5.1 (FIGURE 4A and FIGURE 5A). Thus, while prM maturation does not require the presence of functional furin in LoVo cells, these results suggest that furin may be playing some other direct or indirect role in the DENV-1 lifecycle, such that the absence of furin negatively impacts the biosynthesis or secretion of DENV-1 proteins.

TABLE 9: shows DENV prM maturation levels show serotype-specific differences in Huh- 7.5.1 and furin-deficient LoVo cells. A range is used where one of the peptide concentration values obtained is below LOQ. Error values represent SD across two independent experiments.

In agreement with prior work in the field (11, 23, 24), we found that DENV-2 prM was moderately cleaved during Huh-7.5.1 infection, with maturation efficiency around 56% among extracellular prM molecules (FIGURE 4B and TABLE 9). Moreover, the predominantly immature character of DENV-2 prM in LoVo cells that has been described by others (11) was confirmed by our results, wherein levels of the labelled mature M (2-M-Ac) peptide in LoVo-derived cell culture supernatant were below the assay LOQ, representing a prM maturation rate of no more than 24%. Other studies have reported that the levels of genome-containing virus particles remain relatively unchanged in LoVo cells compared to other cell types when infected with DENV-2 (11). In contrast, we observed a reduction of about 54% in the total amount of mature and immature M was found outside LoVo cells compared to Huh-7.5.1 cells (FIGURE 4B). Levels of extracellular DENV-2 NSlwere also reduced below the assay LOQ in LoVo cells (FIGURE 5B).

Similar to DENV-2, we found that 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). In comparing DENV-3 -infected Huh-7.5.1 and LoVo cell culture supernatants, 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%. As with DENV-2, this suggests a significant role played by furin in the maturation of DENV-3 prM. Total extracellular viral protein levels again exhibited a dramatic reduction for LoVo compared with Huh-7.5.1 cells, including a 71-92% reduction in the total amount of mature and immature M and an 82% reduction in NS1 (FIGURE 4C and FIGURE 5C). This is reminiscent of the effect seen for DENV-1 and DENV-2: a robust inhibition of viral protein secretion among structural (M) and non- structural (NS1) proteins in furin-deficient LoVo cells.

Finally, it was observed that DENV-4 prM maturation was the least efficient of the four serotypes, with prM maturation efficiency in Huh-7.5.1 cells a mere 25% despite relatively high extracellular viral protein abundance (FIGURE 4D and TABLE 9). Unfortunately, the furin dependency of DENV-4 proteolytic maturation could not be directly elucidated by the assay; although extracellular mature M (4-M-Ac) peptide abundance was reduced below the assay LOQ in LoVo versus Huh-7.5.1 cell culture supernatant, a concomitant decrease in immature prM (4-M) peptide levels means that maturation efficiency could be anywhere from 0 to 52% (FIGURE 4D and TABLE 9). This, however, highlights the other key observation: that extracellular viral protein levels are severely reduced in DENV-4-infected LoVo media, with an 83-92% reduction in the total amount of mature and immature M, and with NS1 levels below the LOQ of the assay (FIGURE 4D and FIGURE 5D). This is the strongest effect on viral protein levels observed among the four DENV serotypes. It therefore seems that the DENV-4 lifecycle is the most sensitive to furin deficiency, although it remains undetermined whether furin deficiency results in a reduction in the maturation efficiency prM.

EXAMPLE 3: WUHAN CORONA VIRUS PROTEOTYPIC PEPTIDES

The strain analyzed: 2019-nCoV (YP_009724390.1), S glycoprotein only (new nomenclature by WHO: 2019-nCoV => SARS-CoV-2). 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)).

In summary, uniquely among DENV-1-4, we found that 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.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range.

In the following peptide tables, 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.

TABLE 10A - DENV-1 PEPTIDES

TABLE 10B - DENV-2 PEPTIDES

TABLE IOC - DENV-3 PEPTIDES

TABLE 10D - DENV-4 PEPTIDES

TABLE 11- ZIKV PEPTIDES

TABLE 12 - WNV PEPTIDES

TABLE 13 - POWV PEPTIDES

TABLE 14 - CORONA VIRUS PEPTIDES

*This peptide could be used to monitor proteolysis by NTAc-MRM (i.e. SEQ ID NO: 119).

The word“comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms“a”, “an” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

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Claims

CLAIMS:

1. A method for determining the maturation status of a flavivirus in a human biological sample, the method comprising:

(a) N-terminal acetyl labelling of the human biological sample;

(b) Trypsin digestion of the human biological sample;

(c) Adding stable isotope labelled flavivirus peptides;

(d) Detecting and quantifying the amount of a flavivirus protein fragments in the human biological sample, using mass spectrometry; and

(e) 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.

