WO2022167498A1

WO2022167498A1 - Mass-spectrometric method for pathogen detection

Info

Publication number: WO2022167498A1
Application number: PCT/EP2022/052509
Authority: WO
Inventors: Thomas Hankemeier; Amy C HARMS; Nicolas FP DROUIN
Original assignee: Universiteit Leiden
Priority date: 2021-02-02
Filing date: 2022-02-02
Publication date: 2022-08-11

Abstract

The invention relates to a mass-spectrometry method for detecting pathogens. The method is highly sensitive and is therefore particularly useful for detecting peptides that are present in only low concentrations, such as pathogen peptides in a swab sample obtained from a subject.

Description

Mass-spectrometric method for pathogen detection

Field of the invention

Background art

To manage outbreaks of infectious diseases, such as viral pandemics, there is a need for fast and reliable diagnostics. Diagnostic tests are developed to recognize unique molecular signatures from pathogens. Either small molecules or macromolecules (nucleic acid chains, proteins, carbohydrates, metabolites, or lipids) can be detected. PCR is a very common method and detects DNA and RNA, which it can amplify to enhance a signal. In present daily practice a very high number of PCR tests are clinically performed. PCR is efficient and automatable, and does not require prohibitively expensive reagents. Since the outbreak of COVID-19, testing has mainly been performed with real-time reverse transcriptase quantitative PCR (RT-qPCR). Where RT- qPCR is still considered the gold standard, this test is relatively expensive, dependent on sometimes scarce reagents, and takes several hours to conduct. Several alternatives to RT-qPCR tests - notably antigen, breath, spectroscopic, and RT-LAMP PCR tests - have been or are being developed to overcome these limitations. Although (some of) these alternative testing options have aided in increasing testing capacity, they in some cases offer inferior selectivity or specificity, may not be reliable as stand-alone alternatives, require specific reagents, or are even less cost-efficient. Also PCR tests are complex to validate, and can lead to false results that can be difficult to detect.

Mass-spectrometry is an attractive method for detecting compounds, but its use for screening applications also has many requirements. It has to be sufficiently fast, robust, sensitive, and cost-effective. The COVID-2019 pandemic has increased the demand for such techniques. Mass-spectrometry has been suggested for predicting immunological response of subjects to COVID-19 (US2020386766).

Sensitivity of an assay is important. In April 2020 specific peptides were identified that result from digesting proteins of SARS-CoV-2 into smaller peptides that are well-detectable using mass spectrometry (see medRxiv 2020.11.18.20231688). Based on these results, an initiative was started involving more than 20 academic or clinical labs and companies around the world with the goal to evaluate the possibility of mass spectrometry to supplement PCR testing for COVID19 in nasopharyngeal swabs or other samples of large number of subjects. Using liquid chromatographymass spectrometry (LC-MS or LCMS) and multiple reaction monitoring (MRM, also known as selective reaction monitoring or SRM), from a negative swab sample spiked with recombinant proteins and subsequently digested, the developed method was able to detect down to 0.5 ng loaded into the liquid chromatography (LC) column. However, this method was limited to detect threshold cycle (Ct, also called quantification cycle or Cq) values up to 25. In consequence, the detection capacities of the virus were lower than the antigenic test, which can detect with certitude a sample with a Ct value of 26, and with lower fidelity Ct values down to 30 (see DOI: 10.1016/j.eclinm.2020.100677). The correlation between Ct values and MRM signal intensity was established by Van Puyvelde et al. (see medRxiv 2020.1 1 .18.20231688).

Later, a UK consortium tried to reproduce these results and to optimize the analytical workflow, especially the sample preparation. However, this did not bring the desired results, and only minor improvements were obtained. The UK consortium explored also the use of antibodies for enriching SARS-CoV-2 specific peptides after digestion, but that also did not deliver the required sensitivity to detect SARS-CoV-2 peptides in nasopharyngeal swabs samples at a Ct value of 30 nor the desired throughput. Accordingly, the maximum attainable sensitivity was achieved using a method where the sample preparation was ice-cold acetone precipitation (7 volumes) on 175 pl of saline medium in 1.5 ml Eppendorf vials, resuspending this in 35 pl 50 mM TEABC 0.5 pg trypsin/LysC and 5% acetonitrile with 10ng Cov-MS QConCAT standard, digesting for 15 minutes at 37°C, followed by addition of 3.5pl of 10% formic acid (end concentration 1 %) with LC-MRM detection. Also, it was concluded that background, i.e. matrix, and not instrumental limitations, is currently the limiting factor for increasing the sensitivity (see medRxiv 2020.11 .18.20231688).

Like many other virus pathogens, SARS-CoV-2 infection yields virus-like particles (VLPs) that comprise polypeptides, but no RNA. These unique targets for diagnostics have been historically targeted using antibodies. However, antibodies do not reduce the cost of diagnostic testing, can be less sensitive than mass spectrometry, and are complexto produce. Mass spectrometry is attractive here, because it has been shown to be able to detect such peptides, and sample preparation for polypeptide detection can be faster at large scales (under 20 minutes) and cheaper (below $5 worth of reagents) than that for oligonucleotides (hours per sample at about $25).

One issue for mass spectrometry can be that the substance of interest has to be provided in a solution, which for a very large part consists of a carrier medium. Different carrier media are generally used with swabs to transport e.g. viral signatures from the collection to the analysis. For instance, media commonly in use for SARS-CoV-2 diagnosis sampling were primarily designed for PCR or culture and therefore are often formulated to keep microorganisms such as bacteria intact so as to be able to cultivate and identify them. However, many of the added components interfere with MS detection.

For instance, the universal transport medium (UTM (trademark) available from COPAN ITALIA SPA, Brescia, Italy) consists of modified Hank's balanced salt solution supplemented with bovine serum albumin, cysteine, gelatin, sucrose, and glutamic acid. The pH is buffered with HEPES buffer. Because it contains gelatin in addition to proteins such as serum albumins in very high concentration, UTM can lead to column clogging. It can also cause ion suppression during ionization in the mass spectrometer, and it can interfere with the digestion step described later. Two other media are frequently used, (i) the eSwab (trademark) media (available from COPAN Diagnostics, CA, USA) which contains high concentrations of non-volatile salts and causes interferences with the electrospray ionization used for the MS detection of the peptide and (ii) the GLY-media (Glucose-Lactalbumin-Yeast) made of a yeast extract comprising proteins, which consequently interferes with peptide detection.

Another issue is the acquisition mode of the MS method. When optimizing an LC method, often there is a tradeoff between selectivity and speed. When using fast chromatography, coeluting interferences need to be compensated for by a more selective and/or sensitive detection method.

MS² methods (also known as MS/MS methods, sometimes referred to as tandem MS methods) can select a specific fragment in a first analyzer (often a quadrupole) of the mass spectrometer, which will be fragmented in a collision cell. Then a specific fragment can be monitored (often using a third quadrupole). This approach represents the current gold standard for sensitive targeted analysis in mass spectrometry (see e.g. DOI: 10.1016/j.cbpa.2008.07.024 and DOI: 10.1126/science.1124619). However, in complex samples, noise limits the sensitivity of the measurement. To improve sensitivity, MS³ methods were developed, comprising an additional fragmentation step (Olsen and Mann, PNAS September s, 2004 101 (37) 13417-13422). However, MS³ methods are generally considered to be slow, for instance because the number of compounds that can be tracked in a single analysis is low, which can hinder implementation in a high-throughput setting.

Alternatively, high resolution mass spectrometry can be used for sensitive analysis of biomolecules, and quadrupole-Time-of-Flight MS and especially Orbitrap detectors have been used next to MS² using triple quadrupole for instance to detect the SARS-CoV-2 antigen peptide NCAP, or for its Spike peptides, after digestion.

In mass spectrometry analysis, a scan can cover a range of masses such as in a full scan method or a very narrow range such as in selected ion monitoring. A typical scan can take from 10 ms to 1 second. LC/MS analysis comprises many scans and usual practice suggests 12 scans across an eluting peak gives good reliability for quantitative analysis. A mass spectrometer can scan faster, increasing the number of scans across a peak, or scan for a longer period of time, increasing scan time. An increase in the number of scans is generally recommended to improve quantitation, and it is common to reduce scan time such as by reducing accumulation time to increase the number of scans (see Olsen and Mann cited above). Other methods to improve mass spectrometric detection of pathogen peptides that have been suggested are to focus on sample preparation, particularly on enrichment of samples via for instance immunoprecipitation (Dhaenens, 15 September 2020, The Analytical Scientist).

Diagnostic tests with sufficient sensitivity, specificity, throughput capacity, and rapid turnaround time are sorely needed, at lower cost( preferably per sample), and that function with easily obtained reagents and instrumentation. A key consideration in the development of such a diagnostic tool is the required sensitivity, which should be determined based on the viral load at which individuals remain infectious. Recently Van Kampen et al. reported a study to investigate exactly this (DOI: 10.1101/2020.06.08.20125310). While it remains difficult to define a clear cut-off value, currently experts suggest a Ct value of 28-30 (as determined by RT-qPCR as a reference method) as the level at which individuals can be still contagious. Therefore, a rapid diagnostic should preferably demonstrate good sensitivity at least for Ct values up to 30.

The inventors identified the use of mass spectrometry as a new tool in the arsenal that overcomes existing weaknesses of the diagnostic options discussed herein. A strength of mass spectrometry is its inherent selectivity and sensitivity: individual molecules are quantified based on their mass/charge ratio, and very low abundance molecules can be detected next to very highly abundant molecules. Furthermore, as mass spectrometry is a flexible technology able to detect a wide range of molecules, the proposed diagnostic platform is adaptable to viral mutations.

