Methods and materials for measuring complement activation

Abstract

The present disclosure provides, e.g., signature peptide compounds corresponding to various full-length protein components of the complement enzymatic cascade (e.g., C1q, C1s, C3, C3p, C4, C4p, C5, C5p, FB, and FBp). In one aspect, the invention relates to using these signature peptides for measuring and / or monitoring activation of the complement pathway, e.g., in clinical samples, such as plasma, serum, CSF, aqueous humor, or vitreous humor.

Description

CLAIM OF PRIORITY This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 549,404, filed on Feb 2, 2024; U.S. Provisional Patent Application Serial No. 63 / 634,445, filed on Apr 15, 2024; and U.S. Provisional Patent Application Serial No. 63 / 654,849, filed on May 31, 2024, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD This invention relates to signature peptides corresponding to various fulllength protein components of the complement enzymatic cascade. In one aspect, the invention relates to using these signature peptides for measuring and / or monitoring activation of the complement pathway. BACKGROUND There are numerous deadly diseases (including inflammatory, autoinflammatory, and neurodegenerative disorders) affecting the current human population. Prevention, early detection and diagnosis, and effective treatment (including personalized treatment options) help reduce the socioeconomic burden caused by healthcare spending as well as lost productivity. However, the pace of progress in medical research directly depends on techniques that allow for reliable, quantitative, sensitive, fast, inexpensive, and miniaturized experimental methods. To that end, medical systems and devices that allow multiplexing of scant sample volumes greatly enhance monitoring of clinical trial progression to expedite drug product approval, and significantly improve preventive and diagnostic patient care. SUMMARY The complement pathway entails a signaling network of proteins that become processed and / or activated sequentially, activating the immune system to fight a foreign antigen. However, over-activation and / or dysregulation of complement cascade are known contributors to inflammation, autoimmunity, transplant rejections, cancer, neurodegeneration (e.g.. Alzheimer’s disease), infectious diseases, and ocular diseases like age-related macular degeneration and geographic atrophy. Therapeutics blocking complement activity have been approved and additional therapeutics are being evaluated in the clinic. Hence, direct measurement of the complement pathway activity allows for, e.g., clinical diagnosis as well as monitoring treatment and interpreting results of clinical studies. The highly sensitive, multiplex methods and materials provided in this document advantageously allow' to accurately quantify complement with minimal sample volumes of blood, plasma, serum, cerebrospinal fluid (“CSF”), brain, aqueous humor, and / or vitreous humor. The present disclosure provides, inter alia, a sensitive high-throughput multiplexed LC-MS / MS method to measure complement activity by monitoring subcomponents of complement component 1 (Cl) complex (e.g., total and active), intact complement component 2 (C2), processed C2 (C2p), intact complement factor B (CFB), processed CFB (CFBp), intact complement component 3 (C3), processed C3 (C3p), intact complement component 4 (C4), processed C4 (C4p), intact complement component 5 (C5), and processed C5 (C5p). As described herein, the methods within the present claims use “signature peptides” that both overlap the natural cleavage sites and are immediately adjacent to such cleavage sites advantageously allowing to simultaneously track the intact complement proteins and the endogenously cleaved complement protein fragments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the present application will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIG. 1 provides a schematic illustration of complement pathway (See Seminars in Immunopathology (2021) 43:757-771). FIG. 2A contains amino acid sequence of Complement Cis (subcomponent of Complement Cl complex) (UniPort sequence entry no. P09871). FIG. 2B contains amino acid sequence of Complement Clq subcomponent subunit B (subcomponent of Complement Cl complex) (UniPort sequence entry no. P02746). FIG. 2C Complement Clq subcomponent subunit C (subcomponent of Complement Cl complex) (UniPort sequence entry no. P02747). FIG. 2D contains a schematic representation showing LC-MS detection of Clq, Cis total, and Cis active following either Clq or Cis immunoprecipitation. FIG. 3A contains a bar graph showing ratio of the levels of active (cleaved) Cis over total Cis measured in human CSF of Alzheimer’s disease patients (AD, n = 28) and healthy volunteers (HV, n = 10) liquid chromatography mass spectrometry (LC-MS) analysis of surrogate peptides (sequences). P-value of Welch’s t-test for statistical significance of the difference of the means is provided as inset. FIG. 3B contains a plot of the difference between the mean of the ratio of active / total