2. A method for measuring the level of the flavivirus protein in a human biological sample, comprising 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.

3. A method for measuring the level of the DENV protein in a human biological sample, comprising 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.

4. A method for detecting the presence and measuring the level of DENV protein and truncated DENV protein in a human biological sample, comprising 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.

5. The method of claim 4, wherein the flavivirus peptides are one or more of the peptides selected from SEQ ID NOs: 4-13, 21-25, 31-37 and 48-54, which are indicative of DENV serotype.

6. A method for measuring the level of the ZIKV protein in a human biological sample, comprising 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.

7. A method for detecting the presence and measuring the level of ZIKV protein and truncated ZIKV protein in a human biological sample, comprising 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.

8. A method for measuring the level of the WNV protein in a human biological sample, comprising 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.

9. A method for detecting the presence and measuring the level of WNV protein and truncated WNV protein in a human biological sample, comprising 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.

10. A method for measuring the level of the POWV protein in a human biological sample, comprising 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.

11. A method for detecting the presence and measuring the level of POWV protein and truncated POWV protein in a human biological sample, comprising 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.

12. A purified oligopeptide, wherein the oligopeptide is selected from one or more of those presented in TABLES 10A-10D and 11-13.

13. The oligopeptide of claim 12, wherein the oligopeptide is labelled oligopeptide.

14. The oligopeptide of claim 13, wherein the oligopeptide is a stable isotope labelled oligopeptide.

15. A method of diagnosing a flavivirus infection in a subject, the method comprising: measuring the level of the flavivirus protein in a human biological sample, comprising 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.

16. The method of claim 15, wherein the flavivirus peptide is one or more of the peptides of those presented in TABLES 10A-10D.

17. The method of claim 15 or 16, wherein the flavivirus peptide is one or more of the peptides of those presented in TABLES 10A.

18. The method of claim 15 or 16, wherein the flavivirus peptide is one or more of the peptides of those presented in TABLES 10B.

19. The method of claim 15 or 16, wherein the flavivirus peptide is one or more of the peptides of those presented in TABLES IOC.

20. The method of claim 15 or 16, wherein the flavivirus peptide is one or more of the peptides of those presented in TABLES 10D.

21. A method for determining the maturation status of a flavivirus in a human biological sample, the method comprising:

(a) N-terminal acetyl labelling of the human biological sample;

(b) Trypsin digestion of the human biological sample;

(c) Adding stable isotope labelled flavivirus prM, pr and M peptides;

(d) Detecting and quantifying the amount of a flavivirus prM, pr and M protein

fragments in the human biological sample, using mass spectrometry; and

(e) Calculating the level of the flavivirus prM, pr and M protein in said sample; wherein the flavivirus prM, pr and M peptides are one or more of the peptides selected from SEQ ID NOs: l, 2, 3, 18, 19, 20, 29, 30, 43, 44, 45, 46, 47, 61, 62, 63, 64, 74, 75, 76, 77 90,

91 and 92, and wherein said amount is a relative amount or an absolute amount.

22. The method of claim 21, wherein the flavivirus prM, pr and M peptides are 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.

23. A method for testing an attenuated flavivirus vaccine sample, the method comprising:

(a) N-terminal acetyl labelling of the attenuated flavivirus vaccine sample;

(b) Trypsin digestion of the attenuated flavivirus vaccine sample; (c) Adding stable isotope labelled flavivirus peptides;

(d) Detecting and quantifying the amount of flavivirus protein fragments in the

attenuated flavivirus vaccine sample, using mass spectrometry; and

(e) 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 and is an indication of the maturation status of the attenuated flavivirus vaccine.

24. A method for determining the maturation status of a coronavirus in a human biological sample, the method comprising:

(a) N-terminal acetyl labelling of the human biological sample;

(b) Trypsin digestion of the human biological sample;

(c) Adding stable isotope labelled coronavirus peptides;

(d) Detecting and quantifying the amount of a coronavirus protein fragments in the human biological sample, using mass spectrometry; and

(e) 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.

25. A method for measuring the level of the coronavirus protein in a human biological sample, comprising 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.

26. A method for measuring the level of the SARS-CoV-2 protein in a human biological sample, comprising detecting and quantifying the amount of a 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.

27. 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, comprising 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

28. A purified oligopeptide, wherein the oligopeptide is selected from one or more of those presented in TABLE 14.

29. The oligopeptide of claim 28, wherein the oligopeptide is labelled oligopeptide.

30. The oligopeptide of claim 29, wherein the oligopeptide is a stable isotope labelled oligopeptide.

31. A method of diagnosing a coronavirus infection in a subject, the method comprising: measuring the level of the coronavirus protein in a human biological sample, comprising 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.