Unfortunately there is no clinical method that uses mass spectrometry for detecting SARS- CoV-2 peptides, while there is a general expectation that MS could be employed to provide faster and cheaper diagnosis. There is a need for high-throughput pathogen detection. There is a need for more sensitive detection of pathogens. There is a need for pathogen detection that does not rely on PCR. There is a need for improved detection of viral peptides.

Summary of the invention

The inventors surprisingly found that excellent sensitivity and specificity for substances such as peptides in complex samples can be achieved when using MS³ mass spectrometry, wherein the accumulation time for each MS acquisition scan is at least 125 ms. Accumulation time is illustrated in Fig. 8. Thus the invention provides a method for identifying the presence of a polypeptide associated with a pathogen in a sample, the method comprising the steps of: i)subjecting the sample to enzymatic digestion to obtain a digested sample; ii) analyzing the digested sample using MS/MS/MS (MS³) mass spectrometry, wherein the accumulation time for each MS acquisition scan is at least 125 ms. Preferably the sample is a swab sample previously obtained from a subject, preferably wherein it is a nasopharyngeal swab sample. Preferably the sample has a carrier medium and wherein the carrier medium of the sample is water, preferably ultrapure water. Preferably the digestion of step i): - is performed using a thermostable protease, preferably a thermostable trypsin, and/or - is performed at a temperature of at least about 60 °C, preferably of at least about 70 °C; and/or - is performed for at most 20 minutes, preferably for about 15 minutes. In preferred embodiments the pathogen polypeptide is a SARS-CoV-2 polypeptide, and step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 80% sequence identity with any one of SEQ ID NOs: 3-16 or 18- 34, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14, most preferably with any one of SEQ ID NOs: 3, 4, 1 1 , and 14.

Preferred embodiments are those wherein the analysis in step ii) is liquid chromatography (LC) MS³ analysis, wherein the LC comprises use of a gradient of at most 5 minutes, preferably of at most 60 seconds, more preferably of at most 45 seconds, even more preferably of at most 30 seconds. Alternately the analysis in step ii) is MS³ analysis with direct infusion, wherein optionally the sample has been subjected to solid phase extraction prior to the direct infusion. Preferably scan time is at least about 150 ms, more preferably at least about 200 ms, even more preferably at least about 250 ms; and/or at most 6 scans, preferably at most 5 scans, even more preferably at most 4 scans, most preferably at most 3 scans are measured per peptide. The MS³ mass spectrometry is preferably performed using a mass spectrometer having a first mass analyser and a second mass analyser, wherein the first mass analyser is an ion trap or a quadrupole, preferably a quadrupole, and wherein the second mass analyser is an ion-trap or an orbitrap or an ion cyclotron resonance (ICR)-MS, preferably an ion trap such as a linear ion-trap. Preferably the sample has not been treated to enrich its pathogen polypeptide content using affinity- based techniques. With preferred methods according to the invention the pathogen is detected with a Ct value of at least 25, preferably 26, even more preferably 27, most preferably 30; or the pathogen is detected with over 90% sensitivity, preferably over 95% sensitivity, or with over 90% specificity, preferably over 95% specificity. In a preferred method, the sample is a nasopharyngeal swab sample previously obtained from a subject, wherein the sample has a carrier medium and wherein the carrier medium of the sample is ultrapure water, wherein the digestion of step i) is performed using a thermostable trypsin at a temperature of about 70 °C and for at most 15 minutes, wherein the pathogen polypeptide is a SARS-CoV-2 polypeptide, and wherein step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 90% sequence identity with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14, more preferably with any one of SEQ ID NOs: 3, 4, 11 , and 14, wherein the scan time is at least about 200 ms, and wherein at most 5 scans are measured per peptide.

Also provided is a method for diagnosing the presence of SARS-CoV-2 in a subject, the method comprising the steps of: a) subjecting a sample previously obtained from a subject to a method as defined above; b) identifying the presence of at least one peptide having at least 80% sequence identity with any one of SEQ ID NOs: 3-16 or 18-34; and c) diagnosing the presence of SARS-CoV- 2 when the presence of the at least one peptide has been identified in step b). Preferably step b) comprises identifying the presence of the peptides represented by SEQ ID NOs: 3 and 4, preferably of SEQ ID NOs: 3, 4, 11 , and 14, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14.

Detailed description of the invention

The inventors surprisingly found that excellent sensitivity and specificity for substances such as peptides in complex samples can be achieved when using MS³ mass spectrometry, wherein the accumulation time for each MS acquisition scan is at least 125 ms. Accumulation time is illustrated in Fig. 8. On the time scale of MS³ protocols, accumulation time is rather time consuming, and conventionally this time is shortened to allow for more acquisition scans to take place (Olsen and Mann, PNAS September 14, 2004 101 (37) 13417-13422). The inventors found that improved results could instead be obtained by acquiring a lower number of scans, when those scans followed an accumulation time of at least 125 ms. The method can be used for the detection of biomarkers and substances of interest, such as drugs, metabolites, pesticides, lipids, and polypeptides, and thus the method is useful in clinical testing. Attractive results were obtained when detecting polypeptides. Accordingly the invention provides a method for identifying the presence of a polypeptide associated with a pathogen in a sample, the method comprising the steps of: i) subjecting the sample to sample preparation such as enzymatic digestion to obtain a digested sample; ii) analyzing the digested sample using MS/MS/MS (MS³) mass spectrometry, wherein the accumulation time for each MS acquisition scan is at least 125 ms.

Such a method is referred to hereinafter as a method according to the invention. Preferably the steps are performed in numerical order.

Analysis of the sample is performed using MS³ mass spectrometry, which can also be referred to as simply MS³. MS³ is an extension on established MS/MS or MS² technology. In MS² an ion is selected from a sample using a mass analyser, after which the selected ion is fragmented, for instance through collision with inert gas such as N2. Then, the resulting fragments are identified. MS³ expands on this, by isolating the first fragment, and once again fragmenting it. After this second round of fragmentation, the product ions are detected. MS² and MS³ are known as such.

The goal of analysis is to identify whether specific masses can be detected in the sample, or whether those masses cannot be detected. The presence of a mass is correlated with the presence of an analyte having that mass. Accordingly, when the mass of a peptide is detected, the presence of that peptide in the sample can be established. It is inherent in MS² and MS³ that the presence of a fragment can only be identified if the precursor peptide was present. This is because in earlier steps, the precursor peptide is selected, which then fragments, after which a product ion is selected, which in MS³ is then fragmented again, after which a second generation product ion is detected. Accordingly, when analysis reveals the mass of a second generation product, the product and hence the precursor must also have been selected by the earlier mass analysers. This allows identification of peptides via identification of fragments or second generation product ions. In general, reference can be made to detection of a mass, but also to detection of a peptide, if this peptide has the mass that is detected. This is routine in the field.

Mass analysers in mass spectrometers that are used in mass spectrometry separate the ions according to their mass-to-charge ratio. The final element of a mass spectrometer is a detector. The detector records either the charge induced or the current produced when an ion passes by or hits a surface. In a scanning instrument, the signal produced in the detector during the course of the scan versus where the instrument is in the scan (at what m/Q) will produce a mass spectrum, a record of ions as a function of m/Q. Such an MS acquisition scan results in a mass spectrum, which can be interpreted.

Accumulation time (AT) is a known parameter in MS³ and is sometimes referred to as fill time. As illustrated in Fig. 8, it is the time during which product ions from the first fragmentation are collected, which Fig. 8 demonstrates in an embodiment using a linear ion trap. In MS³, accumulation time is generally followed by selection of a product ion, after which the selected product ion is fragmented (which is the second fragmentation), subsequently followed by a scan for secondary product ions, which is preferably a full scan. During the accumulation time the first fragmenter can continuously emit product ions from the first precursor ion. In other words, accumulation time is the time between the start of the first fragmentation and the selection of a first product ion. Fig. 8 provides a visual representation of accumulation time in a particular experimental setup. An increase in accumulation time would increase the number of product ions that can be selected from, but extension of accumulation time is also known to cause an adverse impact on the detection of analytes. Excessive fragmentation has been reported to occur after extending the accumulation time (Sannes-Lowery et al., Rapid Comm. Mass Spect. Volume 12, Issue 23, 1998, Pages 1957- 1961). In general, accumulation times are kept short to prevent loss of ion signal of the targeted peptide. A short accumulation time is generally under 75 ms, but most often much shorter accumulation times are used, such as 50 ms or lower. Additionally, in the art, scan times are generally kept short to allow an increase in the number of scans.

Surprisingly the inventors found that for digested samples, a long accumulation time instead resulted in good detection, with a good signal-to-noise ratio. The accumulation time is at least 125 ms, but can be anywhere in the range of 125 to 1000 ms, such as 125, 130, 135, 140, 145, 150,

155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,

250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380,

390, 400, 450, or 500 ms. Preferred accumulation times are at least 125, 130, 135, 140, 145, 150,

155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, or 250 ms. Good results were obtained with 125 ms and with 250 ms, of which 250 ms is most preferred.

Preferably the accumulation time is at most 1000 ms, more preferably at most 500 ms, still more preferably it is at most 450 ms, still more preferably at most 400 ms, still more preferably 350 ms, still more preferably 300 ms, most preferably it is at most about 250 ms.

In preferred embodiments of the method according to the invention, the scan time is at least about 150 ms, more preferably at least about 200 ms, even more preferably at least about 250 ms; and/or preferably at most 6 scans, more preferably at most 5 scans, even more preferably at most 4 scans, most preferably at most 3 scans are measured per peptide. For direct injection MS, more than 6 scans can be preferred, such as 12, 11 , 10, 9, 8, or 7 scans.