Cis in ADC vs HV patients. FIG. 3C contains a bar graph showing LC-MS quantitation of the active Cis levels in CSF of AD and HV patients, reported in nanomolar concentration calculated using a signal calibration equation determined with a heavy-labeled peptide standard. P-value of Welch’s t-test of statistical significance is provided as inset. FIG. 3D contains a bar graph showing LC-MS quantitation of the total Cis levels in CSF of AD and HV patients, reported in nanomolar concentration calculated using a signal calibration equation determined with a heavy-labeled peptide standard. P-value of Welch’s t-test of statistical significance is provided as inset. FIG. 3E contains a line plot showing correlation of active Cis vs total Cis in AD patient group, with R2, P-value and Pearson r provided as inset. FIG. 3F contains a line plot showing correlation of active Cis vs total Cis in HV patient group, with R2, P-value and Pearson r provided as inset. FIG. 3G contains a line plot showing correlation between area ratio and concentration ratio. Calibration for Cis IIGGSDADIK, +2y8 light: y=2.90736, x+ / -0.00443 (r=0.99982, r2=0.99964)(weighting 1 / x). FIG. 3H contains a line plot showing correlation between area ratio and concentration ratio. Calibration for Cis total. LLEVPEGR, +2y6 light: y=3.29801, X+ / -0.00231 (r=0.99804, r2=0.99607)(weighting 1 / x2). FIG. 31 contains a plot showing human complexed Cis readout of AD and CN groups CSF. Statistically significant differences were observed between % complexed Cis in AD vs CN group upon anti-Clq capture. FIG. 3J contains a plot showing human complexed Cis readout of AD and CN groups CSF. Results show that the differences are mostly driven by Clq changes, with no significant differences observed between AD and CN Cis readouts. FIG. 4A contains a schematic representation for the signature peptide prediction for the full length and fragment proteins. FIG. 4B contains DDA analysis showing chymotryptic peptides coverage in complement proteins. DDA peptide sequence coverage of Factor B, C3, C4, and C5 is shadowed. Bold arrows in each sequenced indicate cleavage sites. FIG. 5A schematically exemplary shows aqueous humor sample preparation method. FIG. 5B schematically shows exemplary aqueous humor sample preparation method. FIG. 5C contains a schematic example of aqueous humor sample preparation (80 pL and 100 pL scale). The exemplary assays use less than a third of sample volume required for immunoassays, increase robustness and sensitivity. FIG. 6 contains a total ion chromatogram for complement peptide standards in buffer analyzed using a 15-minute multiplexed LC-MS / MS method. FIG. 7A contains line plots showing alternative complement activation using zymosan in aqueous humor samples. FIG. 7B contains line plots showing classical complement activation using HAGG in aqueous humor samples. FIG. 8A contains protein sequence for Factor B. FIG. 8B contains protein sequence for complement component C2. FIG. 8C contains protein sequence for complement component C3. FIG. 8D contains protein sequence for complement component C4. FIG. 8E contains protein sequence for complement component C5. FIG. 9 shows C5 protein coverage by DDA with and without PNGase F. No peptides detected for the full length C5 protein in the region of interest (circled). DDA analysis indicates presence of a glycosylation site on 741 asparagine. Deglycosylation treatment with PNGase F allows for peptide coverage in region of interest. DETAILED DESCRIPTION The complement pathway (which is also known as the complement system, complement cascade, and / or the complement) is a part of the mammalian immune system. Generally, the complement maximizes (and thus complements) the ability of other parts of the immune system (e.g., antibodies, phagocytic cells) to provide protection from microbes and / or external pathogens as well as to clear foreign material and damaged cells from a mammalian tissue. For example, the complement stimulates the phagocytes, increases inflammatory responses, and activates the membrane-attack complex (“MAC”) thereby lysing the invading pathogen. Several dozen of various proteins, peptides, and fragments thereof make up the complement, and there are three distinct biochemical pathways to activate the complement system. Referring to Figure 1, those pathways are the classical, the lectin, and the alternative pathways. The classical complement pathway is activated when Clq within the Cl complex binds to antigen-antibody complexes. This binding triggers activation of serine protease Cis by the enzyme Clr. The active Cis, in turn, cleaves complement component C4 (to produce protein fragments C4a and C4b) and complement component C2 (to produce protein fragments C2a and C2b), thereby forming the classical pathway C3 convertase C4b2b. The C4b2b can cleave C3 into its fragments, e.g., C3a, C3b, and / or C3c. The C3b fragment, along with the C4b2b, may form the classical pathway C5 convertase C4b2b3b. The C5 convertase then cleaves C5 into its fragments (anaphylatoxins), e.g., C5a and C5b. The C5b fragment is the nucleus