Cardiovascular disease

Abstract

The invention relates to cardiovascular disease (CVD), and particularly, although not exclusively, to coronary artery disease (CAD). The invention is especially concerned with CVD- and CAD-associated biomarkers, and to methods and kits for the detection and measurement of such biomarkers in biological samples for diagnosing or prognosing CVD or CAD. The invention also relates to methods of sample preparation and optimisation of a mass spectrometry assay to accurately measure the biomarker concentration in samples.

Description

[0001]Cardiovascular disease The present invention relates to cardiovascular disease (CVD), and particularly, although not exclusively, to coronary artery disease (CAD). The invention is especially concerned with CVD- and CAD-associated biomarkers, and to methods and kits for the detection and measurement of such biomarkers for diagnosing or prognosing disease. The invention relates to the quantitative measurement of biomarkers in biological samples for diagnosing or prognosing CVD or CAD. The invention also extends to methods of sample preparation and optimisation of a mass spectrometry assay to accurately measure the biomarker concentration in samples. The protein Sushi, von Willebrand factor type A, EGF and Pentraxin Domain Containing 1 (SVEP1) is a large extracellular matrix protein encoded by the gene, Svep1, first discovered by Shur and colleagues in 2006. SVEP1 is ubiquitously expressed in human tissues and is thought to be involved in cell adhesion and epidermal differentiation.1, 2Not much is known about the function of SVEP1, but genetic variants of SVEP1 have been associated with Coronary Artery Disease (CAD), blood pressure,3and platelet aggregation.4SVEP1 has been found to interact with integrins α9β15and α4β1,6matrix-binding proteins linked to increased blood pressure and vasoconstriction.6A recent study by Elenbaas et al. (2023) also found that SVEP1 is a ligand for platelet and endothelial aggregation receptor 1 (PEAR1), which has been implicated previously in platelet aggregation, CAD, and related outcomes.7Furthermore, elevated levels of SVEP1 have increasingly been associated with CAD. Jung et al. (2021) found by Mendelian randomisation (MR) that increased SVEP1 protein was causally related to increased CAD, hypertension, and type 2 diabetes.8Elenbaas et al. confirmed these findings, reporting that plasma concentrations of SVEP1 is positively associated with the risk of cardiovascular disease.7However, despite the strong association of SVEP1 with heart disease and the reported interactions of SVEP1 with integrins and PEAR1 representing potentially targetable interactions to treat hypertension and CAD, antibodies that reliably recognize SVEP1 in situ have not been developed.7Therefore, reliable and quantitative measurement of SVEP1 in biological systems is still an onerous task. Work by Jung et al. and Elenbaas et al. to measure SVEP1 in plasma has relied on data collected using the SomaScan aptamer platform in previous large-scale studies, such as the INTERVAL study.9 Designed to measure large multiplexed protein panels at a cost of several hundred dollars per sample with a 4-6 week wait on results, the SomaScan platform is not a practical option for many researchers to measure SVEP1 as a particular protein of interest. In view of the above, therefore, there is an unmet need to reliably identify robust biomarkers of coronary calcification in order to prevent coronary events. Accordingly, there is a need to provide improved methods, assays and apparatuses for quantitatively measuring SVEP1 in biological samples for diagnosing and prognosing cardiovascular disease, and especially coronary artery disease. In order to overcome the limitations of the existing methods used to quantitatively measure SVEP1 in plasma, the inventors performed an agnostic proteomics discovery study using liquid chromatography coupled to mass spectrometry (LC-MS) on the prospective observational Biomedical Research Informatics Centre for Cardiovascular Sciences (BRICCS) cohort (ClinicalTrials.gov Identifier: NCT04388943). They identified SVEP1 in extracellular vesicles (EV) isolated from human plasma and surprisingly found that the EV-SVEP1 levels were significantly increased in subjects exhibiting high coronary artery calcium scores (CACS) vs. healthy controls (p = 0.0374, see Figure 1). Following these observations, the inventors then developed a fully validated, highly specific, and high throughput-targeted LC-MS assay to measure SVEP1 in human plasma using a selected reaction monitoring (SRM) approach. In their assay, the inventors were able to identify several unique quantotypic peptides which were generated following a trypsin digestion of SVEP1, and that these peptides, which were specific to SVEP1, could be detected in mass spectrometry. Using this assay, the inventors also surprisingly identified an association between SVEP1 with coronary artery calcification in the same BRICCS sub-cohort, demonstrating the use of SVEP1 as a robust blood biomarker for coronary artery classification. Accordingly, in a first aspect of the invention, there is provided a method for diagnosing a subject suffering from cardiovascular disease, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the method comprising: (i) digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; and (ii) analysing the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, and comparing this concentration with a reference concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is suffering from cardiovascular disease, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is not suffering from cardiovascular disease, or does not have a pre-disposition thereto, or provides a positive prognosis of the subject’s condition. In a second aspect, there is provided a method of determining the efficacy of treating a subject suffering from cardiovascular disease with a therapeutic agent, the method comprising: (i) digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; and (ii) analysing the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, and comparing this concentration with a reference concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, suggests that the treatment regime with the therapeutic agent is ineffective, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, suggests that the treatment regime with the therapeutic agent is effective. In a third aspect, there is provided an apparatus for diagnosing a subject suffering from cardiovascular disease, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the apparatus comprising:- (i) means for digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; (ii) means for determining the concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof; and (iii) a reference for the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is suffering from cardiovascular disease, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is not suffering from cardiovascular disease, or does not have a pre-disposition thereto, or provides a positive prognosis of the subject’s condition. In a fourth aspect, there is provided a method of treating a subject suffering from cardiovascular disease, the method comprising: (i) digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; (ii) analysing the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, and comparing this concentration with a reference concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is suffering from cardiovascular disease, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition, and (iii) administering, or having administered, to the test subject, a therapeutic agent, wherein the therapeutic agent, reduces or delays progression of cardiovascular disease. An important feature of any useful biomarker used in disease diagnosis and prognosis is that it exhibits high sensitivity and specificity for a given disease. As explained in the examples, the inventors have surprisingly demonstrated that a number of different quantotypic peptides found in blood from test subjects serve as robust biomarkers for cardiovascular disease (CVD), and in particular coronary artery disease (CAD), and can therefore be used for the detection and prognosis of these diseases. In addition, the inventors have shown that using such quantotypic peptides or fragments thereof as biomarkers for disease employs an assay which is simple, reproducible, quantitative and inexpensive, and with minimal inconvenience