The amount of scans measured per peptide refers to the amount of scans a detector makes for detecting each mass. This is particularly important when target ions are offered to a detector only during a limited time frame, such as in LCMS when a particular substance is eluted during a narrow time frame only. It is generally assumed that an increased number of scans leads to improved results as this would by increasing accuracy. More scans generally allows better integration of a peak because peaks are generally considered to require at least 6 scans to define and integrate the peak. Surprisingly, the inventors found that a lower number of scans still leads to excellent results such as highly accurate results, which allows incorporation of the longer ion accumulation times into a protocol. In some embodiments at most 8 scans are performed, or at most 7, 6, 5, 4, 3, 2, or 1 scans. In some embodiments only a single scan is performed per peptide, or in other words only a single scan is performed per mass. In some embodiments, at least 2 scans are performed. In LC-MS³, the inventors surprisingly found exceptionally low levels of noise, and at least two subsequent scans, such as scans with a signal above a predefined threshold, proved sufficient to detect the presence of a polypeptide. MS can be performed using known machines, a skilled person can implement the method of the invention using equipment that is generally available in laboratories that offer mass spectrometry services. In preferred embodiments is provided the method according to the invention, wherein the MS³ mass spectrometry is performed using a mass spectrometer having a first mass analyser and a second mass analyser, wherein the first mass analyser is an ion trap or a quadrupole, preferably a quadrupole, and wherein the second mass analyser is an ion-trap or an orbitrap or an ion cyclotron resonance (ICR)-MS, preferably an ion trap. Preferred ion traps are linear ion traps. Alternatively the first fragmentation can be achieve by in-source fragmentation. The first fragmentation can for instance be mass selective fragmentation, or can comprise fragmenting all ions, or fragmenting a group of ions wherein the group is selected by ion mobility.

As is known, various ways exist for injecting samples into an MS. This is not critical to the invention. Liquid chromatography (LC) MS is attractive because it separates the sample prior to mass detection, which improves the MS signal. A disadvantage is that LC is time consuming. The inventors found that nonetheless short LC protocols could still yield good results. Accordingly the invention provides the method according to the invention, wherein the analysis in step ii) is liquid chromatography (LC) MS³ analysis, wherein the LC comprises use of a gradient of at most 5 minutes, preferably of at most 60 seconds, more preferably of at most 45 seconds, even more preferably of at most 30 seconds. As is known, LC uses a gradient of two solvents to effect separation over a column such as a separation column. The term gradient should not be so narrowly construed as to exclude a flat elution with no change in solvent ratio. A skilled person will appreciate that the gradient refers to the effective separation time on a column. Similarly, column equilibration steps or washing steps are preferably not encompassed by the gradient time. Furthermore the gradient need not be continuous or linear, as a step gradient or non-linear gradient can also be envisaged. The gradient is preferably at most 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 minute, or 55, 50, 45, 40, 35, or 30 seconds or even less.

Another option is direct infusion. Direct infusion can be preferred because it allows scanning any amount of transitions and thus detecting any amount of different ions. Direct infusion also allows very long accumulation times. Accordingly when more than three, preferably more than four, more preferably more than five peptides are to be detected, direct infusion is preferred. It offers the possibility to scan. Lacking the LC step, for direct infusion it is preferred that the sample is otherwise pre-treated. Provided is the method according to the invention, wherein the analysis in step ii) is MS³ analysis with direct infusion, wherein optionally the sample has been subjected to pretreatment such as solid phase extraction prior to the direct infusion. Pre-treatment can be any step that removes impurities from the sample. For instance solid phase extraction, ion exchange, reverse phase chromatography, hydrophilic interaction liquid chromatography, precipitation, or washing, or any combination thereof can be performed. Preferred pre-treatments are solid-phase extraction and precipitation. Precipitation can conveniently be performed by for instance addition of a water- miscible organic solvent such as acetone that causes precipitation of dissolved polypeptides. Decantation of the supernatant can then remove impurities such as lipids. Subsequent resuspension of the precipitated pellet leads to a purified polypeptide sample. Such methods are known in the art. In the case of SARS-CoV-2, the polypeptide sample will comprise polypeptides associated with that pathogen, such as NCAP and spike protein. Selectivity can also be improved by using ion mobility using for instance high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) before injection into the MS. This can thus be used as an alternative to sample pretreatment, or as a complement.

Methods of the invention are highly sensitive. Provided is the method according to the invention, wherein the pathogen is detected with a Ct value of at least 25, preferably 26, even more preferably 27, most preferably 30; or wherein the pathogen is detected with over 90% sensitivity, preferably over 95% sensitivity, or with over 90% specificity, preferably over 95% specificity. Sensitivity and specificity are known in the art, and can be calculated as defined in the section on general definitions. Preferably, sensitivity is over 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99%, or it is 100%, more preferably it is over 98, 99%, or it is 100%. Preferably, specificity is over 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99%, or it is 100%, more preferably it is over 95%, even more preferably it is over 96%, most preferably it is over 98% such as 100%.

Ct value refers to threshold cycle (Ct, also called quantification cycle or Cq). It refers to a parameter from qPCR experiments. The correlation between Ct values and MRM signal intensity was established by Van Puyvelde et al. (see medRxiv 2020.11.18.20231688) and is also demonstrated in the examples. Preferably the pathogen is detected with a Ct value of at least 25,

26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36, preferably at least 26, even more preferably at least

27, most preferably at least 30 such as 31 . A Ct value can be determined by correlating the peptide MS signal (for instance the Iog2(peak area)) to Ct values based on known correlations. Ct value is preferably Ct value as determined by PCR, more preferably as determined by qPCR.

The polypeptide of which the presence is identified can be any polypeptide. It is particularly useful to identify the presence of a polypeptide associated with a pathogen, as this can contribute to detecting or diagnosing the presence of the pathogen, or diagnosing a disease or condition associated with the pathogen. A pathogen is preferably an infectious microorganism, wherein for instance a virus is also considered a microorganism. In other words, a pathogen is preferably an infectious microorganism or a virus. Accordingly preferred pathogens are a virus, a bacterium, a protozoan, a prion, a viroid, and a fungus. A bacterium and a virus are highly preferred, and a virus is most preferred. In other embodiments the polypeptide is associated with a disease or condition in that it is an aberrant polypeptide, or an unduly expressed polypeptide, or a peptide that is expressed in response to, or as a result of, a disorder.

A skilled person can implement the present method to identify the presence of any pathogen provided that information is known about the polypeptides associated with said pathogen. A polypeptide associated with a pathogen can be a polypeptide that is part of the pathogen, that is produced by the pathogen, or that is produced by a host in response to the presence of the pathogen. For instance the polypeptide can be a viral capsid polypeptide, it can be a polymerase encoded by a viral genome, and it can be an antibody or other host response to the pathogen. Preferably the polypeptide is a polypeptide that is part of the pathogen, such as a viral capsid protein or a bacterial protein antigen. It should be noted that the polypeptide that is detected is likely to be a fragment of a precursor polypeptide, which is in turn a fragment of an earlier precursor. That earlier precursor can be the polypeptide as present in the sample, or it can be a digestion product of the polypeptide as present in the sample.

Preferred viral pathogens are respiratory viral pathogens, such as RSV, a pneumovirus, an influenza virus, and a coronavirus. More preferably, the respiratory viral pathogens is a coronavirus, such as SARS-CoV or SARS-CoV-1 , MERS-CoV or SARS-CoV-2. Most preferably, the respiratory viral pathogens is SARS-CoV-2, i.e. can cause COVID-19.

Sample and optional treatment thereof

The sample in which the presence of a polypeptide is identified can be any sample. The method is of particular use when the sample is a sample that has previously been obtained from a subject, preferably a subject at risk of comprising the pathogen, or at risk of being infected with the pathogen, or suspected of comprising the pathogen, or suspected of being infected with the pathogen. A subject comprising a pathogen is often described as carrying a pathogen. Optionally multiple samples can be obtained from a single subject at the same time, for instance for performing parallel detection techniques. Additional samples can be obtained at a later moment to monitor the progression of a disease or condition. A sample can also be a food sample or a water sample or an excipient sample or any other sample.

The method for obtaining the sample is dependent on what the origin of the sample is. Examples of samples can be bodily fluids, skin samples, hair samples, urine orfecal samples, saliva samples, plasma samples, mouthwash samples, mucus samples, and swab samples. Mouthwash samples and swab samples are preferred for having a low invasive nature. For samples obtained from a subject, a swab sample is particularly convenient. Swab samples can be skin swabs, mouth swabs, nasal swabs, nasopharyngeal swabs, surface swabs, and so forth. Accordingly the invention provides the method according to the invention, wherein the sample is a swab sample previously obtained from a subject, preferably wherein it is a nasopharyngeal swab sample. Swabs can conveniently be stored dry.

Swabs are commonly known. For example, traditional-type swabs are constituted by a rod and an end, at which end there is a collecting element, for example constituted by a cotton fibre wound about the rod or by a sponge or the like, for defining a collecting portion adapted for absorbing internally thereof the sample to be collected. A further type of swab is known from W02004086979 or US9173779, which describes flocked swabs comprising an elongate support body and a plurality of flocked fibres arranged at an end of the support body for defining a collecting portion for the analytes or biological samples. Swabs are generally elongate rods which are in general made of plastic materials.

Preferred swabs are single-piece, single-material swabs manufactured by injection molding, and knitted polymer swabs such as knitted polyester swabs. Preferred swabs are dry swabs. Single-piece, single-material swabs manufactured by injection molding, particularly dry swabs of this type, are most preferred. Single-material swabs are preferably polyester swabs. A skilled person can select a useful swab for the present methods. Preferred swabs do not contain constituents that interfere with the analytical method, such as leeching constituents or products of swab degradation. A skilled person can determine whether a swab interferes with the method by testing blank samples or known reference samples, for instance using different swabs.