of the membrane attack complex (MAC, C5b-9), which is formed when C5b binds to C6, C7, C8, and C9 proteins of the complement system to produce the lytic core. In addition, C5a and C3a induce chemotaxis and inflammatory responses via binding to their receptors (e.g., C5aR and C3aR) on various immune cells (e.g., infiltrating macrophages). Referring again to Figure 1, the lectin pathway is similar to the classical pathway, except that the recognition components, mannan-binding lectin (MBL) and the MBL-associated serine proteases, are activated by interaction with microbial carbohydrate structures. Once activated, the MBL-associated proteases cleave C4 and C2 to form the C3 convertase complex C4b2b, thereby triggering the remaining steps of the complement cascade (as described for the classical pathway). Finally, the alternative pathway of complement activation results from spontaneous cleavage of the abundant plasma protein C3 into anaphylotoxin (C3a) and opsonin (C3b) fragments to induce effector functions. The C3b production is amplified via the factor D mediated cleavage of factor B and subsequent generation of the alternative pathway C3 convertase C3bBb. The production of the C3 cleaving enzyme C3bBb induces further C3 cleavage and concomitant generation of the alternative pathway C5 convertase C3bBb3b. But no matter which pathway activates the complement, the 5 activation results in enhanced inflammatory response, including the death of pathogens by the MAC, production of reactive oxygen species, and / or secretion of pro-inflammatory anaphylatoxins such as C4a, C3a, and C5a. Signature peptides In one general aspect, the present disclosure provides a peptide (e.g., a 10 “signature peptide”) selected from any one of the following sequences: Table 1 IIGGSDADIK Active-Cis LLEVPEGR Total Cis LQVIFK Total Cis AARLPVAPLRK Total Cis YQSVFTVAR Total Clq LEQGENVFLQATDK Total Clq VVTFCGHTSK Total Clq LSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIY B-full length LSSLTETIEGVDAEDGHGPGEQQKR Ba KIVLDPSGSMNIY Bb QKDAPDHQELNLDVSLQLPSRSSKITHRIHW C3-full length RSEETKENEGF C3-full length TSSSGQQTAQRAELQCPQPAA C3b SEETKENEGF C3g QKDAPDHQELNLDVSLQLPSR C3d AESLRKKSRDKGQAGLQRAL C4-full length IASHTTEERGLNVTLSSTGRNGF C4-full length AESLRKKSRDKGQAGLQR C4b IASHTTEERGLNVTLSSTGR C4d GRKIQIQRSGH C2-full length GRKIQIQRS C2b TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY C5-full length TECCVVASQLRANISHKDMQLGR C5a LHMKTLLPVSKPEIRSY C5b or a salt thereof. In some embodiments, the peptide comprises the indicated sequence. In some embodiments, the peptide consists essentially of the indicated sequence. In some 15 embodiments, the peptide consists of the indicated sequence. Methods to diagnose and / or treat complement-associated disorders On another general aspect, the present disclosure provides a method of diagnosing a complement-associated disorder (e.g., any one of the disorder described herein) in a subject. In some embodiments, the method comprises: (a) treating a sample obtained from the subject with a protease (e.g.. trypsin, chymotrypsin, or LyC) to obtain a processed sample; (b) determining an amount of at least one “signature peptide” (e.g.. 1, 2, or all of the foregoing signature peptides in Table 1) in the processed sample; and (c) comparing the amount determined in step (b) to a reference amount of the peptide, wherein an increase in the amount of the peptide in the processed sample relative to the reference amount is indicative of the subject having the complement-associated disorder. In some embodiments, the step (b) comprises determining an amount of at least one Active-Cis, pBa, pBb, pC3b, pC3g, pC3d, pC4b, pC4d, pC2b, pC5a, or pC5b “signature peptide” provided in Table 1, or any combination thereof. In some embodiments, the reference amount is an amount of the “signature peptide” determined in a processed sample derived from a healthy subject. In some embodiments, the increase is about 1.1 fold, about 1.2. fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 2 fold, about 3 fold, about 5 fold, about 7 fold, or about 10 fold. In some embodiments, the present disclosure provides a “signature peptide” for a full-length (FL) complement protein (e.g.. Cis), the “signature peptide” selected from any one of the following sequences: LLEVPEGR Cls-total LQVIFK Cls-total AARLPVAPLRK Cls-total or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a processed (p) complement protein, the “signature peptide” having the following sequence: IIGGSDADIK Cls-active or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a full-length (FL) complement protein (e.g., Factor B or CFB), the “signature peptide” having the following sequence: LSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIY B or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a processed (p) complement protein (pB, e.g., Ba or Bb), the “signature peptide” having any one of the following sequences: LSSLTETIEGVDAEDGHGPGEQQKR Ba KIVLDPSGSMNIY Bb or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a full-length (FL) complement protein (e.g., C2), the “signature peptide” having