to the patient. Advantageously, the methods and apparatus of the invention provide a simple means for diagnosing cardiovascular disease from a simple biological sample, such as blood. The method according to the first aspect is useful for enabling a clinician to make decisions with regards to the best course of treatment for a subject who is currently suffering, or who may suffer, from cardiovascular disease, preferably CAD. It is preferred that the method of the first aspect is useful for enabling a clinician to decide how to treat a subject who is currently suffering from cardiovascular disease. In addition, the methods of the first and second aspects are useful for monitoring the efficacy of a putative treatment for the cardiovascular disease. Treatment for CVD and CAD includes lifestyle intervention, such as changes to exercise and diet. Pharmacological treatments include cholesterol-lowering drugs (statins). Aspirin may also be used to thin the blood and prevent blood clots and myocardial infarction. Beta blockers, calcium channel blockers and angiotensin-converting enzyme (ACE) and angiotensin II receptor blockers (ARBs) can also be used to lower blood pressure. If these treatments are unsuccessful or diagnosis occurs too late for early treatment, surgical interventions may be performed such as with angioplasty, stent replacement, or coronary artery bypass graft surgery (CABG). Hence, the apparatus according to the third aspect is useful for providing a prognosis of the subject’s condition, such that the clinician can carry out the treatment according to the fourth aspect. The apparatus of the third aspect may also be used to monitor the efficacy of a putative treatment for the CVD, preferably CAD. The methods and apparatus are therefore very useful for guiding a treatment regime for the clinician, and to monitor the efficacy of such a treatment regime. The clinician may use the apparatus of the invention in conjunction with existing diagnostic tests to improve the accuracy of diagnosis. Preferably, the apparatus is configured to perform the methods described herein. The skilled person will know that the protein Sushi, von Willebrand factor type A, EGF and Pentraxin Domain Containing 1 (SVEP1) is a large extracellular matrix protein encoded by the gene, Svep1. In one embodiment, the amino acid sequence of human SVEP1 can have an amino acid sequence referred to herein as SEQ ID No: 1, as follows: MWPRLAFCCWGLALVSGWATFQQMSPSRNFSFRLFPETAPGAPGSIPAPPAPGDEAAGSR VERLGQAFRRRVRLLRELSERLELVFLVDDSSSVGEVNFRSELMFVRKLLSDFPVVPTAT RVAIVTFSSKNYVVPRVDYISTRRARQHKCALLLQEIPAISYRGGGTYTKGAFQQAAQIL LHARENSTKVVFLITDGYSNGGDPRPIAASLRDSGVEIFTFGIWQGNIRELNDMASTPKE EHCYLLHSFEEFEALARRALHEDLPSGSFIQDDMVHCSYLCDEGKDCCDRMGSCKCGTHT GHFECICEKGYYGKGLQYECTACPSGTYKPEGSPGGISSCIPCPDENHTSPPGSTSPEDC VCREGYRASGQTCELVHCPALKPPENGYFIQNTCNNHFNAACGVRCHPGFDLVGSSIILC LPNGLWSGSESYCRVRTCPHLRQPKHGHISCSTREMLYKTTCLVACDEGYRLEGSDKLTC QGNSQWDGPEPRCVERHCSTFQMPKDVIISPHNCGKQPAKFGTICYVSCRQGFILSGVKE MLRCTTSGKWNVGVQAAVCKDVEAPQINCPKDIEAKTLEQQDSANVTWQIPTAKDNSGEK VSVHVHPAFTPPYLFPIGDVAIVYTATDLSGNQASCIFHIKVIDAEPPVIDWCRSPPPVQ VSEKVHAASWDEPQFSDNSGAELVITRSHTQGDLFPQGETIVQYTATDPSGNNRTCDIHI VIKGSPCEIPFTPVNGDFICTPDNTGVNCTLTCLEGYDFTEGSTDKYYCAYEDGVWKPTY TTEWPDCAKKRFANHGFKSFEMFYKAARCDDTDLMKKFSEAFETTLGKMVPSFCSDAEDI DCRLEENLTKKYCLEYNYDYENGFAIGPGGWGAANRLDYSYDDFLDTVQETATSIGNAKS SRIKRSAPLSDYKIKLIFNITASVPLPDERNDTLEWENQQRLLQTLETITNKLKRTLNKD PMYSFQLASEILIADSNSLETKKASPFCRPGSVLRGRMCVNCPLGTYYNLEHFTCESCRI GSYQDEEGQLECKLCPSGMYTEYIHSRNISDCKAQCKQGTYSYSGLETCESCPLGTYQPK FGSRSCLSCPENTSTVKRGAVNISACGVPCPEGKFSRSGLMPCHPCPRDYYQPNAGKAFC LACPFYGTTPFAGSRSITECSSFSSTFSAAEESVVPPASLGHIKKRHEISSQVFHECFFN PCHNSGTCQQLGRGYVCLCPLGYTGLKCETDIDECSPLPCLNNGVCKDLVGEFICECPSG YTGQRCEENINECSSSPCLNKGICVDGVAGYRCTCVKGFVGLHCETEVNECQSNPCLNNA VCEDQVGGFLCKCPPGFLGTRCGKNVDECLSQPCKNGATCKDGANSFRCLCAAGFTGSHC ELNINECQSNPCRNQATCVDELNSYSCKCQPGFSGKRCETEQSTGFNLDFEVSGIYGYVM LDGMLPSLHALTCTFWMKSSDDMNYGTPISYAVDNGSDNTLLLTDYNGWVLYVNGREKIT NCPSVNDGRWHHIAITWTSANGIWKVYIDGKLSDGGAGLSVGLPIPGGGALVLGQEQDKK GEGFSPAESFVGSISQLNLWDYVLSPQQVKSLATSCPEELSKGNVLAWPDFLSGIVGKVK IDSKSIFCSDCPRLGGSVPHLRTASEDLKPGSKVNLFCDPGFQLVGNPVQYCLNQGQWTQ PLPHCERISCGVPPPLENGFHSADDFYAGSTVTYQCNNGYYLLGDSRMFCTDNGSWNGVS PSCLDVDECAVGSDCSEHASCLNVDGSYICSCVPPYTGDGKNCAEPIKCKAPGNPENGHS SGEIYTVGAEVTFSCQEGYQLMGVTKITCLESGEWNHLIPYCKAVSCGKPAIPENGCIEE LAFTFGSKVTYRCNKGYTLAGDKESSCLANSSWSHSPPVCEPVKCSSPENINNGKYILSG LTYLSTASYSCDTGYSLQGPSIIECTASGIWDRAPPACHLVFCGEPPAIKDAVITGNNFT FRNTVTYTCKEGYTLAGLDTIECLADGKWSRSDQQCLAVSCDEPPIVDHASPETAHRLFG DIAFYYCSDGYSLADNSQLLCNAQGKWVPPEGQDMPRCIAHFCEKPPSVSYSILESVSKA KFAAGSVVSFKCMEGFVLNTSAKIECMRGGQWNPSPMSIQCIPVRCGEPPSIMNGYASGS NYSFGAMVAYSCNKGFYIKGEKKSTCEATGQWSSPIPTCHPVSCGEPPKVENGFLEHTTG RIFESEVRYQCNPGYKSVGSPVFVCQANRHWHSESPLMCVPLDCGKPPPIQNGFMKGENF EVGSKVQFFCNEGYELVGDSSWTCQKSGKWNKKSNPKCMPAKCPEPPLLENQLVLKELTT EVGVVTFSCKEGHVLQGPSVLKCLPSQQWNDSFPVCKIVLCTPPPLISFGVPIPSSALHF GSTVKYSCVGGFFLRGNSTTLCQPDGTWSSPLPECVPVECPQPEEIPNGIIDVQGLAYLS TALYTCKPGFELVGNTTTLCGENGHWLGGKPTCKAIECLKPKEILNGKFSYTDLHYGQTV TYSCNRGFRLEGPSALTCLETGDWDVDAPSCNAIHCDSPQPIENGFVEGADYSYGAIIIY SCFPGFQVAGHAMQTCEESGWSSSIPTCMPIDCGLPPHIDFGDCTKLKDDQGYFEQEDDM MEVPYVTPHPPYHLGAVAKTWENTKESPATHSSNFLYGTMVSYTCNPGYELLGNPVLICQ EDGTWNGSAPSCISIECDLPTAPENGFLRFTETSMGSAVQYSCKPGHILAGSDLRLCLEN RKWSGASPRCEAISCKKPNPVMNGSIKGSNYTYLSTLYYECDPGYVLNGTERRTCQDDKN WDEDEPICIPVDCSSPPVSANGQVRGDEYTFQKEIEYTCNEGFLLEGARSRVCLANGSWS GATPDCVPVRCATPPQLANGVTEGLDYGFMKEVTFHCHEGYILHGAPKLTCQSDGNWDAE IPLCKPVNCGPPEDLAHGFPNGFSFIHGGHIQYQCFPGYKLHGNSSRRCLSNGSWSGSSP SCLPCRCSTPVIEYGTVNGTDFDCGKAARIQCFKGFKLLGLSEITCEADGQWSSGFPHCE HTSCGSLPMIPNAFISETSSWKENVITYSCRSGYVIQGSSDLICTEKGVWSQPYPVCEPL SCGSPPSVANAVATGEAHTYESEVKLRCLEGYTMDTDTDTFTCQKDGRWFPERISCSPKK CPLPENITHILVHGDDFSVNRQVSVSCAEGYTFEGVNISVCQLDGTWEPPFSDESCSPVS CGKPESPEHGFVVGSKYTFESTIIYQCEPGYELEGNRERVCQENRQWSGGVAICKETRCE TPLEFLNGKADIENRTTGPNVVYSCNRGYSLEGPSEAHCTENGTWSHPVPLCKPNPCPVP FVIPENALLSEKEFYVDQNVSIKCREGFLLQGHGIITCNPDETWTQTSAKCEKISCGPPA HVENAIARGVHYQYGDMITYSCYSGYMLEGFLRSVCLENGTWTSPPICRAVCRFPCQNGG ICQRPNACSCPEGWMGRLCEEPICILPCLNGGRCVAPYQCDCPPGWTGSRCHTAVCQSPC LNGGKCVRPNRCHCLSSWTGHNCSRKRRTGF [SEQ ID No: 1] Therefore, preferably the method comprises digesting SVEP1 protein which comprises an amino acid sequence substantially as set out in SEQ ID No: 1, or a variant or fragment thereof. The method preferably comprises detecting the one or more SVEP1-derived quantotypic peptide or a fragment thereof, wherein the quantotypic peptide or a fragment thereof is derived from an amino acid sequence substantially as set out in SEQ ID No: 1, or a variant or fragment thereof. Preferably, the enzyme is trypsin. Surprisingly, the inventors have identified and empirically assessed quantotypic peptides that are unique to SVEP1 in the human proteome. Therefore, in one embodiment, the quantotypic peptide is preferably selected from: (i) CALLLQEIPAISYR (SEQ ID No: 2), (ii) QGFILSGVK (SEQ ID No: 3), (iii) GAFQQAAQILLHAR (SEQ ID No: 4), (iv) DVEAPQINCPK (SEQ ID No: 5), (v) YSCVGGFFLR (SEQ ID No: 6), (vi) IVLCTPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), (vii) ISCGPPAHVENAIAR (SEQ ID No: 8), (viii) SLATSCPEELSK (SEQ ID No: 9), (ix) LLQTLETITNK (SEQ ID No: 10), (x) EGYTLAGLDTIECLADGK (SEQ ID No: 11), (xi) TCDIHIVIK (SEQ ID No: 12), and (xii) IGSYQDEEGQLECK (SEQ ID No: 13), or a variant or analogue thereof. In some embodiments, during the digestion of the above proteins into peptides, the inventors break the disulfide bonds between cysteine (C) amino acids and modify them with carbamidomethylation (abbreviated to “cam”) in order to prevent the bonds from reforming. As such, a variant or analogue of any of the peptides described herein may be a carbamidomethylated form of the peptide. As such, any of the quantotypic peptides having a Cysteine may be modified with carbamidomethylation (cam), for example as follows: (i) C(cam)ALLLQEIPAISYR (SEQ ID No: 2), (ii) QGFILSGVK (SEQ ID No: 3), (iii) GAFQQAAQILLHAR (SEQ ID No: 4), (iv) DVEAPQINC(cam)PK (SEQ ID No: 5), (v) YSC(cam)VGGFFLR (SEQ ID No: 6), (vi) IVLC(cam)TPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), (vii) ISC(cam)GPPAHVENAIAR (SEQ ID No: 8), (viii) SLATSC(cam)PEELSK (SEQ ID No: 9), (ix) LLQTLETITNK (SEQ ID No: 10), (x) EGYTLAGLDTIEC(cam)LADGK (SEQ ID No: 11), (xi) TC(cam)DIHIVIK (SEQ ID No: 12), and (xii) IGSYQDEEGQLEC(cam)K (SEQ ID No: 13). The selection of other less quantotypic peptides could affect assay sensitivity. The inventors believe that these SVEP1-derived quantotypic peptides or variant or analogue thereof acting as CVD biomarkers are an important aspect of