Material from the swab can be made available for further analysis using known methods. Exemplary methods comprise transferring a swab associated with an aliquot of the biological sample into a liquid, referred to herein as a carrier medium, wherein a portion or all of the aliquot is released into the carrier medium. This can provide efficient transfer of analyte such as cells or nucleic acid or polypeptide into the carrier medium. After having been transferred from a swab to a carrier medium, the sample can be further processed, or can be measured directly, or can be stored, although storage as a dry swab may be preferable.

A carrier medium can be any suitable liquid that dissolves polypeptides, such as water or ethanol or other lower alcohols or mixtures thereof; a skilled person can identify suitable solvents that can act as a carrier medium. Water is preferred. The invention thus provides the method according to the invention, wherein the sample has a carrier medium and wherein the carrier medium of the sample is water, preferably ultrapure water. More preferably the carrier medium consists of water such as ultrapure water.

In preferred embodiments the sample is not stored or transported in a medium wherein the medium as used comprises non-volatile salts or polypeptides. Additionally, the elegance of the present method allows the analysis of polypeptides while not requiring complex sample preparation steps that involve laborious enrichment of the target polypeptide, such as via affinity- based techniques such as immunoprecipitation. Accordingly in preferred embodiments the sample has not been treated to enrich its pathogen polypeptide content using affinity- based techniques. In other embodiments, such techniques can help to further increase sensitivity of the method according to the invention.

A subject can be human or animal. It is preferably a mammal such as a bat or a pangolin or a mink or a cat or a dog or an ape or a monkey or a human, more preferably it is a primate, most preferably a human. In some embodiments the subject is not human. In some embodiments the sample can be obtained from a cell culture derived from a subject.

In step i) the sample is subjected to enzymatic digestion to obtain a digested sample. Enzymatic digestion is widely used, also in mass spectrometry. When the sequence of a polypeptide is known, and the digestion results are known, then the presence of peptides that are a digestion product can indicate the presence of the undigested polypeptide. Digestion is preferably performed in a digestion medium, which is preferably a buffer. Digestion buffers are well known and their use can depend on the choice of digestion enzyme. Digestion buffers are commercially available, often from the same supplier who can provide a digestion enzyme. Digestion is preferably carried out using a protease, more preferably a thermostable protease, most preferably a thermostable trypsin. Preferred proteases are trypsin and lysobaccter serine proteases such as lys-C protease. An advantage of trypsin is that it has suitable recognition sites where it cleaves its substrate, leading to peptides that are digestion products of which the sequence can be predicted prior to digestion. Digestion is preferably performed for at most 30 minutes, more preferably at most 20 minutes, most preferably at most 15 minutes, such as for about 15 minutes. It can be performed for 120, 90, 60, 45, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12 ,1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. It is preferably performed for at least 5 minutes, more preferably for at least 10 minutes, most preferably for at least 14 or about 15 minutes. In preferred embodiments digestion is performed for about 15 to about 20 minutes.

Digestion is preferably performed at a temperature of at least 40 °C, more preferably of at least 50 °C, still more preferably at least 60 °C, and most preferably at least 70 °C. It is preferably performed at a temperature of at most 90 °C, more preferably at most 80 °C, most preferably of at most about 70 °C. In other embodiments the digestion is performed at 37 °C. Accordingly, in preferred embodiments, the digestion of step i): a) is performed using a thermostable protease, preferably a thermostable trypsin, and/or b) is performed at a temperature of at least about 60 °C, preferably of at least about 70 °C; and/or c) is performed for at most 20 minutes, preferably for about 15 minutes.

In some embodiments a) applies. In some embodiments b) applies. In some embodiments c) applies. In some embodiments a) and b) apply. In some embodiments a) and c) apply. In some embodiments b) and c) apply. In preferred embodiments each of a), b), and c) apply.

Exemplary peptides to be detected

A preferred pathogen is SARS-CoV-2, which can cause COVID-19. Important polypeptides associated with SARS-CoV-2 are its spike protein (SEQ ID NO: 2) and its nucleocapsid (NCAP) protein (SEQ ID NO: 1). Further peptides that are digestion products thereof are shown in the below table S.1 , which also lists the undigested precursor polypeptides. In this table the digestion products are trypsin digestion products. Peptides represented by these SEQ ID NOs were found to be attractive precursor ions, which can produce product ions that can be detected very well.

Table S.1 - sequences used in this application

SEQ ID NO: 2 is:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWF

HAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCE FQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFK

NIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGA

AAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVR

FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT

NVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPAT

VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITP CSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAE HVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTIS VTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVK QIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQK FNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYEN QKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDK VEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIIT TDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEI DRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCS CGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 1 is:

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKE DLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGA NKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRS RNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAA EASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFF GMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQ ALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

SEQ ID NO: 17 is a fragment of a human histone and is used as an internal positive control to establish that material was indeed obtained with the sample.

The method was found to be well suited for detecting SARS-CoV-2 polypeptides, such as those represented by SEQ ID NOs: 1 and 2. Accordingly, in preferred embodiments the pathogen polypeptide is a SARS-CoV-2 polypeptide, and step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 80% sequence identity with any one of SEQ ID NOs: 3-16 or 18-34, optionally with any one of SEQ ID NOs: 3-16, more preferably with any one of SEQ ID NOs: 3, 4, 11 , and 14, even more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14. SEQ ID NOs: 3 and 4 showed particularly attractive signal-to-noise ratios, allowing exceptionally precise detection. SEQ ID NOs: 11 and 14 displayed good signal-to-noise ratios and had attractive elution times relative to SEQ ID NOs: 3 and 4, allowing an unexpected combination of four peptides that can be detected surprisingly well, with low noise and high precision.

SEQ ID NO: 9 was surprisingly found to be detectable for the alpha variant of SARS-CoV- 2, but not for the gamma variant. This variant is known to have certain mutations. The mutation that falls within SEQ ID NO: 9 as present in the alpha variant causes the gamma version of the peptide to not be detected when original SEQ ID NO: 9 is selected for. SEQ ID NOs: 3, 4, 11 , and 14 remain well visible and thus confirm that SARS-CoV-2 is present. Thus when attempting to detect SEQ ID NO: 9, its absence can identify the detected SARS-CoV-2 as being a gamma variant. Its presence can identify the detected SARS-CoV-2 as not being a gamma variant.

Accordingly in preferred embodiments the invention provides a method for confirming that a SARS-CoV-2 is not a gamma variant, the method comprising the step of detecting SEQ ID NO: 9 using a method according to the invention. This method is particularly attractive for identifying alpha variants or omicron variants. In other preferred embodiments the invention provides a method for confirming that a SARS-CoV-2 is a gamma variant, the method comprising the step of screening for SEQ ID NO: 9 using a method according to the invention, and not identifying the presence of a corresponding polypeptide in the sample. In these methods, preferably at least one of SEQ ID NOs: 3, 4, 11 , and 14 are also screened for and are identified, more preferably all four of these are screened for. For confirming that a SARS-CoV-2 is a gamma variant, detection of SEQ ID NO: 21 is useful as it is the analogue of SEQ ID NO: 9 that is present in the gamma variant.

Sequence identity as described herein is preferably over the entire length of the SEQ ID NO. Preferably the detected peptide has the same length as the recited SEQ ID NO. Due to mutations in a pathogen, polypeptides associated with the pathogen can change. When a mutation is known, or has become known, the present method can be used to detect these mutants. Accordingly, the recited SEQ ID NOs and variations that have 80% sequence identity with these SEQ ID NOs can be detected using the present invention, and can help identify present and future mutants of the pathogen. In other embodiments, the sequence identity is preferably at least 90%. Highly preferably only a single or two substitutions or deletions are present as compared to the recited SEQ ID NO. Most preferably only a single substitution or deletion is present.

The method of the invention can thus allow the identification of mutant pathogens. For such methods step ii) preferably further comprises the step of identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments has at least one known mutation as compared to another variant of the pathogen.

When both the mutant and the original sequence are screened for, absence of the mutant signal and presence of the original signal can confirm that the sample comprises an original pathogen (see for instance Fig. 3B). Presence of the mutant signal and absence of the original signal can confirm that the sample comprises a mutant pathogen (see for instance Fig. 3C). Preferably at least one additional peptide is detected as a control peptide, wherein the control peptide is known to not be mutated across the original and the mutant polypeptide. Preferred peptides are conserved peptides. For SARS-CoV-2, suitable control peptides are at least one of SEQ ID NOs: 3, 4, 11 , and 14, preferably 2, 3, or 4 thereof. These SEQ ID NOs are present in at least the alpha, beta, gamma, and omicron variants.

The invention provides a method for identifying the presence of variants of a pathogen, preferably SARS-CoV-2, in a sample. Preferably, step ii) of these methods comprise identifying the presence of at least one conserved peptide fragment of the pathogen polypeptide, wherein the peptide fragments preferably have at least 90%, preferably 100%, sequence identity with any one of SEQ ID NOs: 3, 4, 11 , and 14. Preferably at least two of these fragments are detected, more preferably at least three, most preferably all four.

In embodiments, the method is for identifying the alpha variant, wherein step ii) comprises identifying the presence of at least one peptide fragment of the alpha variant, wherein the peptide fragments preferably have at least 80% sequence identity, preferably 100% sequence identity, with any one of SEQ ID NOs: 18-20.

In embodiments, the method is for identifying the gamma variant, wherein step ii) comprises identifying the presence of at least one peptide fragment of the gamma variant, preferably wherein the peptide fragments have at least 80% sequence identity, preferably 100% sequence identity, with any one of SEQ ID NOs: 21 , 22, or 24-26.