the following sequence: GRKIQIQRSGH C2 or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a processed (p) complement protein (pC2, e.g., C2b), the “signature peptide” having the following sequence: GRKIQIQRS C2b or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a full-length (FL) complement protein (e.g., C3), the “signature peptide” selected from any one of the following sequences: QKDAPDHQELNLDVSLQLPSRSSKITHRIHW C3 RSEETKENEGF C3 or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a processed (p) complement protein (pC3, e.g., C3b, C3g, or C3d), the “signature peptide” selected from any one of the following sequences: TSSSGQQTAQRAELQCPQPAA C3b SEETKENEGF C3g QKDAPDHQELNLDVSLQLPSR C3d or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a full-length (FL) complement protein (e.g., C4), the “signature peptide” selected from any one of the following sequences: AESLRKKSRDKGQAGLQRAL C4 IASHTTEERGLNVTLSSTGRNGF C4 or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a processed (p) complement protein (pC4, e.g., C4b or C4d), the “signature peptide” having any one of the following sequences: AESLRKKSRDKGQAGLQR C4b IASHTTEERGLNVTLSSTGR C4d or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a full-length (FL) complement protein (e.g., C5), the “signature peptide” having the following sequences: TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY C5 or a salt thereof. In some embodiments, the present disclosure provides a “signature peptide” for a processed complement protein, the “signature peptide” having the following sequence: TECCVVASQLRANISHKDMQLGR C5a LHMKTLLPVSKPEIRSY C5b or a salt thereof. In some embodiments, the present disclosure provides a method of diagnosing a complement-associated disorder in a subject, the method comprising: determining an amount of at least one “signature peptide” for a full-length (FL) complement protein (e.g., Cls-total, B, C2, C3, C4, or C5, as described herein) in a processed sample obtained by treating a sample obtained from the subject with a protease; wherein a decrease in the amount of the peptide in the processed sample relative to a reference amount of the peptide is indicative of the subject having the complement-associated disorder. In some embodiments, the decrease is about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 2 fold, about 3 fold, about 5 fold, about 7 fold, or about 10 fold. In some embodiments, the method comprises (a) obtaining the sample from the subject; (b) treating the sample with a protease to obtain the processed sample; and (c) determining the amount of the at least one “signature peptide” (e.g., signature peptide for full-length Cls-total, B, C2, C3, C4, or C5, as described herein) in the processed sample. In some embodiments, the present disclosure provides a method of diagnosing a complement-associated disorder in a subject, the method comprising: (a) determining an amount of at least one “signature peptide” for a processed (p) complement protein (e.g., Cls-active, pB, pC2, pC3, pC4, or pC5, as described herein) in a processed sample obtained by treating a sample obtained from the subject with a protease; wherein an increase in the amount of the peptide in the processed sample relative to a reference amount of the peptide is indicative of the subject having the complement-associated disorder. In some embodiments, the increase is about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 2 fold, about 3 fold, about 5 fold, about 7 fold, or about 10 fold. In some embodiments, the method comprises (a) obtaining the sample from the subject; (b) treating the sample with a protease to obtain the processed sample; and (c) determining the amount of the at least one “signature peptide” (e.g., signature peptide for Cls-active, pB, pC2, pC3, pC4, or pC5, as described herein) in the processed sample. In some embodiments, the present disclosure provides a method of diagnosing a complement associated disorder in a subject, the method comprising determining (i) a first amount of at least one “signature peptide” for a processed (p) complement protein (e.g., Cls-active, pB, pC2, pC3, pC4, or pC5, as described herein) and (ii) a second amount of at least one “signature peptide” for a full-length (FL) complement protein (e.g., Cls-total, B, C2, C3, C4, or C5, as described herein) in a processed sample, the signature peptides obtained by treating a sample obtained from the subject with a protease; wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder. In some embodiments, the increase is about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 2 fold, about 3 fold, about 5 fold, about 7 fold, or about 10 fold. In some embodiments, the method comprises (a) obtaining the sample from the subject; (b) treating the sample with a protease to obtain the processed sample; and (c) determining the first and the second amounts of the “signature peptides” in the processed sample as described herein. Identifying / determining steps In some embodiments, any of the methods described herein include a step of obtaining a sample from the subject. In some embodiments, the