the invention. Hence, in a fifth aspect, there is provided use of a peptide, or a variant or analogue thereof, wherein the peptide is selected from a group of peptides consisting of: CALLLQEIPAISYR (SEQ ID No: 2), QGFILSGVK (SEQ ID No: 3), GAFQQAAQILLHAR (SEQ ID No: 4), DVEAPQINCPK (SEQ ID No: 5), YSCVGGFFLR (SEQ ID No: 6), IVLCTPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), ISCGPPAHVENAIAR (SEQ ID No: 8), SLATSCPEELSK (SEQ ID No: 9), LLQTLETITNK (SEQ ID No: 10), EGYTLAGLDTIECLADGK (SEQ ID No: 11), TCDIHIVIK (SEQ ID No: 12), and IGSYQDEEGQLECK (SEQ ID No: 13), as a biomarker for diagnosing a subject suffering from cardiovascular disease, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition. Variants or analogues of any of the peptides described herein may comprise carbamidomethylated forms of any of the peptides, as discussed above. Advantageously, and preferably, the quantotypic peptides, CALLLQEIPAISYR (SEQ ID No: 2) and QGFILSGVK (SEQ ID No: 3), are excised efficiently by trypsin to be observed consistently in biological matrices. They are not prone to aggregation, degradation or variable modifications, do not contain missed cleavage sites or dibasic content, and are long enough to have good specificity without being too long and hydrophobic. Therefore, in a preferred embodiment, the quantotypic peptide is CALLLQEIPAISYR (SEQ ID No: 2). Preferably, the peptide is C(cam)ALLLQEIPAISYR (SEQ ID No: 2). In another preferred embodiment, the quantotypic peptide is QGFILSGVK (SEQ ID No: 3). In an embodiment, the cardiovascular disease which may be diagnosed, prognosed or treated, as described herein, may be coronary artery disease (CAD). In another embodiment, the cardiovascular disease may be or comprise coronary artery calcification. In yet another embodiment, the cardiovascular disease may be or comprise plaque build-up. Detection of the quantotypic peptides of a fragment thereof may be carried out by chromatography, such as liquid chromatography (LC), ultra performance liquid chromatography (UPLC), or gas chromatography (GC), which is preferably coupled to mass spectrometry (MS) or time-of-flight (TOF). Preferably, however, separation of the digested peptides may be achieved by mass spectrometry (MS) or tandem mass spectrometry (MS / MS) using a selected reaction monitoring (SRM) approach for ion selection. Preferably, in one embodiment, LC-MS is used. However, in another embodiment UPLC-MS or UPLC-MS / MS (i.e. tandem mass spectrometry) may be used. Preferred SRM assay parameters may be substantially as shown in Table 1. Preferably, fragments of a sequence are generated by collision-induced dissociation (CID). A preferred LC gradient programme that is used may be substantially as shown in Table 2. Preferably, an LC gradient of H2O and acetonitrile with increasing acetonitrile is used. Preferred calibration curve concentrations may be substantially as shown in Table 3. Preferably, the calibration curve uses a “ratio-to-heavy” approach. Accordingly, preferably the invention comprises digesting the SVEP1 into one or more SVEP1-derived quantotypic peptides, and then detecting the one or more SVEP1- derived quantotypic peptide or a fragment thereof. Preferably, the one or more SVEP1- derived quantotypic peptides are ionised into fragments, and it is these fragments which are detected, for example by LC-MS, or UPLC-MS / MS. For example, in one embodiment, the quantotypic peptide is CALLLQEIPAISYR (SEQ ID No: 2), which is ionised into the following three fragments: CALL (SEQ ID No: 14), CALLL (SEQ ID No: 15) and QEIPAISYR (SEQ ID No: 16). Preferably, the quantotypic peptide is C(cam)ALLLQEIPAISYR (SEQ ID No: 2), which is ionised into the following three fragments: C(cam)ALL (SEQ ID No: 14), C(cam)ALLL (SEQ ID No: 15) and QEIPAISYR (SEQ ID No: 16). Accordingly, the detection step comprises detection of at least one, two or all three of the fragments derived from CALLLQEIPAISYR (SEQ ID No: 2) or C(cam)ALLLQEIPAISYR (SEQ ID No: 2). In another embodiment, the quantotypic peptide is QGFILSGVK (SEQ ID No: 3), which is ionised into the following three fragments: LSGVK (SEQ ID No: 17), ILSGVK (SEQ ID No: 18), and FILSGVK (SEQ ID No: 19). Accordingly, the detection step comprises detection of at least one, two or all three of the fragments derived from QGFILSGVK (SEQ ID No: 3). The above peptide fragments are generated when the larger peptides are fragmented using collision inducted dissociation (CID) using the collision energies (CE) shown in Table 1. The fragments for each peptide co-elute (i.e. are measured at the same retention times) also shown in Table 1. Thus, the one or more SVEP1-derived quantotypic peptide or a fragment thereof may be detected by its corresponding retention time in the chromatographic column. Retention time is a measure of the time a compound (in this case the fragment of the SVEP1-dervied peptide) spends in a chromatographic column, and is dependent upon its volatility and affinity for the column. More volatile peptides or fragments thereof will have a lower retention time, while less volatile peptides or fragments thereof will have a higher retention time. The retention time (RT) range used is shown in Table 1. The one or more SVEP1-derived quantotypic peptide or fragment thereof is measured across the entire RT range, i.e. is unscheduled. In preferred embodiments, each of the various aspects of the invention may comprise determining the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof; and comparing the concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof with a reference for the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is suffering from cardiovascular disease, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is not suffering from cardiovascular disease, or does not have a pre- disposition thereto, or provides a positive prognosis of the subject’s condition. Thus, it will be appreciated that, in their most preferred embodiments, the aspects involve detecting an increase and / or decrease of the one or more SVEP1-derived quantotypic peptide or a fragment thereof. The subject may be any animal of veterinary interest, for instance, a cat, dog, horse etc. However, it is preferred that the subject is a mammal, such as a human, either male or female. Preferably, a sample is taken from the subject, and the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the bodily sample is then measured. The sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, tissue, and / or CSF, or combinations thereof. The inventors have surprisingly detected SVEP1 in extracellular vesicles (EV) isolated from blood. Thus, preferably the bodily sample comprises extracellular vesicles (EV). Preferably, the extracellular vesicles are isolated from blood, more preferably blood plasma. Therefore, preferably the sample is blood. Preferably, the extracellular vesicles (EV) comprise, contain or encapsulate the SVEP1. The apparatus may comprise a sample collection container for receiving the extracted bodily sample. “Fresh” blood samples may be assayed immediately after being digested with the enzyme. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before being subjected to the enzyme. The sample may then be de-frosted and analysed at a later date. Detection of the one or more SVEP1-derived quantotypic peptide or a fragment thereof may be carried out on whole blood. Preferably, the blood sample comprises blood serum. Most preferably, however, the blood sample comprises blood plasma. The blood may be further processed before the assay is performed. For instance, an anticoagulant, such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Thus, the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction, which may be used for analysis. Hence, it is preferred that the one or more SVEP1- derived quantotypic peptide or a fragment thereof is analysed or assayed in a blood plasma or a blood serum sample. It is especially preferred that the one or more SVEP1- derived quantotypic peptide or a fragment thereof is measured in vitro from a blood serum sample or a plasma sample taken from the subject. Preferably, the biological sample is blood plasma. The apparatus may comprise sample extraction means for obtaining the sample from the test subject. The sample extraction means may comprise a needle or syringe or the like. The apparatus may comprise a sample collection container for receiving the extracted sample, which may be liquid, gaseous or semi-solid. Accordingly, in a preferred