In embodiments, the method is for identifying the beta variant, wherein step ii) comprises identifying the presence of at least one peptide fragment of the beta variant, wherein the peptide fragments preferably have at least 80% sequence identity, preferably 100% sequence identity, with SEQ ID NO: 23.

In embodiments, the method is for identifying the omicron variant, wherein step ii) comprises identifying the presence of at least one peptide fragment of the omicron variant, wherein the peptide fragments preferably have at least 80% sequence identity, preferably 100% sequence identity, with any one of SEQ ID NOs: 27-34.

In embodiments, the method is for identifying the variant is not an alpha variant, wherein step ii) comprises identifying the absence of peptide fragments known to be unique to alpha variants, preferably it comprises identifying the absence of peptide fragments have at least 80% sequence identity, preferably 100% sequence identity, with SEQ ID NOs: 18-20.

In embodiments, the method is for identifying the variant is not a beta variant, wherein step ii) comprises identifying the absence of peptide fragments known to be unique to beta variants, preferably it comprises identifying the absence of peptide fragments have at least 80% sequence identity, preferably 100% sequence identity, with SEQ ID NOs: 23.

In embodiments, the method is for identifying the variant is not a gamma variant, wherein step ii) comprises identifying the absence of peptide fragments known to be unique to gamma variants, preferably it comprises identifying the absence of peptide fragments have at least 80% sequence identity, preferably 100% sequence identity, with SEQ ID NOs: 21 , 22, or 24-26.

In embodiments, the method is for identifying the variant is not an omicron variant, wherein step ii) comprises identifying the absence of peptide fragments known to be unique to omicron variants, preferably it comprises identifying the absence of peptide fragments have at least 80% sequence identity, preferably 100% sequence identity, with SEQ ID NOs: 27-34.

When for example SEQ ID NO: 4 is detected in step ii), this identifies the presence of SEQ ID NO: 1 in the sample, because SEQ ID NO: 4 is a digestion and fragmentation product of SEQ ID NO: 1. In a preferred embodiment the method according to the invention is provided, wherein the sample is a nasopharyngeal swab sample previously obtained from a subject, wherein the sample has a carrier medium and wherein the carrier medium of the sample is ultrapure water, wherein the digestion of step i) is performed using a thermostable trypsin at a temperature of about 70 °C and for at most 15 minutes, wherein the pathogen polypeptide is a SARS-CoV-2 polypeptide, and wherein step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 90% sequence identity with any one of SEQ ID NOs: 3, 4, 11 , and 14, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14, wherein the scan time is at least about 200 ms, and wherein at most 5 scans are measured per peptide.

In an even more preferred embodiment the method according to the invention is provided, wherein the sample is a nasopharyngeal swab sample previously obtained from a subject, optionally having used an injection-moulded swab, wherein the sample has a carrier medium and wherein the carrier medium of the sample is ultrapure water, wherein the digestion of step i) is performed using a thermostable trypsin at a temperature of about 70 °C and for about 15 minutes, wherein the pathogen polypeptide is a SARS-CoV-2 polypeptide, and wherein step ii) comprises identifying the presence of at least two, optionally four peptide fragments of the pathogen polypeptide, wherein the peptide fragments have at least 90% sequence identity with at least two, optionally all four of SEQ ID NOs: 3, 4, 1 1 , and 14, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14, wherein the scan time is about 250 ms, and wherein at most 5 scans are measured per peptide.

Diagnostic methods

The method according to the invention being useful for identifying the presence of pathogens, it can also contribute to diagnosis of such presence. Such a diagnostic method can comprise the steps of: a) subjecting a sample previously obtained from a subject to a method according to the invention as defined above; b) identifying the presence of at least one peptide having at least 80%, optionally 100% sequence identity with a peptide known to be associated with the pathogen; and c) diagnosing the presence of the pathogen when the presence of the at least one peptide has been identified in step b).

For instance, the invention provides a method for diagnosing the presence of SARS-CoV-2 in a subject, the method comprising the steps of: a) subjecting a sample previously obtained from a subject to a method according to the invention as defined above; b) identifying the presence of at least one peptide having at least 80% sequence identity with any one of SEQ ID NOs: 3-16 or 18-34, preferably 3-16; and c) diagnosing the presence of SARS-CoV-2 when the presence of the at least one peptide has been identified in step b).

In step b) it is preferred that 100% sequence identity is detected, although an amount of sequence identity in the range of 80 to 100% can allow for the detection of mutants of the pathogen. Preferably, the presence of a peptide having at most two substitutions or deletions, more preferably at most 1 substitution or deletion is detected.

Preferably, step b) comprises identifying the presence of the peptides represented by SEQ ID NOs: 3 and 4, preferably of SEQ ID NOs: 3, 4, 11 , and 14, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14. Preferably, step a) comprises subjecting the sample to a method wherein the sample is a nasopharyngeal swab sample previously obtained from a subject, wherein the sample has a carrier medium and wherein the carrier medium of the sample is ultrapure water, wherein the digestion of step i) is performed using a thermostable trypsin at a temperature of about 70 °C and for at most 30 or 20 minutes, most preferably at most 15 minutes, wherein the pathogen polypeptide is a SARS-CoV-2 polypeptide, and wherein step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 90% sequence identity with any one of SEQ ID NOs: 3, 4, 11 , and 14, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14, wherein the scan time is at least about 200 ms, and wherein at most 5 scans are measured per peptide.

The method according to the invention can be used with mouth flush or saliva as a sample, and the platform is flexible to be modified in case of potential new requirements. Finally, the technology platform can also be used to monitor other (future) infections such as viral infections, and can be rapidly adapted in case of emerging viruses to prevent future pandemics. It should further be appreciated that the present invention is not limited to any particular virus or pathogen, as long as polypeptides are associated with it. In any event, antibodies formed in response to a pathogen could be detected. Those skilled in the art will understand that the methods disclosed herein can be used to identify signatures for, and assess, other diseases, including those not specifically mentioned herein. The present invention is also not limited to use of particular types of mass spectrometry as long as the characteristics of the invention are comprised in the method.

General Definitions

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of” meaning that a combination or a composition as defined herein may comprise additional components) than the ones specifically identified, said additional components) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37° C (from about 20° C to about 40° C), and a suitable concentration of buffer salts or other components.

In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value, optionally more or less 5%.

The meanings of the terms “sensitivity”, “specificity”, “positive predictive value” and “negative predictive value” are typically known in the art and are defined in the context of the present invention according to the “Predictive Value Theory”, as established by the University of Iowa, USA. In this theory, the diagnostic value of a procedure is defined by its sensitivity, specificity, predictive value and efficiency. Sensitivity of a test is the percentage of all patients with disease present who have a positive test. (TP/(TP+FN)) x 100=Sensitivity (%) where TP=Test Positive; FN=False Negative. Specificity of a test is the percentage of all patients without disease who have a negative test. (TN/(FP+TN))xi 00=Specificity (%) where TN=Test Negative; FP=False Positive. Predictive value of a test is a measure (%) of the times that the value (positive or negative) is the true value, i.e. the percent of all positive tests that are true positives is the Positive Predictive Value ((TP/(TP+FP))'100=Predictive Value of a Positive Result (%); ((TN/(FN+TN))x 100= Predictive Value Negative Result (%)).

Each embodiment as identified herein may be combined together unless otherwise indicated. The invention has been described above with reference to a number of embodiments. A skilled person could envision trivial variations for some elements of the embodiments. These are included in the scope of protection as defined in the appended claims. All patent and literature references cited are hereby incorporated by reference in their entirety.

Short description of drawings

Fig. 1 - Influence of accumulation time on signal intensity of peptide K using LC-MS³.

Fig. 2A - 0.05 ng of recombinant protein in a negative swab loaded on the column and analyzed in MS² mode.

Fig. 2B - as for Fig. 2A, but analyzed in MS³ mode instead. Fig. 3A - LC-MS³ analysis of target NCAP peptides from a viral culture of SARS-CoV-2 omicron variant showing limit of detection; 8250 viral copy equivalents loaded on the column. Peptides R, A, K, and N were screened for.

Fig. 3B - LC-MS³ analysis of target NCAP peptides from a viral culture of SARS-CoV-2 alpha variant showing the detection of peptide GQG in addition to the four main peptide targets. Peptides R, A, K, N, and GQG were screened for.

Fig. 3C - LC-MS³ analysis of target NCAP peptides from a viral culture of SARS-CoV-2 gamma variant showing the non-detection of peptide GQG due to a mutation. Peptides R, A, K, N, and GQG were screened for.

Fig. 3D - LC-MS³ analysis of specific target SPIKE peptides of SARS-CoV-2 alpha variant from the recombinant protein. Peptides D, V, and Q were screened for.

Fig. 4 - Peptide-MS result across Ct values. Association between Ct-value as obtained by RT- qPCR and the result of the present MS method. Dots reflect SARS-CoV-2 infected samples as determined by PCR, with the x-axis indicating the Ct value; filled dots indicate samples identified positive by MS method, open dots as negative.

Fig. 5 - Correlation of MS signal of NCAP protein (peak area) using a first nasopharyngeal swab and Ct value of PCR of a second combined throat/nasopharyngeal swab of the same individual.

Fig. 6 - LC-MS³ analysis of four target peptides of NCAP of SARS-CoV-2 in a nasal swab with a Ct-value of 33 (by PCR of twin routine GGD swabs).

Fig. 7 - High throughput LC-MS³ analysis of four target peptides of NCAP of SARS-CoV-2 in a nasal swab.