sample is selected from blood, plasma, serum, cerebrospinal fluid (CSF), aqueous humor, vitreous humor, brain homogenate, and eye homogenate. Additional examples of a sample obtained from a subject include hair, urine, saliva, sputum, feces, sweat, fingernail clippings, and various other bodily fluids. In some embodiments, the subject is a patient suspected of having a complement-associated disorder (e.g., geographic atrophy). The sample may be collected by any appropriate means. Some samples may be collected as the body naturally eliminates them. For other sample collection, minor surgical procedures and anesthesia may be used (e.g., using needles, syringes, or laparoscopy). The sample used for testing is often determined by the purpose of the particular test. For example, a sample of aqueous humor (AH) or vitreous humor (VH) may be collected from a subject, e.g., suspected of having an ocular complement-mediated disorder (e.g., AMD, GA, etc). In another example, a CSF sample may be collected from a subject, e.g., suspected of having a neurological disorder (e.g., Alzheimer’s or Parkinson’s disease). In some embodiments, the method includes treating the sample obtained from the subject with a protease to obtain a processed sample. Examples of proteases include cysteine protease, threonine protease, serine protease, aspartic protease, and metalloprotease. In some embodiments, the protease is trypsin, chymotrypsin, elastase, papain, calpain, cathepsin, pepsin, rennin, hermolysin, and carboxypeptidase. In some embodiments, the protease is selected from trypsin, chymotrypsin, lysC, and gluC, or a combination thereof. One example of a suitable protease is chymotrypsin. Without being bound by any particular theory or speculation, chymotrypsin produces “signature peptides” to monitor cleaved / processed (active) complement proteins of interest (e.g., B, C3, C4, and C5). Chymotrypsin cleaves the carboxyl side of aromatic amino acids, such as tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp), while also cleaving peptide bonds at a lower rate at leucine (Leu) and methionine (Met). Another example of a suitable protease is trypsin or LysC, or a combination thereof. In some embodiments, determining an amount of “signature peptide” in the processed sample is carried out using a mass spectrometry technique. In some embodiments, determining the amount is carried out using an immunoassay. Quantifying the amount of “signature peptide” in the processed sample can be carried out suing any suitable analytical technique or process, e.g., using calibration curves or similar methods. Exemplary and illustrative embodiments and methodology for determining amount of “signature peptide” in the processed sample are described in the “Examples” section and drawings herein. Treatment step In some embodiments, any of the methods described herein may include a step comprising administering to the subject a therapeutic agent in an effective amount to treat the complement-associated disorder. For example, the therapeutic agent is an inhibitor of complement, e.g., cinryze, berinert, ruconest, sutimlimab, pegcetacoplan, eculizumab, ravulizumab, avacincaptad, pozelimab, zilucoplan, iptacopan, danicopan, or avacopan. In another example, the therapeutic agent is a nonsteroidal antiinflammatory drug (NSAID), e.g., diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, or tolmetin. In some embodiments, the therapeutic agent is a steroidal anti-inflammatory drug, e.g., bethamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, or dexamethasone. The choice of the therapeutic agent may be determined by the treating physician on a case-by-case basis depending on the complement-associated disorder, the severity of the disease, the route of administration, the sex, age and general health condition of the subject. In some embodiments, the complement-associated disorder is ophthalmic disease or disorder. In some embodiments, the ophthalmic disorder is age-related macular degeneration (AMD), geographic atrophy (GA), retinopathy, uveitis, glaucoma, Stargardt disease, retinal occlusive vasculitis, intraocular neovascular syndrome, corneal inflammation (e.g., acute or chronic), intraocular inflammation (e.g., acute or chronic), or choroidal neovascularization (CNV), retinal vein occlusion, dry eye syndrome, age-related maculopathy (e.g., early to intermediate dry AMD in which GA has not developed), posterior keratitis (e.g., unilateral posterior interstitial keratitis), or non-ulcerating inflammation of the corneal stroma. In some embodiments, the complement-associated disorder is neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is selected from Alzheimer’s disease, schizophrenia, frontotemporal dementia, Huntington’s disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome (GBS), Neuromyelitis Optica (NMO), Myasthenia gravis (MG), Multifocal Motor Neuropathy (MMN), Cold agglutinin disease (CAD), and Immune thrombocytopenic purpura (ITP). In some embodiments, the complement-associated disorder is paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (MG), or neuromyelitis