embodiment, the concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof from a blood plasma sample is analysed. In some embodiments, preferably the concentration of CALLLQEIPAISYR or C(cam)ALLLQEIPAISYR (SEQ ID No: 2), QGFILSGVK (SEQ ID No: 3), GAFQQAAQILLHAR (SEQ ID No: 4), DVEAPQINCPK or DVEAPQINC(cam)PK (SEQ ID No: 5), YSCVGGFFLR or YSC(cam)VGGFFLR (SEQ ID No: 6), IVLCTPPPLISFGVPIPSSALHFGSTVK or IVLC(cam)TPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), ISCGPPAHVENAIAR or ISC(cam)GPPAHVENAIAR (SEQ ID No: 8), SLATSCPEELSK or SLATSC(cam)PEELSK (SEQ ID No: 9), LLQTLETITNK (SEQ ID No: 10), EGYTLAGLDTIECLADGK or EGYTLAGLDTIEC(cam)LADGK (SEQ ID No: 11), TCDIHIVIK or TC(cam)DIHIVIK (SEQ ID No: 12), and / or IGSYQDEEGQLECK or IGSYQDEEGQLEC(cam)K (SEQ ID No: 13), or a fragment thereof, is detected. Preferably, the concentration of CALLLQEIPAISYR or C(cam)ALLLQEIPAISYR (SEQ ID No: 2) or QGFILSGVK (SEQ ID No: 3), or a fragment thereof, is detected. The difference in concentration of signature compound in the methods of the first aspect or the apparatus of the third aspect may be an increase or a decrease compared to the reference. The inventors monitored the concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof in patients who suffered from cardiovascular disease, and compared them to the concentration of these same one or more SVEP1-derived quantotypic peptide or a fragment thereof in individuals who did not suffer from cardiovascular disease (i.e. reference or controls). They demonstrated that there was a statistically significant increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the patients suffering from cardiovascular disease. It will be appreciated that the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in patients suffering from cardiovascular disease is highly dependent on a number of factors, for example how far the condition has progressed, and the age and gender of the subject. It will also be appreciated that the reference concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in individuals who do not suffer from cardiovascular disease may fluctuate to some degree, but that on average over a given period of time, the concentration tends to be substantially constant. In addition, it should be appreciated that the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in one group of individuals who suffer from cardiovascular disease may be different to the concentration of that compound in another group of individuals who do not suffer from cardiovascular disease. However, it is possible to determine the average concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in individuals who do not suffer from cardiovascular disease, and this is referred to as the reference or ‘normal’ concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof. This normal concentration corresponds to the reference values discussed above. In one embodiment, the methods of the invention preferably comprise determining the ratio of SVEP1-derived quantotypic peptides or a fragment thereof within the sample, such as a blood plasma sample (i.e. using other components within it as a reference), and then compare these markers to the disease to show if they are elevated or reduced. The inventors have demonstrated that the one or more SVEP1-derived quantotypic peptide or a fragment thereof can be reliably detected using LC-MS. The reference values may be obtained by assaying a statistically significant number of control samples (i.e. samples from subjects who do not suffer from cardiovascular disease). Accordingly, the reference (iii) according to the apparatus of the third aspect of the invention may be a control sample (for assaying). The apparatus preferably comprises a positive control (most preferably provided in a container), which corresponds to the one or more SVEP1-derived quantotypic peptide or a fragment thereof. The apparatus preferably comprises a negative control (preferably provided in a container). In a preferred embodiment, the apparatus may comprise the reference, a positive control and a negative control. The apparatus may also comprise further controls, as necessary, such as “spike-in” controls to provide a reference for concentration, and further positive controls for each of the one or more SVEP1-derived quantotypic peptide or a fragment thereof. Accordingly, the inventors have realised that the difference in concentrations of the one or more SVEP1-derived quantotypic peptide or a fragment thereof between the reference normal (i.e. control) and increased / decreased levels, can be used as a physiological marker, suggestive of the presence of cardiovascular disease in the test subject. It will be appreciated that if a subject has an increased / decrease concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof which is considerably higher / lower than the ‘normal’ concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof in the reference, control value, then they would be at a higher risk of having cardiovascular disease, or a condition that was more advanced, than if the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof was only marginally higher / lower than the ‘normal’ concentration. The inventors have noted that the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof referred to herein in the test individuals was statistically more than the reference concentration (as calculated using the method described in the Example). This may be referred to herein as the ‘increased’ concentration of the signature compound. The skilled technician will appreciate how to measure the concentrations of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in a statistically significant number of control individuals, and the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the test subject, and then use these respective figures to determine whether the test subject has a statistically significant increase / decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, and therefore infer whether that subject is suffering from cardiovascular disease. In the method of the second aspect and the apparatus of the third aspect, the difference in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the bodily sample compared to the corresponding concentration in the reference is indicative of the efficacy of treating the subject’s cardiovascular disease with the therapeutic agent. The difference may be an increase or a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the bodily sample compared to the reference value. In this embodiment, the reference sample is a sample taken from the subject at an earlier time point. The reference sample may have been taken from the subject prior to commencing treatment. Accordingly, the method and / or apparatus may show if an improvement has occurred in the subject since the start of treatment. Alternatively, or additionally, the reference sample may comprise a sample taken from the subject subsequent to commencing treatment. In some embodiments, the reference sample may comprise a plurality of samples taken from the subject at different time points subsequent to commencing treatment. For example, the plurality of samples may be one or more days apart, one or more weeks apart, one or more months apart, or even one or more years apart. For example, samples may be taken from the subject at least once, twice or three times every week, every month or every year. The samples may be taken at evenly spaced intervals or a randomly spaced intervals. The plurality of samples may also include a sample taken from the subject prior to commencing treatment, or after treatment has started. Accordingly, the method of the second aspect and the apparatus of the third aspect can determine if an improvement is ongoing. In embodiments where the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the bodily sample is lower than the corresponding concentration in the reference, then this would indicate that the therapeutic agent is successfully treating the cardiovascular disease in the test subject. Conversely, where the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof in the bodily sample is higher than the corresponding concentration in the reference, then this would indicate that the therapeutic agent is not successfully treating the cardiovascular disease. The inventors were interested to see if SVEP1 is implicated in any other subsets of cardiovascular disease. Therefore, as described in the Examples, the inventors have identified SVEP1 in extracellular