Fig. 8 - Visual representation of an embodiment of MS³ with an indication of the accumulation time (indicated with 1) which here is preceded by emission of product ions from the first fragmentation, and followed by the fragment selection that precedes the second fragmentation.

Examples

Example 1 -sample preparation

Sample provision: in this example the pathogen SARS-CoV-2 is the pathogen of interest. Our approach involves collecting dry swabs which are resuspended in water. In tests comparing the efficiency of recovering viral signal from swabs using common media formulations, we observed water such as ultrapure water didn't lead to significant difference in terms of protein recovery from the swabs in comparison to the other carrier media. In addition, the use of water avoids the addition of components known to interfere with a mass-spectrometry workflow. This led to more efficient digestion of the proteins and lower signal background in the mass-spectrometry data.

We further evaluated standard flocked swabs such as nylon flocked or cotton swabs, or swabs with knitted polymer fibres. We also used single-piece swabs with certain features at the surface to sample sufficient mucus. The present method worked with these different types of swabs. Knitted polymer fibre swabs were found to allow extraction of viral particles containing the SARS-CoV-2 proteins particularly well. Digestion: an initial protocol (DOI: 10.1101/2020.11.18.20231688) aimed at digestion with sequencing grade trypsin at 37 °C, with the hope that a protocol could be developed wherein digestion was required for only 15 min. The present inventors found that such short durations and even shorter ones could in fact be achieved when an enzyme stable at 70 °C was used, for less than 20 min or for 20 or 15 min. Thermostable trypsin from Promega (Promega Corporation, Wl, USA) gave good results. Although the digestion time could be the same in both protocols, heating the sample at 70 °C was found to unfold the target proteins, making action of the thermostable trypsin more efficient. Thus, digestion at 70 °C was found to improve digestion by allowing a shorter duration, or by obtaining better digestion at the full duration, or a combination of both.

Example 2 - MS acquisition mode

In our method, an extra fragmentation step was added as compared to reported analysis of SARS- CoV-2 peptides after digestion (see DOI: 10.4155/bio.14.108). Ion traps are able to perform MSⁿ and this was utilized to gain additional selectivity in the MS detection method. Briefly, after selection of the m/z corresponding to the peptide of interest, it is fragmented, and a specific fragment is then selected, fragmented again and specific 2^nd generation fragments are monitored. This approach, called MS³ has several advantages. Firstly, the selectivity of the signal monitored is greatly improved, giving a trace nearly free of noise. The second advantage is the accumulation in the trap allowed for higher sensitivity. The last generation hybrid Q-TRAP mass spectrometer was used due to its high ion capacity and outstanding sensitivity. A demonstration of the benefits seen when accumulating ions in the trap giving a proportional increase of signal intensity is presented in Fig.

1. Longer acquisition times in combination with the elimination of noise contribute to achieving sufficient sensitivity to detect peptides such as SARS-CoV-2 peptides shown here. Surprisingly, the combination of the high selectivity and the ion trapping resulted in a signal/noise ratio increase (or sensitivity) close to 1000 times in comparison of the MRM mode (Fig. 2).

Example 3 - detection of SARS-CoV-2 peptides

For developing a high-throughput assay, a choice of SARS-CoV-2 peptides obtained after digestion was made. This choice was achieved using the following criteria:

1 . Selectivity of the peptides compared to other possible peptides occurring in the sample

2. Low noise in reconstructed LC-MS³ chromatograms

3. High signal of the peptides in LC-MS³

4. Peptides with similar hydrophobicity to enable shallow reverse phase gradients for separation and elution, or solid phase extraction (SPE) and subsequent direct infusion MS, or elution from SPE directly into the MS;

Currently, the separation method used involve long LC separation with a gradient of about 3-7 min. Here, we have increased the throughput of the separation by a factor 10 using a gradient of only 30 seconds. To achieve this, we have used a short column. The method can be further accelerated with a switching column method, so that the separation on one column is transferred to the MS while the other column is washed and conditioned for the next analysis. A careful selection of the targeted peptides further contributed to the throughput. This selection was made based on the MS³ performance of the peptides and their elution windows.

A challenge of using this fast-LC method are the very sharp peaks (3s), which do not give sufficient time to acquire many data points (more than 4 data point per peak) when using 250 ms filling time in MS³ mode (Fig. 1). This could lead to less accurate quantification if quantification is achieved by integrating a peak. For more accurate quantification, lower accumulation time can be used (125- 150ms), but this leads to a proportional loss of sensitivity. However, as it was surprisingly found that there is hardly any noise in the MS³ mode as performed here, a different strategy than integrating a peak can be used to determine whether a polypeptide is present. For instance if a signal is in an MS³ trace above a certain threshold for at least two, preferably at least three, subsequent scans then a polypeptide can be considered to be present rather than integrating a peak. However, given that an important goal is detection of the presence of specific SARS-CoV-2 peptides, this can be acceptable.

To achieve throughputs higher than those possible with the fast-LC- MS³ method described above, analysis of target peptides using direct infusion is possible. A direct infusion method allows for the analysis of several samples per minute, and because this sample introduction method does not have the sharp elution peaks present in fast LC, it allows for the inclusion of more peptide targets. Indeed, the long accumulation times required for highly sensitive MS³ measurements reduce the number of data points acquired per second, which was found to lead to undersampling of the LC- MS peaks and, which made quantification by fast-LC less accurate (peak width ~3s). Direct infusion of the samples for about 10s was found to provide sufficient acquisition time for accurate quantification for multiple targets, and thus to to give more flexibility for the selection of peptide MS³ transition. It was also found to simplify the addition of new target peptides in case of protein sequence changes due to mutations, such as those from reported virus variants. Direct infusion was achieved using a continuous flow and injection of plugs of samples into the flow. It was also achieved using acoustic injection such as by using the Echo MS of Sciex (a division of Danaher corporation, DC, USA), or via a SPE column such as the Rapid Fire system of Agilent (Agilent technologies inc., Santa Clara, CA, USA). Selectivity can be improved using sample preparation such as solid phase extraction, or by using ion mobility using e.g. high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) in front of the MS.

3. 1 detailed protocol

3. 1. 1 Sample collection

Nasopharyngeal swab samples were collected at the testing site of the Public Health Service of Amsterdam (Gemeentelijke Gezondheidsdienst, GGD) at the location RAI and at the location Zuidoost (Amsterdam, the Netherlands). Forthis, study volunteers visiting the GGD testing facility for a routine SARS-CoV-2 diagnostic test (using PCR) were approached and asked whether they are willing to participate in this study. Only volunteers of 18 years and older were included in this study. Volunteers signed an informed consent form and an additional nasopharyngeal swab, using injection-moulded polyester (Delft, the Netherlands ) was taken for MS analysis from these volunteers. The volunteers were not asked whether they had symptoms. The swab was put into an empty 15 mL centrifuge tube, barcoded, and stored in a -20°C freezer at the GGD test facility until transport to the lab in Leiden, and stored at -80°C in Leiden in the lab until the MS analysis. The routine GGD swab, in polypropylene with a flocked tip, was transferred in a vial with buffer and sent for RT-qPCR analysis by the lab of InBiome (Amsterdam, the Netherlands). They used a PerkinElmer SARS-CoV-2 Real-Time RT PCR Assay the N- gene and Orfl ab, and an internal control on human RNA target.

3. 7.2 Sample preparation

500 pL of ultrapure water was added to the dry swab, directly into their storing tube. Then the tubes were quickly and individually vortexed and sonicated in a sonication both for 10 min prior agitation for 10 min on an orbital shaking plate. 150 pL of the aqueous solution were transferred to a 1.5 mL tube and 1050 pL of ice cold acetone was added. After agitation for 10 min and then the samples were centrifuged at 13000G at 4 °C for 10 min. A maximum of the supernatant is manually discarded, the pellet is saved and the remaining acetone is left for evaporation during few minutes. 30 pL of Rapid Digest Buffer purchased from Promega is then added to pellet. The sample is vortexed until dissolution of the pellet and 10 pL of Rapid Trypsin/LysC (Promega) at 0.1 pl/pl in 50 mM of acetic acid are added. The samples are incubated at 70 °C and 900 rpm for 20 min. Finally the digestion is stopped by adding 4 pL of 10% formic acid to the sample and briefly centrifuged at 13000G at 4 °C for 10 min to remove any particles in suspension before transfer into LC vials.

3. 1.3 Liquid chromatography

A Nexera X2 ultra-high pressure liquid chromatography (UHPLC) system from Shimadzu (Duisburg, Germany) was used for fast LC-MS/MS and MS³ experiments. Separations were performed with a Waters Acquity™ Premier Peptide column (CSH, 2.1 mm x 100 mm, 1.7 pm) maintained at 50°C. The mobile phases consisted of 0.1 % formic acid (A) and MeCN (B). The injection volume was 10 pL and the flow rate of the mobile phase was set at 0.6 mL/min. The separation was carried out using the following gradient: 3 to 40% B in 5 min, then increased to 90% in 2 min and maintained at 90 for 0.9 min. Then the initial conditions are restored in 0.1 min and hold for reconditioning fori .9 min. The total analysis time was 10 min.

3. 1.4 Fast-LC Nexera X2 ultra-high pressure liquid chromatography (UHPLC) system from Shimadzu (Duisburg, Germany) was used for fast LC-MS/MS and MS³ experiments. Separations were performed with a Waters Acquity™ Premier Peptide column (CSH, 2.1 mm x 50 mm, 1.7 pm) maintained at 50°C. The mobile phases consisted of 0.1 % formic acid (A) and MeCN (B). The injection volume was 10 pL and the flow rate of the mobile phase was set at 1.2 mL/min. The separation was carried out using the following gradient: 20 to 30% B in 0.3 min, then increased to 90% in 0.14 min and maintained for 0.04 min. Then the initial conditions are restored in 0.1 min and hold for reconditioning for 0.51 min. The total analysis time was 1 min.