optical spectrum disorder (NMOSD). In some embodiments, the present disclosure provides a method of treating any of the complement-associated disorders described herein. In some embodiments, the method of treating the disorder comprises (i) diagnosing a complement-associated disorder in a subject as described herein; and (ii) administering to the subject a therapeutic agent in an effective amount to treat the complement-associated disorder. In some embodiments, the method of treating the disorder comprises (i) diagnosing a complement-associated disorder in a subject as described herein; and (ii) selecting a therapeutic agent for treatment of the complement-associated disorder; and (iii) administering to the subject the therapeutic agent selected in step (ii) in an effective amount to treat the complement-associated disorder. EXAMPLES Example 1 - Direct measurement of Cis (total and active) and Clq in Human / Cyno CSF and plasma / serum samples The present example provides peptide-based MS assays for monitoring complement pathway. The assays, designed for minimal sample volumes of blood, CSF, and brain, offer pharmacodynamic and biomarker readouts, e.g., supporting therapeutic programs where complement monitoring is needed. Materials and methods: The assays employed, e.g., complement protein standards (e.g., Cis, Clr, Clq), biotinylated antibodies, Trypsin, Trypsin / LysC or LysC kits. With variable volumes (5-200 pL), plasma and CSF were used. Assays involved protein denaturation, reduction, alkylation, and trypsin / LysC or LysC digestion. For some assays, samples underwent incubation with capture antibodies and elution. Experiments were conducted on Sciex 7500, Waters Acquity UPLC, T3 column, 1.8 pm, 2.1x100 mm, with quantitation ranges at 0.05-24 pg / mL for plasma and 0.005-3.6 pg / mL for CSF. Data analysis was performed with Skyline. For example, the following materials were used: DYNABEADS MYONE Streptavidin T1 beads; Genentech biotinylated antibody (50 pg / mL) (Clq antibody or Cis antibody depending on the application, each antibody either made in-house or procured commercially), 50 mM Tris-HCl buffer (pH 7.5), tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide (IAM), Trypsin (Promega catalog no. V5071), LysC (Promega catalog, no. VAI 170), formic acid, CaCh (30 mM in water), PBST (phosphate-buffered saline + 0.1% Tween 20) with 1 mM CaCh. Cl complement proteins: Cyno / human Clq protein (procured from Complement Tech) 100 L x 1 mg / mL in 10 mM HEPES, 300 mM NaCl, pH 7.2; Lot 2. 10 pg / mL Clq protein solution in 0.2% Bovine serum albumin (“BSA”) in water Cyno / human Cis Pro protein (Complement Tech or in-house generated) Cyno / human Cis Active protein (in house generated) Isotopic ally labeled* peptide standards (internal standards, “IS”) (15 pM stock in 10% methanol, IS working solution, 100 nM in water): Total Cis LLEVPEGR* Total Clq YQSVFTVAR* Pro-Cis QRIIGGSDADIK* Active-Cis IIGGSDADIK* Total Cis LQVIFK* Total Cis AARLPVAPLRK* Total Clq LEQGENVFLQATDK* *Isotopic label at C-terminal amino acid, with heavy R* = 13C6, 15N4; heavy 5 K* = 13C6, 15N2. Note that there are 6xC and 4xN isotopes used in the R label; and 6xC and 2xN isotopes in the K label. Preparation of complement protein standards: Cis Pro and QC* for plasma study 4% BSA / H2O, 0.1-20 pg / mL Cis Pro for CSF study water, 0.01-0.4 pg / mL Clq and QC for plasma study 4% BSA / H2O, 0.1-100 pg / mL Clq for CSF study water, 0.025-1 pg / mL Cis Act and QCs for plasma study 4% BSA / H2O, 0.1-20 pg / mL Cis Act for CSF study water, 0.01-0.4 pg / mL *Quality control (“QC”) samples refer to standards spiked into bio-matrix or BSA / H2O buffer at a known concentration not already tested in the calibration curve. 10          Anti-Clq capture experiment: Immunoprecipitation step: in a 2 mL 96-well plate, added 10 pL of each CSF, serum, or plasma, or calibration standard. Added 300 pL 4% BSA-PBST with 1 mM CaCh, mixed for 10 minutes. Added 40 pL (2 pg) of 50 pg / mL Genentech Biotinylated antibody in 4% BSA-PBST with ImM CaCh. Incubated plate at 25 °C for 1.5 hr at 600 15 rpm. Added 25 pL washed T1 beads into each well and incubated at 25 °C for 1 hr at 950 rpm. Prepared wash plates (2 ml Kingfisher plate, available from ThermoFisher) and elution plate (2 ml protein LoBind plate): Plate 1 (300 pL of 0.05% chaps in IX PBS + 10 pL 30 mM CaCl2), Plate 2 (300 pL of 0.05% chaps in IX PBS+ 10 pL 30 mM CaCl2), Plate 3 (100 pL of 0.05% chaps in IX PBS, and 200 pL of IX PBS + 10 pL 30 mM CaCl2), Elution plate (150 pL of 25 mM HC1). Performed immunoprecipitation (“IP” or affinity capture) on KingFisher with the predefined method. In sum, in order to achieve greater sensitivity in complex bio-matrices, Clq and / or Cis may be immunoprecipitated (immune-captured) prior to LC-MS analysis. Additionally, capturing Clq with subsequent LC-MS detection of Cis (or vice versa) may inform on the complexation status of these subunits within the Cl complex. In other words, co-enrichment of both Clq and Cis upon IP directly indicates whether or not these subunits are non-covalently associated