vesicles (EV) isolated from human plasma and found that the EV-SVEP1 levels were significantly increased in those subjects exhibiting high coronary artery calcium scores (CACS) vs. healthy controls (p = 0.0374). Calcification reflects an active, dynamic stage of disease associated with inflammation. Extensive investigation has established the value of identifying and quantifying coronary calcium in coronary artery disease (CAD), and an increasing body of evidence has suggested that calcification has underappreciated facets that modulate future event risk. Whether the presence of calcium in the plaque is indicative of a stable or unstable plaque is complex, as certain stages of calcification are more atherogenic than others. Microcalcification is often observed early in disease and is thought to originate from smooth muscle cell (SMC) apoptosis, with cell death providing a phospholipid-rich environment to nucleate apatite, a key component in calcified lesions. Macrophage- derived vesicles also play a key role in calcification, typically resulting in larger calcifications compared to SMC apoptosis. Over time, microcalcifications can coalesce to form larger speckles and fragments of calcification, occurring from the outer rim of the necrotic lipid core and into the surrounding collagenous matrix. Further progression leads to the formation of calcified sheets or plates, which can fracture and lead to nodular calcification. Plaque rupture has been shown to correlate with the number of spotty calcifications and inversely with the number of large calcified sites. Protuberant nodules extending into the lumen and leading to fibrin deposition and disruption of the endothelial lining are known to lead to 2-7 % of acute coronary events. Thus, in view of these findings, in a sixth aspect of the invention, there is provided the use of SVEP1, or a variant or fragment thereof, as a biomarker for coronary artery calcification or plaque build-up. Furthermore, in a seventh aspect of the invention, there is provided method of treating coronary artery calcification and / or plaque build-up in an artery in a subject in need of treatment thereof, the method comprising detecting the presence of SVEP1, or a variant or fragment thereof, in a biological sample from the subject, and administering a plaque-reducing agent to the subject if the concentration of SVEP1, or a variant or fragment thereof, is above a reference threshold. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms “substantially the amino acid / nucleotide / peptide sequence”, “variant” and “fragment”, can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with any of the sequence identified herein. Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment. Preferably, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N / T)*100. Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45ºC followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65ºC. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from any of the sequences described herein. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids. All of the features described herein (including any accompanying claims, abstracts and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:- Figure 1 shows a violin plot showing the abundance for extracellular vesicle SVEP1 (EV-SVEP1) in clinically confirmed cases (CACS ≥ 200) and control (CACS < 10) groups. Figure 2 shows a flow chart of one embodiment of a high throughput targeted LC- MS to measure identified peptides which are unique to SVEP1 in human plasma to diagnose or prognose cardiovascular disease (CVD), in particular coronary artery disease (CAD). Figure 3A shows a 9-point calibration curve (2 fmol – 500 fmol) for QGF[…] peptide standard. Figure 3B represents a bar plot showing the peak areas (x106) for each of the light standards and their counterparts heavy standards held constant at 50 fmol. Figure 3C shows a chromatogram demonstrating 50 fmol heavy QGF[…] spiked into digested patient plasma to measure their SVEP1 concentration. Examples The inventors have developed a novel, fully validated, quantitative, and highly specific assay for diagnosing coronary artery disease (CAD). The assay uses high throughput targeted LC-MS to measure identified quantotypic peptides which are unique to SVEP1 in human plasma using a selected reaction monitoring (SRM) approach, coupled with a reproducible plasma protein digestion preparation protocol in 96-well plate format derived from previous work by Mbasu et al.10. The assay is amenable to automation with liquid handling systems and has been fully validated to regulatory body guidelines (ICH / FDA / EMA), addressing hurdles associated with bioanalytical assay validation for subsequent clinical application. Using the assay the inventors were able to verify the association of SVEP1 with coronary calcification in the prospective observational Biomedical Research Informatics Centre for Cardiovascular Sciences (BRICCS) cohort (ClinicalTrials.gov Identifier: NCT04388943), thereby clearly demonstrating the use of SVEP1 as a potential blood biomarker for CAD, and the utility of the assay. Materials and methods Sample Preparation In Eppendorf twin-tec semi-skirted 250 μL 96-well plates (Eppendorf, Hamburg, Germany), starting with 10 μL of undepleted plasma, 75 μL of AmBic 50 mM pH 7.4 was added along with 5 μL of 1% RapiGest solution and the plate incubated 80oC for 1 hour in a Thermo Scientific Hybaid PX2 thermal cycler (Thermo Fisher Scientific, Loughborough, UK). Reduction was then carried out by addition of dithiothreitol (DTT) to a final concentration of 5 mM followed by incubation for 30 minutes at 60oC. The plate was cooled to room temperature and alkylation carried out by addition of iodoacetamide (IAA) to a final concentration of 10 mM. The plate was incubated in the dark at room temperature (RT) for 30 minutes.20 μL of 1 mg / mL trypsin was added and proteins digested overnight for 18 hours at 37oC.13 μL trifluoroacetic acid (TFA) 10% (v / v) was added to a final concentration of approximately 1%. The plate was centrifuged at 4000 g for 30 minutes using an Eppendorf Centrifuge 5810R (Eppendorf, Stevenage, UK) and the supernatant collected and transferred into Waters QuanRecovery 700 µL Plates (Waters, Milford, USA) for analysis by mass spectrometry. Peptide Standards Unlabelled and stable isotope-labelled peptide standards were obtained from Peptide Synthetics (Peptide Protein Research Ltd., Southampton, UK) for the quantotypic SVEP1 peptides C(cam)ALLLQEIPAISYR [SEQ ID NO: 2] and QGFILSGVK [SEQ ID NO: 3]. Stable isotope-labelled standards (SILS) were and15N labelled on the last amino acid, either Lysine K (C6, N2) or Arginine R (C6, N4) as appropriate. These peptide standards have been empirically assessed as quantotypic peptides, are unique to SVEP1 in the human proteome, and are efficiently excised by trypsin to be observed consistently in biological matrices. The selected peptide standards are not prone to aggregation, degradation or variable modifications, do not contain missed cleavage sites or dibasic content and are long enough to have good specificity without being too long and hydrophobic. LC-MS and SRM Assays Parameters Data were obtained using a Waters Acquity LC system coupled to a Xevo TQ-XS mass spectrometer equipped with an Acquity Premier Peptide BEH C18 analytical column, 300 Å, 1.7 µm, 2.1 x 50 mm. Mobile phase A was H2O + 0.1% FA. Mobile phase B was acetonitrile (MeCN) + 0.1% formic acid (FA). The seal wash was made of H2O + 10% methanol (MeOH), the weak needle wash was made of H2O + 0.1% FA, and the strong needle wash was MeCN + 0.1% FA. The flow rate was 0.6 mL / min. The autosampler temperature was 8 °C and the column temperature was 40 °C. The Xevo TQ-XS was equipped with a Waters Zspray LockSpray in electrospray ionisation (ESI) positive mode. The cone voltage was set to 35 V and the capillary voltage was set to 0.6 kV. The parameters for the selected reaction monitoring (SRM) assay are described in Table 1. Table 1 - SRM assay parameters for the SVEP1 light and heavy peptides PeptidePrecursor m / zFragment m / z CE RT Window (Light / Heavy)Fragment (Light / Heavy) (eV) (mins) QGFILSGVK 474.8 / y5 503.3 / 511.3 15(+2) 478.83.85 – 5.85y6 616.4 / 624.4 17 y7 763.5 / 771.5 17 C(cam)ALLLQE b4 458.2 / 458.2 25 IPAISYR 824.0 / b 571.3 / 571.3 82 5 23 7.17 – 9.17 (+2) 9.0 y9 1076.6 / 1086.6 23 The LC gradient used for this assay was the 12.5 min programme described in Table 2. Table 