3. 1.5 Mass spectrometry The UHPLC system was hyphenated with an AB Sciex 7500 Q-Trap MS (AB Sciex,

Concord, ON, Canada) equipped with an Optiflow Pro ion source. MS experiments were performed in the positive ionization mode using cubic-selected reaction-monitoring (SRM³). The details of the SRM³ transition that were monitored for each peptide, as well as the respective collision energies and Q0 dissociation in simple mode, are reported in Table 3.1 .5.1 . MS³ fragment are extracted with a mass error of ± 0.5 Da. The accumulation time for every SRM³ was set to 250 ms and the MS experiments were scheduled without overlap to maximize the number of data points without compromising sensitivity. The curtain gas, ion source gas 1 and 2 pressures were fixed at 40 , 40 and 65 psi, respectively. Source temperature was set at 450 °C and the CAD at 12. The spray voltage and EP, were adjusted to 2000 V and 10V, respectively, for each SRM³ experiment. Data acquisition, instrument control and data treatment were done using SciexOS (AB Sciex, Concord ON, Canada).

Table 3. 1.5.1 - data related to detected peptides and polypeptides

A single gradient is used for SST and for SARS-CoV-2 peptide analysis, with a gradient from 3% to 40% B in 5 minutes, followed by a plateau at 90% B for 45 seconds, followed by a recalibration at 3% B until 7 minutes have passed in total. The MS valve switches to the MS after 0.1 min (position A) and to the waste after 5.7 min (position B). Injection volume SST is 1 pL, digest analysis is 10 pL. Good results were also obtained with MS analysis between 1 and 4 minutes, which reduces wear of the MS.

Advantages of the above are that the following evaluation criteria are satisfied:

1 . A sensitivity higher than 95% for Ct values up to 30

2. A specificity of at least 98%

3. A lead time of maximum 60 min (meaning the time from the moment the sample reaches the lab towards a validated result)

4. A scalable test, meaning it can do perform thousands of tests per day.

Mass spectrometry (MS) allows for the very specific detection of amino acid sequences, e.g., those of SARS-CoV2 proteins. MS is inherently highly sensitive, it can detect femtomoles of biomolecules and even below. Interestingly, these readings are unaffected by most mutations, especially when combining multiple peptides in one test as we describe here. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR, using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments.

MS builds upon a method that detects proteolytically digested SARS-CoV-2 proteins. For this example we chose two viral peptide targets AYNVTQAFGR (SEQ ID NO: 4) and KQQTVTLLPAADLDDFSK (SEQ ID NO: 3) that we demonstrated can be detected at high sensitivity and specificity using MS. They were obtained from the NCAP protein (SEQ ID NO: 1) and isolated from a tryptic digest of nasal swabs to identify viral load. In-silico analysis further demonstrated that these peptides are unique for SARS-CoV-2.

The detection of peptides unique to the virus offers good specificity, however, the LC-MS² method developed and published earlier was not sensitive and fast enough for implementation for reliable SARS-CoV-2 diagnostics. Therefore, we made pivotal changes to improve the sensitivity and throughput. We improved mass spectrometry detection utilizing advanced MS/MS/MS (MS³) methods so that there was hardly any noise when detecting the specific viral peptide fragments (see Figure 3C). As a result, we can now accurately detect SARS-CoV-2 infections in nasal swabs with a viral load up to Ct values of 30 as determined by RT-qPCR, on average within 60 minutes. Our approach also uses temperature stable enzymes that enable speeding up digestion of the sample prior to MS³.

Our method has some key (logistical) advantages as described below. At the sampling site:

• The sampling is not different than current sampling practices other than using a singlematerial one-piece injection moulded swab.

• No special skills or delicate pre-processing steps are required from the sampling personnel. Moving the sample to the lab:

• During the study, samples were stored at -80°C to ensure long stability of samples. When implemented, the storage requirements are expected to be limited to keeping samples at room temperature for a limited time, or in refrigerator for max 2 days; this can be validated, which is planned.

• Lab setups are relatively straightforward, meaning they can be set up at the site of sampling or in a trailer as a mobile lab.

In the lab:

• MS uses different devices and reagents from PCR which are widely available.

• MS is expected to run flawlessly on for instance Sciex Qtrap MS, which is readily available.

• The lab analysis can be fully automated, further increasing speed and further reducing the need for specialised personnel.

Promega is the chosen supplier of trypsin because they have heat-stable enzymes allowing for much faster digestion. For the depreciation the costs are estimated to be 3-5 Euro/sample, assuming depreciation during 4 months and full use of the possible throughput. 3.2 sample acquisition and preparation

The present method was designed to work with dry swabs, and single-piece single-material injection moulded swabs were used to avoid supply problems and background contamination as seen in other sampling devices. No manipulation was required by the personnel other than putting the swab in a container which can be stored in a -20°C freezer and sent under dry ice to the processing laboratory. There, swabs were resuspended in 500 pL of ultrapure water using vortex mixing and sonication and were aliquoted and stored at -80°C at the lab until further analysis.

To 150 pL of the aqueous swab extract, 1050 pL of ice-cold acetone is added for a volume ratio aqueous phase to acetone of 1 to 7. This was agitated shortly at 4°C and centrifuged at 16 000G for 15 min at 4°C. The protein pellet was resuspended in 30 pL of Rapid Digest Buffer (promega) and 10 pL of the protease solution (0.1 ug/ul of Trypsin/LysC in 50 mM acetic acid) was added to the sample and incubated at 70 °C for 20 min. The reaction was stopped by adding 4 pL of formic acid (10 vol.-% in water) to the digest and transferred to liquid chromatography vials.

3.3 MS analysis

The LC-MS/MS system used for the MS³ included a Shimadzu LC coupled to a Sciex 7500 Qtrap. The separation was achieved using a 2.1 x 100 mm reversed phase column (Acquity Premier, Peptide, CSH, 1.7 urn, Waters) with a 5-minute gradient. Electrospray ionization in positive mode resulted in doubly charged peptides that were fragmented in an MRM 3 method with 250 ms accumulation time. Peak areas reflect viral protein concentration including protein from viron and virus-like particles in the swab samples.

Tests done on a viral culture of omicron variant with known number of digital copies indicated we can detect down to 1 ,65e5 copies/mL of sample, corresponding to the actual digestion of 33 000 virus particles and the injection of 8250 (Fig. 3A). In addition, we have shown our targets are highly conserved and can be used for the detection of both alpha and gamma variants (Fig 3B and C). The detection of a specific peptide can be based on the presence of a certain peptide due to the mutation (so changed peptide sequence), or due to the absence of a peptide due to a mutation. Based on the genomic information of the variants of concern, these peptide targets can also be used to detect delta and omicron variants. As highlighted in Fig 3D with the case of the alpha variant, our LC-MRM3 approach is able to detect SPIKE specific mutant peptides . Finally, the method could be accelerated using a 1 min gradient (Fig. 7).

3.4 analysis of four peptides

We identified four viral peptide targets (RGPEQTQGNFGDQELIR (SEQ ID NO: 14) AYNVTQAFGR (SEQ ID NO: 4), KQQTVTLLPAADLDDFSK (SEQ ID NO: 3) and NPANNAAIVLQLPQGTTLPK (SEQ ID NO: 11)) that we found can be detected at high sensitivity and specificity using our method. They were obtained from the NCAP protein and isolated from a tryptic digest of dry nasal swabs to identify viral load. In-silico analysis further demonstrated that these peptides are unambiguous for SARS-CoV-2. The low noise is demonstrated in Fig. 6. As a result, we can now consistently detect SARS-CoV-2 infections in nasal swabs with a viral load of Ct values of 31 as determined by RT- qPCR, and with lower sensitivity also at higher Ct values, on average within 60 minutes. The sample preparation comprises extraction with water from the swab, a protein isolation step based on precipitation with acetone, rapid digestion of the proteins, and subsequent LC-MS³ analysis. The throughput of the LC-MS method can be increased to more than 50 samples/hour (Fig. 7).

Example 4 - case study

Approach: in 160 subjects sampled at GGD Amsterdam, the Netherlands (of which 49 with positive PCR and of which 26 with Ct < 30), the present method was compared to PCR testing to evaluate its accuracy.

Evaluation criteria: the evaluation criteria were defined as follows:

1 . A sensitivity higher than 95% for Ct values up to 30

2. A specificity of at least 98% (for the present method)

4. A scalable test, meaning it can be easily scaled to perform thousands of tests per day.

Results:

• The present method showed 100% sensitivity in the group with positive PCR with Ct < 30 (n=26)

• The present method showed sub-100% sensitivity in the group with positive PCR with Ct between 30 and 34 (n=8; 75% sensitivity)

• Specificity was 98%; in 111 PCR-negative tests, the present method found 2 positive samples.

• In our lab setting, the sample preparation workflow was designed to prepare a batch of 96 samples in 30-40 minutes for MS analysis. One MS instrument could analyze more than 100 samples per hour. The throughput time is hence on average currently 60 minutes (time to result) at an estimated cost price of ~€ 3-4 (chemicals and reagents excluding depreciation of equipment). The throughput can be further increased with more automation, parallelization and better integration of devices down to approximately 30-40 minutes.

Conclusions: the present method meets the criteria outlined above and qualifies as a highly accurate and rapid COVID test. Our initial results suggest a significantly higher accuracy than the antigen tests currently being used for rapid testing at a price point well below for instance the loop- mediated isothermal amplification (LAMP) test.