with each other within the Cl complex. By pulling down Clq, any detectable Cis corresponds to Cis in the Cl complex. When pulling down Cis, any detectable Clq corresponds to the relative Clq:Cls fraction in the Cl complex. See Figure 2D Digestion step: Neutralized the elution plate with 20 pL of IM pH 8.0 Tris-HC1 to each well. Added 10 pL of 100 mM dithiothreitol (“DTT”) in IM pH 8.0 Tris-HC1 to achieve a final concentration of about 5.6 mM. Incubated with shaking at 850 rpm at 37 °C for 45 min. After cooling down, added 10 pL of 240 mM IAM in H2O to achieve final concentration of 13 mM. Incubated in dark with shaking 850 rpm at room temperature for 30 min. Exposed the plate under light at room temperature for 20 min. For trypsin digestion: added 15 pL of 0.1 pg / pL of Trypsin to each well. For LysC digestion: Added 10 pL of 0.1 pg / pL of LysC to each well. Incubated with shaking at 850 rpm at 37 °C overnight. Added 30 pL of 10% FA to stop the reaction. Added 10 pL of IS working solution to each well. Proceeded to LC-MS analysis (see below). Direct digestion experiment: Reduction: Added 10 pL of STD, QC, plasma, serum, or CSF sample to a protein LoBind plate. Added 80 pL of Rapid Trypsin / LysC digestion buffer to each well. Added 10 pL of 45 mM TCEP to each well. Centrifuged briefly. Incubated the plate at 60 °C for 1 hour while shaking (600 RPM) on a ThermoMixer. Alkylation: Removed the plate from the incubator and allow to cool at RT for 5 mins. Centrifuged briefly. Added 10 pL of 140 mM IAM to each tube. (Note: Final cone is approximately 12 mM IAM). Centrifuged briefly. Incubated the plate at RT for 30 mins with shaking at 900 RPM, protect from light. After incubation, put the plate under light for 20 minutes Digestion (in case of Trypsin / LysC): Added 10 pL of 0.35 mg / mL rapid Trypsin / LysC to each well. Centrifuged briefly. Incubated the plate at 60 °C for 90 min, 5 with shaking at 900 RPM. Digestion (in case of LysC only): Added 20 pL of 0.1 mg / mL LysC to each well. Centrifuged briefly. Incubated the plate at 37 °C overnight, with shaking at 900 RPM. Signature peptides: Target Peptide Sequence Enzyme Total Cis LLEVPEGR Trypsin / LysC Total Clq YQSVFTVAR Trypsin / LysC Pro-Cis QRIIGGSDADIK LysC Active-Cis IIGGSDADIK LysC Total Cis LQVIFK LysC Total Cis AARLPVAPLRK LysC Total Clq LEQGENVFLQATDK LysC Total Clq VVTFCGHTSK LysC 10          Acidification: Removed the plate from the incubator and allowed to cool to RT for 5 mins. Centrifuged briefly. Added 10 pL of 20% FA to each well (final concentration approximately 1 % FA). Added 10 pL of IS working solutions to each sample except blank without IS. Proceeded to LC-MS analysis. LC-MS analysis: Mass Spectrometer: Sciex 7500. Liquid chromatography and 15 column information Waters Acquity UPLC, T3 column, 1.8 pm, 2.1x100 mm. Type of analysis: MRM (multiple reaction monitoring). Transitions monitored for quantitation: Peptide Sequence Enzyme Qi1 Q32 CE3 CXP4 Active Cis - light IIGGSDADIK LysC 494.769 762.363 26.7 22 Active Cis - heavy IIGGSDADIK* LysC 498.769 770.363 26.7 22 Clq - light YQSVFTVAR Trypsin / LysC 535.785 779.441 28.1 22 Clq - heavy YQSVFTVAR* Trypsin / LysC 540.785 789.441 28.1 22 Cis total - light LLEVPEGR Trypsin / LysC 456.761 686.347 24.7 22 Cis total - heavy LLEVPEGR* Trypsin / LysC 461.765 696.356 24.7 22 Cis total - light LQVIFK LysC 374.241 506.333 17.3 22 Cis total - heavy LQVIFK* LysC 378.248 514.347 17.3 22 Cis total - light AARLPVAPLRK LysC 397.927 513.350 17.1 30 Cis total - heavy AARLPVAPLRK* LysC 400.599 521.364 17.1 30 Clq - light LEQGENVFLQATDK LysC 796.401 822.435 38 30 Clq - heavy LEQGENVFLQATDK * LysC 800.408 830.449 38 30 Pro-Cis - light QRIIGGSDADIK LysC 636.848 762.362 33.2 22 Pro-Cis - heavy QRIIGGSDADIK* LysC 640.856 770.377 33.2 22 ^1: precursor isolation. 2Q3: detection of predefined fragment (transition). 3CE: collision energy. 4CXP: cell exit potential (parameter that controls intensity of product / fragment ions). *: denotes site of isotopic label (see table above with information about isotopically labeled peptide standards). Summary of results: the Example provides highly sensitive assays for complement pathway quantitation. For the various protein targets, recombinant constructs covering active and zymogen forms of Cis were generated. Peptide mapping experiments were performed to identify high intensity signature peptides for the protein targets and selective to their processing status. Using the recombinant protein standards, signal calibration curves were generated in buffer with BSA, establishing linear quantitation for all tested peptides with r2 >0.99 in the range of 0.005-3.6 pg / mL in CSF and 0.05-24 pg / mL in serum or plasma. Precision and accuracy of the assays were assessed by spiking-in known concentrations of recombinant standards in the appropriate matrices (serum, plasma or CSF) at levels representative of endogenous amounts. Known ratios of the processed vs intact forms of the protein standards were also spiked-in into the matrix. This assay developed in this example can be used, e.g., to diagnose and / monitor