2 - 5 min gradient programme used on the Waters Acquity UPLC system to measure the SVEP1 peptides Time (min) % A% B0.00 9550.50 9553.50 55453.51 5954.40 5954.41 9555.00 955Calibration Curves Fresh stock solutions of the unlabelled QGF[…] and CAL[…] standards were made to 4 pmol / µL using diluted pooled plasma digest as the matrix and combined in a 1:1 ratio to a final concentration of 2 pmol / µL.1:2 serial dilutions were performed to construct a calibration curve as described in Table 3. Table 3 - The serial dilutions and concentrations of each point in the final calibration curves for the SVEP1 peptides QGF and CAL Serial Dilution of Light StandardFinal concentration (fmol / µL)Standard DilutionLight concentrationCAL[…] (fmol / µL) QGF[…]A LS no dilution 2000 Not used1000 L + 50 HB1:2 A in matrix 1000 500 L + 50 H500 L + 50 HC 1:2 B in matrix 500 250 L + 50 H250 L + 50 HD1:2 C in matrix 250 125 L + 50 H125 L + 50 HE1:2 D in matrix 125 62.5 L + 50 H62.5 L + 50 HF1:2 E in matrix 62.5 31.25 L + 50 H31.25 L + 50 H15.63 L + 50 H G 1:2 F in matrix 31.25 15.63 L + 50 H [LLOQ] H1:2 G in matrix 15.625 7.81 L + 50 HNot usedI1:2 H in matrix 7.8125 3.91 + 50 HNot usedJ1:2 I in matrix 3.906251.95 + 50 HNot used [LLOQ]LZero – matrix +0 0 L +0 L + 50 H HS 50 HM Blank – matrix 0 0 L + 0 H0 L + 0 HA stock solution of 4 pmol / µL was prepared for the QGF[…] and CAL[…] heavy standards in diluted plasma. These were combined in a 1:1 ratio to give a combined stock of 2 pmol / µL, which was diluted 1:20 in diluted plasma to give a combined 100 fmol / µL heavy standard. This heavy standard was added in a 1:1 ratio to the light serial dilutions to give the final calibration curve concentrations shown in Table 3. Calibration curves were analysed in triplicate across 8 analytical runs. The calibration range used for QGF[…] was a 9-point calibration curve from 1.953 fmol lower limit of quantitation, LLOQ) to 500 fmol. The calibration range used for CAL[…] was a 7- point calibration curve from 15.625 fmol (≈ LLOQ) to 1000 fmol. The calibration curves and appropriate quality control (QC) samples underwent full bioanalytical assay validation including assessment of assay recovery (matrix effects), precision, accuracy, and stability according to the acceptance criteria outlined by the FDA and ICH Bioanalytical Method Validation guidance.11, 12Results Example 1 – The novel assay The inventors performed a proteomics discovery study using liquid chromatography coupled to mass spectrometry (LC-MS) on the prospective observational Biomedical Research Informatics Centre for Cardiovascular Sciences (BRICCS) cohort (ClinicalTrials.gov Identifier: NCT04388943). From this work, they identified SVEP1 in extracellular vesicles (EV) isolated from human plasma and surprisingly found that found the EV-SVEP1 levels were significantly increased in subjects exhibiting high coronary artery calcium scores (CACS) vs. healthy controls (p = 0.0374, see Figure 1). The inventors then developed a fully validated, highly specific, and high throughput targeted LC-MS assay to measure SVEP1 in human plasma using a selected reaction monitoring (SRM) approach. Referring now to Figure 2, there is shown a flowchart of one embodiment of a process 2 for detecting SVEP1 from blood, for the purpose of diagnosing cardiovascular disease (CVD), in particular coronary artery disease (CAD). A blood sample 4 is taken from a test subject, and then treated as described in the materials and methods section to obtain the blood plasma 6. The SVEP1 is then isolated 8 from the plasma 6. In some embodiments, the assay 2 could be further coupled with a prior enrichment step 10 using molecularly imprinted polymers (MIPs) designed to target and attach to the SVEP1 protein. In other words, MIPs can act as a synthetic antibody specific to the SVEP1 protein, or they could be developed as “anti-peptide antibodies” to allow for enrichment of the SVEP1 peptides in the SRM assay workflow, increasing the sensitivity of the assay. A trypsin digestion step 12 is then performed on the SVEP1 isolate 8 (or enrichment 10) to create a plurality of shorter peptides derived from SVEP1, one or more of which are unique and quantotypic 14, i.e. accurately represent the level or concentration of the peptide in the sample. The inventors have found the following quantotpic peptides based on SVEP1, as follows:- (i) CALLLQEIPAISYR (SEQ ID No: 2), (ii) QGFILSGVK (SEQ ID No: 3), (iii) GAFQQAAQILLHAR (SEQ ID No: 4), (iv) DVEAPQINCPK (SEQ ID No: 5), (v) YSCVGGFFLR (SEQ ID No: 6), (vi) IVLCTPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), (vii) ISCGPPAHVENAIAR (SEQ ID No: 8), (viii) SLATSCPEELSK (SEQ ID No: 9), (ix) LLQTLETITNK (SEQ ID No: 10), (x) EGYTLAGLDTIECLADGK (SEQ ID No: 11), (xi) TCDIHIVIK (SEQ ID No: 12), and (xii) IGSYQDEEGQLECK (SEQ ID No: 13), or a variant or analogue thereof. During the digestion of the above proteins into peptides, the inventors broke the disulfide bonds between cysteine (C) amino acids and modified them with carbamidomethylation (abbreviated to cam) in order to prevent the bonds from reforming. Accordingly, the above peptides were modified to: (i) C(cam)ALLLQEIPAISYR (SEQ ID No: 2), (ii) QGFILSGVK (SEQ ID No: 3), (iii) GAFQQAAQILLHAR (SEQ ID No: 4), (iv) DVEAPQINC(cam)PK (SEQ ID No: 5), (v) YSC(cam)VGGFFLR (SEQ ID No: 6), (vi) IVLC(cam)TPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), (vii) ISC(cam)GPPAHVENAIAR (SEQ ID No: 8), (viii) SLATSC(cam)PEELSK (SEQ ID No: 9), (ix) LLQTLETITNK (SEQ ID No: 10), (x) EGYTLAGLDTIEC(cam)LADGK (SEQ ID No: 11), (xi) TC(cam)DIHIVIK (SEQ ID No: 12), and (xii) IGSYQDEEGQLEC(cam)K (SEQ ID No: 13). Preference, however, was given to C(cam)ALLLQEIPAISYR (SEQ ID No: 2) and QGFILSGVK (SEQ ID No: 3). The inventors believe that these two peptides in particular are not prone to aggregation, degradation or variable modifications, do not contain missed cleavage sites or dibasic content, and are long enough to have good specificity without being too long and hydrophobic. The next step is to carry out liquid chromatography – mass spectrometry (LC-MS) on the sample 16. This involved ionising the above SVEP1-dervied unique peptides into sub-fragments 18, which are then separated on LC and detected by MS. Detecting of the corresponding fragments of the SVEP1-dervied unique peptides is indicative of CVD or CAD 20. Example 2 - Measurement of SVEP1 concentration in patient plasma The inventors assessed the sensitivity and accuracy of the novel quantitative assay 2 for the peptide QGFILSGVK (SEQ ID No: 3). As shown in Figure 3A, a 9-point calibration curve (2 fmol – 500 fmol) for the QGFILSGVK (SEQ ID No: 3) peptide displays the analytical signal (i.e., how the instrumental response changes with the concentration of the peptide). Additionally, the bar chart shown in Figure 3B, shows the peak areas (x106) for each of the light standards and their heavy standard counterparts held constant at 50 fmol. Finally, Figure 3C shows a chromatogram demonstrating that a strong signal of 50 fmol heavy QGFILSGVK (SEQ ID No: 3) spiked into digested patient plasma, thereby providing a quantitative measure of their SVEP1 concentration. The three fragments for QGFILSGVK are the y5 (LSGVK), y6 (ILSGVK), and y7 (FILSGVK) fragments, and for C(cam)ALLLQEIPAISYR are the b4 (C(cam)ALL), b5 (C(cam)ALLL) and y9 (QEIPAISYR) fragments. These are generated when the peptides are fragmented using collision inducted dissociation (CID) using the collision energies (CE) shown in Table 1. With reference to Figure 3C, the fragments for each peptide co- elute (i.e. are measured at the same retention times) also shown in Table 1. Example 3 - Correlation between of SVEP1 concentration in patients’ plasma and coronary calcification An agnostic discovery proteomics study was performed on the prospective observational Biomedical Research Informatics Centre for Cardiovascular Sciences (BRICCS) cohort (ClinicalTrials.gov Identifier: NCT04388943). Subjects attending the Rapid Access Chest Pain Clinic, suspected of having CAD, and referred to have coronary computed tomography angiography (CCTA) were selected for this sub-study. Participants had CACS (coronary artery calcium score) measured before CCTA.50 patients with CACS ≥ 200 Agatston (median 407.5, range 208 – 1841) matched with 50 controls with CACS < 10 Agatston (median 0, range 0 – 8) were selected for a discovery proteomics study. Propensity score was used to match for the demographic variables that were significantly different