Perspectives: the test is ready for automation and implementation in test streets and for testing of children in schools and employees in companies. The test is a valuable addition to the existing toolkit of tests that currently includes PCR, LAMP PCR, breath test, and antigen tests. The high sensitivity and specificity at a competitive price make the present method attractive. Supply conditions of reagents and instrumentation are favourable as this test is fundamentally different from all currently used technologies for COVID-19 testing.

Example 5 - sensitivity and specificity

Persons visiting the GGD test street in Amsterdam for a routine SARS-CoV-2 diagnostic test (by PCR) were asked to participate in this study. Only volunteers of 18 years and older were asked to participate in the study. Volunteers signed informed consent and an additional nasopharyngeal swab was collected. The second swab was transferred in an empty tube, barcoded, and stored in a freezer for inclusion in this study. During the following periods a second swab was collected by the GGD Amsterdam and stored in a -20°C freezer at the GGD till transport to the lab in Leiden, and stored at -80°C in the lab till further analysis. 1318 swabs were collected using knitted polymer swabs. 1262 swabs were collected using single-material single-piece injection moulded swabs.

160 samples (selecting all positive COVID cases from 711 samples from the second sample campaign (part 2B) and matched with an equal number of negative cases) were measured and the values were compared with RT-qPCR (N-gene data used; Orfl ab resulted in similar results) data as obtained from the routine GGD swab.

MS results were analyzed by personnel not knowing the Ct value of corresponding PCR tests (they were informed about the Ct value only after reporting of the MS analysis results). 53 samples had measurable Ct-values for the GGD swab (GGD refers to municipal health authorities in the Netherlands). For 35 samples, a SARS-CoV-2 positive response for the KQQ peptide was obtained with the LC-MS peptide method.

For samples with a Ct value up to 30, the sensitivity of the MS method is close to 100%. As such it outperforms most current antigen tests. The method was found to be very selective in detecting the SARS-CoV-2 NCAP peptide and does not depend on biomolecular interactions or amplification, and (hence) is less sensitive to interferences from the sample matrix. The costs for the test are comparable to the antigen tests, though the manual handling is actually less than for the antigen test. The costs of the test are significantly lower than for the RT-LAMP SARS-CoV-2 test.

Additional results for similar experiments are shown in the tables below:

Table 3 - additional unblinded results

In summary:

• Our method showed 100% sensitivity in the group with positive PCR with Ct < 30 (n=26)

• Our method showed reduced sensitivity in the group with positive PCR with Ct between 30 and 34 (n=8; 75% sensitivity)

• Specificity was 98%; in 111 PCR-negative tests, our method found 2 positive samples in which the PCR test did not find RNA.

• In our lab setting, the sample preparation workflow was designed to prepare a batch of 96 samples in 30-40 minutes for MS analysis. One MS instrument could analyze more than 100 samples per hour. The throughput time is hence on average currently 60 minutes (time to result) at an estimated cost price of ~€ 3-4 (chemicals and reagents excluding depreciation of equipment). The latter strongly suggest this test can be offered at a price point well below the LAMP test.

• The throughput can be further increased with more automation, parallelization and better integration of devices down to approximately 30-40 minutes.

675 additional swabs were collected from volunteers from 3 January 2021 to 10 January 2021 in the same way as above. Samples were collected at location Zuidoost in Amsterdam, and from 5-7 January also at location RAI in Amsterdam. The procedure was exactly the same as for the first validation. Researchers did not have information whether these volunteers had symptoms or not. Researchers did not know which volunteers were infected with SARS-CoV-2. The analysis is ongoing, and the available results of the MS analysis of 349 swabs have been so far been sent to GGD Amsterdam, and then the PCR results of the first swab was sent to the inventor’s lab, and the results of the MS analysis of the second swab was like for the first study compared with the PCR result as golden standard (see Table 4). For these 349 volunteers, 20 cases of SARS-CoV-2 were detected by RT-qPCR as reference standard, resulting in a prevalence of 5.7%.

Table 4 - Additional blinded results of comparing the results of the MS assay with the N-gene RT- qPCR results of routine GGD swab showing the number of positive and negative hits as well as the specificity and sensitivity with the 95% confidence intervals.

In the above, our MS method showed a sensitivity of 100% (95%CI:87%-100%) in the group with positive PCR with Ct values up to 30 (n=12), and a sensitivity of 92% (95%CI: 64%-100%) in the group with positive PCR with Ct values up to 32 (n=13). Specificity was 100% (95%CI: 99%-100%) (329 negative samples).

Please note that this is a different test than all the others. None of the others directly measures the presence of viral proteins using such a specific assay. The quantitative results obtained via PCR reflect RNA measurement, whereas those obtained via MS reflect NCAP presence in viral and viral- like particles; hence, as these tests measure different underlying indicators, even were both tests to achieve perfect measurement from the same swab we still expect differences in results (Fig. 5). Importantly, the MS method can be easily automated. Because of its scalability, automation, accuracy and flexibility, MS can contribute to both the scale and diversity of testing approaches, e.g., it can enable rapid testing of children, employees of companies and those attending events.

In addition, the peptide MS method shows great potential for use with less invasive samples like saliva and mouth flush, i.e. flushing the mouth with 2 ml of water for 10 seconds. The evaluation of these samples has been initiated.

Conclusion: our method meets the criteria outlined above and hence qualifies as a highly accurate and rapid COVID test. We are pleased that our initial results suggest a significantly higher accuracy than the antigen tests currently being used for rapid testing at a price point well below the RT-LAMP SARS-CoV-2 test.

Claims

32 Claims

1 . A method for identifying the presence of a polypeptide associated with a pathogen in a sample, the method comprising the steps of: i) subjecting the sample to enzymatic digestion to obtain a digested sample; ii) analyzing the digested sample using MS/MS/MS (MS³) mass spectrometry, wherein the accumulation time for each MS acquisition scan is at least 125 ms.

2. The method according to claim 1 , wherein the sample is a swab sample previously obtained from a subject, preferably wherein it is a nasopharyngeal swab sample.

3. The method according to claim 1 or 2, wherein the sample has a carrier medium and wherein the carrier medium of the sample is water, preferably ultrapure water.

4. The method according to any one of claims 1-3, wherein the digestion of step i):

- is performed using a thermostable protease, preferably a thermostable trypsin, and/or

- is performed at a temperature of at least about 60 °C, preferably of at least about 70 °C; and/or

- is performed for at most 20 minutes, preferably for about 15 minutes.

5. The method according to any one of claims 1-4, wherein the pathogen polypeptide is a SARS- CoV-2 polypeptide, and wherein step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 80% sequence identity with any one of SEQ ID NOs: 3-16 or 18-34, more preferably with any one of SEQ ID NOs: 3, 4, 9, 11 , and 14, most preferably with any one of SEQ ID NOs: 3, 4, 11 , and 14.

6. The method according to any one of claims 1-5, wherein the analysis in step ii) is liquid chromatography (LC) MS³ analysis, wherein the LC comprises use of a gradient of at most 5 minutes, preferably of at most 60 seconds, more preferably of at most 45 seconds, even more preferably of at most 30 seconds.

7. The method according to any one of claims 1-5, wherein the analysis in step ii) is MS³ analysis with direct infusion, wherein optionally the sample has been subjected to solid phase extraction prior to the direct infusion.

8. The method according to any one of claims 1 -7 wherein the scan time is at least about 200 ms, preferably at least about 250 ms; and/or wherein at most 6 scans, preferably at most 5 scans, even more preferably at most 4 scans, most preferably at most 3 scans are measured per peptide. 33

9. The method according to any one of claims 1-8, wherein the MS³ mass spectrometry is performed using a mass spectrometer having a first mass analyser and a second mass analyser, wherein the first mass analyser is an ion trap or a quadrupole, preferably a quadrupole, and wherein the second mass analyser is an ion-trap or an orbitrap or an ion cyclotron resonance (ICR)-MS, preferably an ion trap such as a linear ion-trap.

10. The method according to any one of claims 1-9, wherein the sample has not been treated to enrich its pathogen polypeptide content using affinity-based techniques.

11. The method according to any one of claims 1-10, wherein the pathogen is detected with a Ct value of at least 25, preferably 26, even more preferably 27, most preferably 30; or wherein the pathogen is detected with over 90% sensitivity, preferably over 95% sensitivity, or with over 90% specificity, preferably over 95% specificity.

12. The method according to claim 1 , wherein the sample is a nasopharyngeal swab sample previously obtained from a subject, wherein the sample has a carrier medium and wherein the carrier medium of the sample is ultrapure water, wherein the digestion of step i) is performed using a thermostable trypsin at a temperature of about 70 °C and for at most 15 minutes, wherein the pathogen polypeptide is a SARS-CoV-2 polypeptide, and wherein step ii) comprises identifying the presence of at least one peptide fragment of the pathogen polypeptide, wherein the peptide fragments have at least 90% sequence identity with any one of SEQ ID NOs: 3, 4, 11 , and 14, wherein the scan time is at least about 200 ms, and wherein at most 5 scans are measured per peptide.

13. A method for diagnosing the presence of SARS-CoV-2 in a subject, the method comprising the steps of: a) subjecting a sample previously obtained from a subject to a method as defined in any one of claims 1-13; b) identifying the presence of at least one peptide having at least 80% sequence identity with any one of SEQ ID NOs: 3-16 or 18-34; and c) diagnosing the presence of SARS-CoV-2 when the presence of the at least one peptide has been identified in step b).

14. The method according to claim 13, wherein step b) comprises identifying the presence of the peptides represented by SEQ ID NOs: 3 and 4, preferably of SEQ ID NOs: 3, 4, 11 , and 14.

15. The method according to claims 13 or 14, wherein step a) comprises subjecting the sample to a method as defined in claim 12.