in a multivariate logistic regression: age, sex, history of high blood pressure and diabetes. Plasma samples were prepared in the Leicester van Geest MultiOmics Facility utilising a novel multiplexed proteomics protocol utilising extraceullar vesicles (EVs) to enrich for low abundance plasma proteins. Evs are a rich source of potential CAD biomarkers due to their involvement in key pathophysiological pathways such as inflammation, endothelial dysfunction and calcification. Samples were processed using a bottom-up proteomics workflow digesting plasma proteins into peptides using trypsin. Samples were analysed in a shotgun proteomics fashion using liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) utilising the Waters Synapt G2-S high- definition MS platform. Protein identification was performed using Progenesis QI software and 136281 peptides corresponding to 4416 proteins were identified at 1% false discovery rate. Data were pre-processed using a novel bioinformatics workflow to filter out statistically redundant features, remove sources of technical variation, and normalise data. Principal component analysis (PCA) was performed on the data which demonstrated discrimination between case and control based on their protein profiles. A linear model adjusted for sex, age, ethnicity, smoking status and diabetes was constructed using the Limma-Voom method. Differential expression analysis was performed using Limma and p-values were adjusted for multiple hypothesis testing by the Benjamini-Hochberg correction.166 proteins were significantly different in case vs. control at adj. p-value ≤ 0.05, including SVEP1 (p = 0.0374). Figure 1 shows a violin plot showing the abundance for extracellular vesicle SVEP1 (EV-SVEP1) in clinically confirmed cases (CACS ≥ 200) and control (CACS < 10) groups, illustrating the upregulation of SVEP1 in subjects with coronary calcification. Following the initial shotgun discovery analysis, a specific, high throughput, quantitative LC-MS / MS selected reaction monitoring (SRM) assay was developed and validated to international regulatory body guidelines to verify the association of the SVEP1 peptides QGF[…] and CAL[…] with coronary calcification in the same cohort. Synthetic peptide standards were obtained from Peptide Synthetics Peptide Protein Research Ltd. (Hampshire, UK). Figure 3A shows a 9-point calibration curve (2 fmol – 500 fmol) for QGF[…] peptide standard. Figure 3B represents a bar plot showing the peak areas (x106) for each of the light standards and their counterparts heavy standards held constant at 50 fmol. Figure 3C shows a chromatogram demonstrating 50 fmol heavy QGF[…] spiked into digested patient plasma to measure their SVEP1 concentration. The study plasma samples from the same cohort were prepared using a high- throughput bottom-up proteomics protocol in 96-well plate format adapted from a method previously developed in the group by Mbasu et al.60Samples were analysed in triplicate using the optimised SRM assay on the Waters Xevo TQXS triple quadrupole MS platform. Raw data were imported into Skyline Targeted Mass Spec Environment for peak integration, quality control and normalisation to an internal standard. The association of SVEP1 with coronary calcification was verified in the same cohort using the targeted assay. Conclusions The inventors have devised a novel, highly effective assay for diagnosing or prognosing CAD, or coronary calcification, and so can be used in patient stratification. They identified SVEP1 in extracellular vesicles (EV) isolated from human plasma and surprisingly found that the EV-SVEP1 levels were significantly increased in subjects exhibiting high coronary artery calcium scores (CACS) vs. healthy controls. As shown in Figure 2, the assay involves taking a small blood sample from the patient, performing a trypsin digestion on the sample to digest the SVEP1 in it, and then detecting unique quantotypic peptides, or ionised fragments thereof, using LC-MS. References 1. Shur I, Socher R, Hameiri M, Fried A and Benayahu D. Molecular and cellular characterization of SEL-OB / SVEP1 in osteogenic cells in vivo and in vitro. J Cell Physiol.2006;206:420-7. 2. Samuelov L, Li Q, Bochner R, Najor NA, Albrecht L, Malchin N, Goldsmith T, Grafi-Cohen M, Vodo D, Fainberg G, Meilik B, Goldberg I, Warshauer E, Rogers T, Edie S, Ishida-Yamamoto A, Burzenski L, Erez N, Murray SA, Irvine AD, Shultz L, Green KJ, Uitto J, Sprecher E and Sarig O. SVEP1 plays a crucial role in epidermal differentiation. Exp Dermatol.2017;26:423-430. 3. Myocardial Infarction G, Investigators CAEC, Stitziel NO, Stirrups KE, Masca NG, Erdmann J, Ferrario PG, Konig IR, Weeke PE, Webb TR, Auer PL, Schick UM, Lu Y, Zhang H, Dube MP, Goel A, Farrall M, Peloso GM, Won HH, Do R, van Iperen E, Kanoni S, Kruppa J, Mahajan A, Scott RA, Willenberg C, Braund PS, van Capelleveen JC, Doney AS, Donnelly LA, Asselta R, Merlini PA, Duga S, Marziliano N, Denny JC, Shaffer CM, El-Mokhtari NE, Franke A, Gottesman O, Heilmann S, Hengstenberg C, Hoffman P, Holmen OL, Hveem K, Jansson JH, Jockel KH, Kessler T, Kriebel J, Laugwitz KL, Marouli E, Martinelli N, McCarthy MI, Van Zuydam NR, Meisinger C, Esko T, Mihailov E, Escher SA, Alver M, Moebus S, Morris AD, Muller-Nurasyid M, Nikpay M, Olivieri O, Lemieux Perreault LP, AlQarawi A, Robertson NR, Akinsanya KO, Reilly DF, Vogt TF, Yin W, Asselbergs FW, Kooperberg C, Jackson RD, Stahl E, Strauch K, Varga TV, Waldenberger M, Zeng L, Kraja AT, Liu C, Ehret GB, Newton-Cheh C, Chasman DI, Chowdhury R, Ferrario M, Ford I, Jukema JW, Kee F, Kuulasmaa K, Nordestgaard BG, Perola M, Saleheen D, Sattar N, Surendran P, Tregouet D, Young R, Howson JM, Butterworth AS, Danesh J, Ardissino D, Bottinger EP, Erbel R, Franks PW, Girelli D, Hall AS, Hovingh GK, Kastrati A, Lieb W, Meitinger T, Kraus WE, Shah SH, McPherson R, Orho-Melander M, Melander O, Metspalu A, Palmer CN, Peters A, Rader D, Reilly MP, Loos RJ, Reiner AP, Roden DM, Tardif JC, Thompson JR, Wareham NJ, Watkins H, Willer CJ, Kathiresan S, Deloukas P, Samani NJ and Schunkert H. Coding Variation in ANGPTL4, LPL, and SVEP1 and the Risk of Coronary Disease. N Engl J Med.2016;374:1134-44. 4. Keramati AR, Chen MH, Rodriguez BAT, Yanek LR, Bhan A, Gaynor BJ, Ryan K, Brody JA, Zhong X, Wei Q, Consortium NT-OfP, Kammers K, Kanchan K, Iyer K, Kowalski MH, Pitsillides AN, Cupples LA, Li B, Schlaeger TM, Shuldiner AR, O'Connell JR, Ruczinski I, Mitchell BD, Faraday N, Taub MA, Becker LC, Lewis JP, Mathias RA and Johnson AD. Genome sequencing unveils a regulatory landscape of platelet reactivity. Nat Commun.2021;12:3626. 5. Sato-Nishiuchi R, Nakano I, Ozawa A, Sato Y, Takeichi M, Kiyozumi D, Yamazaki K, Yasunaga T, Futaki S and Sekiguchi K. Polydom / SVEP1 is a ligand for integrin alpha9beta1. J Biol Chem. 2012;287:25615-30. 6. Morris GE, Denniff MJ, Karamanavi E, Andrews SA, Kostogrys RB, Bountziouka V, Ghaderi- Najafabadi M, Shamkhi N, McConnell G, Kaiser MA, Carleton L, Schofield C, Kessler T, Rainbow RD, Samani NJ and Webb TR. The integrin ligand SVEP1 regulates GPCR-mediated vasoconstriction via integrins alpha9beta1 and alpha4beta1. Br J Pharmacol.2022;179:4958-4973. 7. Elenbaas JS, Pudupakkam U, Ashworth KJ, Kang CJ, Patel V, Santana K, Jung I-H, Lee PC, Burks KH, Amrute JM, Mecham RP, Halabi CM, Aliso A, Di Paola J and Stitziel NO. SVEP1 is an endogenous ligand for the orphan receptor PEAR1. Nature Communications.2023;14. 8. Jung IH, Elenbaas JS, Alisio A, Santana K, Young EP, Kang CJ, Kachroo P, Lavine KJ, Razani B, Mecham RP and Stitziel NO. SVEP1 is a human coronary artery disease locus that promotes atherosclerosis. Sci Transl Med.2021;13. 9. Sun BB, Maranville JC, Peters JE, Stacey D, Staley JR, Blackshaw J, Burgess S, Jiang T, Paige E, Surendran P, Oliver-Williams C, Kamat MA, Prins BP, Wilcox SK, Zimmerman ES, Chi A, Bansal N, Spain SL, Wood AM, Morrell NW, Bradley JR, Janjic N, Roberts DJ, Ouwehand WH, Todd JA, Soranzo N, Suhre K, Paul DS, Fox CS, Plenge RM, Danesh J, Runz H and Butterworth AS. Genomic atlas of the human plasma proteome. Nature.2018;558:73-79. 10. Mbasu RJ, Heaney LM, Molloy BJ, Hughes CJ, Ng LL, Vissers JP, Langridge JI and Jones DJ. Advances in quadrupole and time-of-flight mass spectrometry for peptide MRM based translational research analysis. Proteomics.2016;16:2206-20. 11. (FDA) TUSFaDA. Bioanalytical Method Validation: Guidance for Indsutry.2018:1-37. 12. (EMA) EMA. ICH guideline M10 on bioanalytical method validation and study sample analysis. 2022:5 - 41.

Claims

Claims 1. A method for diagnosing a subject suffering from cardiovascular disease, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the method comprising: (i) digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; and (ii) analysing the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, and comparing this concentration with a reference concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is suffering from cardiovascular disease, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is not suffering from cardiovascular disease, or does not have a pre-disposition thereto, or provides a positive prognosis of the subject’s condition.

2. A method of determining the efficacy of treating a subject suffering from cardiovascular disease with a therapeutic agent, the method comprising: (i) digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; and (ii) analysing the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, and comparing this concentration with a reference concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference,suggests that the treatment regime with the therapeutic agent is ineffective, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, suggests that the treatment regime with the therapeutic agent is effective.

3. The method according to either claim 1 or claim 2, wherein the method comprises digesting SVEP1 protein which comprises an amino acid sequence substantially as set out in SEQ ID No: 1, or a variant or fragment thereof.

4. The method according to any preceding claim, wherein the method comprises detecting the one or more SVEP1-derived quantotypic peptide or a fragment thereof, wherein the quantotypic peptide or a fragment thereof is derived from an amino acid sequence substantially as set out in SEQ ID No: 1, or a variant or fragment thereof.

5. The method according to any preceding claim, wherein the enzyme is trypsin.

6. The method according to any preceding claim, wherein the quantotypic peptide is selected from: CALLLQEIPAISYR (SEQ ID No: 2), QGFILSGVK (SEQ ID No: 3), GAFQQAAQILLHAR (SEQ ID No: 4), DVEAPQINCPK (SEQ ID No: 5), YSCVGGFFLR (SEQ ID No: 6), IVLCTPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), ISCGPPAHVENAIAR (SEQ ID No: 8), SLATSCPEELSK (SEQ ID No: 9), LLQTLETITNK (SEQ ID No: 10), EGYTLAGLDTIECLADGK (SEQ ID No: 11), TCDIHIVIK (SEQ ID No: 12), and, IGSYQDEEGQLECK (SEQ ID No: 13), or a variant or analogue thereof.

7. The method according to any preceding claim, wherein the quantotypic peptide is selected from: C(cam)ALLLQEIPAISYR (SEQ ID No: 2), QGFILSGVK (SEQ ID No: 3), GAFQQAAQILLHAR (SEQ ID No: 4), DVEAPQINC(cam)PK (SEQ ID No: 5), YSC(cam)VGGFFLR (SEQ ID No: 6), IVLC(cam)TPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), ISC(cam)GPPAHVENAIAR (SEQ ID No: 8), SLATSC(cam)PEELSK (SEQ ID No: 9), LLQTLETITNK (SEQ ID No: 10), EGYTLAGLDTIEC(cam)LADGK (SEQ ID No: 11), TC(cam)DIHIVIK (SEQ ID No: 12), and IGSYQDEEGQLEC(cam)K (SEQ ID No: 13).

8. The method according to any preceding claim, wherein the quantotypic peptide is CALLLQEIPAISYR (SEQ ID No: 2), C(cam)ALLLQEIPAISYR (SEQ ID No: 2), or QGFILSGVK (SEQ ID No: 3).

9. The method according to any preceding claim, wherein the cardiovascular disease is coronary artery disease (CAD), coronary artery calcification or plaque build-up.

10. The method according to any preceding claim, wherein detection of the quantotypic peptides of a fragment thereof is carried out by chromatography, such as liquid chromatography (LC), ultra performance liquid chromatography (UPLC), or gas chromatography (GC), which is preferably coupled to mass spectrometry (MS) or time-of-flight (TOF).

11. The method according to any preceding claim, wherein detection of the quantotypic peptides or a fragment thereof is carried out by LC-MS.

12. The method according to any preceding claim, wherein the one or more SVEP1- derived quantotypic peptides are ionised into fragments, which are detected, for example by LC-MS, or UPLC-MS / MS.

13. The method according to any preceding claim, wherein the quantotypic peptide is CALLLQEIPAISYR (SEQ ID No: 2), which is ionised into the following three fragments: CALL (SEQ ID No: 14), CALLL (SEQ ID No: 15) and QEIPAISYR (SEQ ID No: 16), or C(cam)ALL (SEQ ID No: 14), C(cam)ALLL (SEQ ID No: 15) and QEIPAISYR (SEQ ID No: 16).

14. The method according to claim 13, wherein the detection step comprises detection of at least one, two or all three of the fragments derived from CALLLQEIPAISYR (SEQ ID No: 2) or C(cam)ALLLQEIPAISYR (SEQ ID No: 2).

15. The method according to any preceding claim, wherein the quantotypic peptide is QGFILSGVK (SEQ ID No: 3), which is ionised into the following three fragments: LSGVK (SEQ ID No: 17), ILSGVK (SEQ ID No: 18), and FILSGVK (SEQ ID No: 19).

16. The method according to claim 15, wherein the detection step comprises detection of at least one, two or all three of the fragments derived from QGFILSGVK (SEQ ID No: 3).

17. The method according to any preceding claim, the sample is blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, tissue, and / or CSF, or combinations thereof.

18. The method according to any preceding claim, wherein the sample is blood.

19. The method according to claim 19, wherein the blood sample comprises blood plasma.

20. The method according to any preceding claim, wherein the bodily sample comprises extracellular vesicles (EV), optionally wherein the extracellular vesicles (EV) comprise, contain or encapsulate the SVEP1.

21. The method according to any preceding claim, wherein the concentration of CALLLQEIPAISYR or C(cam)ALLLQEIPAISYR (SEQ ID No: 2), QGFILSGVK (SEQ ID No: 3), GAFQQAAQILLHAR (SEQ ID No: 4), DVEAPQINCPK or DVEAPQINC(cam)PK (SEQ ID No: 5), YSCVGGFFLR or YSC(cam)VGGFFLR (SEQ ID No: 6), IVLCTPPPLISFGVPIPSSALHFGSTVK or IVLC(cam)TPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), ISCGPPAHVENAIAR or ISC(cam)GPPAHVENAIAR (SEQ ID No: 8), SLATSCPEELSK or SLATSC(cam)PEELSK (SEQ ID No: 9), LLQTLETITNK (SEQ ID No: 10), EGYTLAGLDTIECLADGK or EGYTLAGLDTIEC(cam)LADGK (SEQ ID No: 11), TCDIHIVIK or TC(cam)DIHIVIK (SEQ ID No: 12), and / or IGSYQDEEGQLECK or IGSYQDEEGQLEC(cam)K (SEQ ID No: 13), or a fragment thereof, is detected.

22. An apparatus for diagnosing a subject suffering from cardiovascular disease, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the apparatus comprising:- (i) means for digesting SVEP1 protein, in a bodily sample obtained from the subject, with an enzyme to thereby form one or more SVEP1-derived quantotypic peptide; (ii) means for determining the concentration of the one or more SVEP1- derived quantotypic peptide or a fragment thereof; and (iii) a reference for the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof from an individual who does not suffer from cardiovascular disease, wherein (a) an increase in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is suffering from cardiovascular disease, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition, or wherein (b) a decrease in the concentration of the one or more SVEP1-derived quantotypic peptide or a fragment thereof, compared to the reference, indicates that the subject is not suffering from cardiovascular disease, or does not have a pre-disposition thereto, or provides a positive prognosis of the subject’s condition.

23. The apparatus according to claim 22, wherein the apparatus the SVEP1-derived quantotypic peptide is as defined in any one of claims 6-8.

24. The apparatus according to either claim 22 or claim 23, wherein the fragment of the one or more SVEP1-derived quantotypic peptide is as defined in any one of claims 13-16.

25. The apparatus according to any one of claims 22-24, wherein the apparatus is configured to perform the method according to any one of claims 1-22.

26. Use of a peptide, or a variant or analogue thereof, wherein the peptide is selected from a group of peptides consisting of: CALLLQEIPAISYR (SEQ ID No: 2), QGFILSGVK (SEQ ID No: 3), GAFQQAAQILLHAR (SEQ ID No: 4), DVEAPQINCPK (SEQ ID No: 5), YSCVGGFFLR (SEQ ID No: 6),IVLCTPPPLISFGVPIPSSALHFGSTVK (SEQ ID No: 7), ISCGPPAHVENAIAR (SEQ ID No: 8), SLATSCPEELSK (SEQ ID No: 9), LLQTLETITNK (SEQ ID No: 10), EGYTLAGLDTIECLADGK (SEQ ID No: 11), TCDIHIVIK (SEQ ID No: 12), and IGSYQDEEGQLECK (SEQ ID No: 13), as a biomarker for diagnosing a subject suffering from cardiovascular disease, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition.

27. Use of SVEP1, or a variant or fragment thereof, as a biomarker for coronary artery calcification or plaque build-up.

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