treatment of neurodegenerative diseases like Alzheimer’s diseases, and to aid clinical development in these indications. Figures 3A-3J provide as example of Cis LC-MS quantitation in human CSF. Example 2 - Direct measurement of complement proteins and fragments (e.g., Factor B, C2, C3, C4, C5; and fragments Bb, C2b, C3b, C4a, C4b, C4c, C5a, and C5b) in human aqueous humor samples Materials: Heat Aggregated Gamma Globulin (HAGG) was purchased from Quidel, preactivated zymosan and purified human complement proteins and fragments (Factor B, C3, C4, C5, fragment Bb, C3b, C4a, C4b, C4c, C5a, and C5b) were purchased from CompTech. Standard and isotopically labeled internal standard peptides were purchased from JPT peptide technologies. Tris hydrochloride was purchased from Fisher. Pierce dithiothreitol (DTT), Pierce iodoacetamide (IAA), trifluoroacetic acid, Pierce BSA protein digest, and n-Dodecyl-P-D-maltoside (DDM) were purchased from Thermo Scientific. Chymotrypsin was purchased from Promega. EDTA, CaCh, and high-performance liquid chromatography-analytical grade solvents acetonitrile, methanol, water, and formic acid were purchased from J.T. Baker. SPEC Pt C18 was purchased from Agilent. PNGase F was purchased from New England Biolabs. Banked healthy volunteer human aqueous humor samples and samples from AMD donors were purchased from BioIVT. Total protein 5 amounts for aqueous humor samples were measured using the NanoOrange manufacturer protocol. Methods In silica analysis to generate complement protein signature peptides: Complement protein sequences (Factor B, C3, C4, and C5) were obtained from 10 UniProtKB. Endogenous cleavage sites that lead to the activation of these proteins are listed in Table 1. An in silica analysis using the Expasy PeptideCutter was performed to identify the protease that would have the best coverage of the endogenous cleavage sites for the proteins of interest. (Gasteiger et al., 2005, Protein Identification and Analysis Tools on the ExPASy Server. In: WalkerJM (ed). The Proteomics Protocols 15 Handbook. Humana Press, pp. 571-607. Reference) These in silica analyses revealed, e.g., chymotrypsin as a protease capable of generating “signature peptides” for tracking proteolytic activation of the complement proteins. Table 1. Endogenous protein activation cleavage sites Protein Cleavage site Fragments Generated Factor B 260 Fragment Ba and Fragment Bb C3 667 C3a and C3b 748 C3b and C3c 1320 C3fandC3g 1303 C3d and C3f C4 756 C4b 675 C4a and C4b 956 C4c and C4d 1336 C4d and C4e C5 749 C5a and C5b Data-dependent acquisition mass spectrometry to confirm generation of the chymotryptic signature peptides upon activation: Given that chymotrypsin has the ability to cleave at several different amino acids, data-dependent acquisition (DDA) was utilized to verify the generation of chymotryptic signature peptides after complement pathway activation. Figure 4A schematically shows signature peptide prediction for the full-length and fragment proteins. The arrow at the center of overlapping peptide (o) indicates the endogenous cleavage site, while the side arrows indicate cleavages as the result of enzymatic digestion. Expasy PeptideCutter was used to determine the best digestive enzyme for Factor B, C3, C4, and C5 to produce signature peptides to monitor the full length and fragment proteins for complement activation. Chymotrypsin (FWY) was selected based on the resultant peptides that met the following criteria: coverage of cleavage site (o) for the full length protein and immediately adjacent to cleavage sites (a) and (b) for the fragment proteins. Figure 4B shows DDA of chemotrypic signature peptide sequence coverage of Factor B, B3, C4, and C5 (shadowed), and the bolded arrows indicate endogenous cleavage sites. A total mix of 10 pg of the human purified full-length proteins (Factor B, C3, C4, and C5) and the active fragments (fragment Bb, C3b, C4a, C4b, C4c, C5a, and C5b) was digested with chymotrypsin as specified below in the sample preparation method section. Peptides were purified and samples were analyzed using a Top 12 method on an Ultimate 3000 online nano-LC system coupled to a Q Exactive HF Orbitrap (ThermoFisher Scientific) operated in data dependent mode. The DDA-MS method included a full MSI scan performed in full scan positive mode, scanning 375 to 1600 m / z, with an MSI resolution of 60,000 Hz, Automatic Gain Control (AGC) target of IxlO6, and a maximum fill time of 20 ms. MS2 resolution was set at 15,000 with the AGC target of IxlO5 and a maximum fill time of 50 ms, with an isolation window of 1.5 m / z and a normalized collision energy (NCE) 30. Peptides were analyzed using an Aurora Ultimate 25 cm x 75 pm Cl8 analytical column at 50 °C at a flow rate of 400 nL / min using a gradient of 2% to 25% over 39 min, then 25% to 50% over 4 min, then 50% to 90% over 1 min. Solvents were composed of 0.1% formic acid (FA) and either 2% acetonitrile (ACN) (solvent A) or 97.9% ACN (solvent B). The loading solvent was 10% methanol. Targeted LC-MS / MS method: LC-MS / MS analysis was performed on a Sciex 6500+ QTRAP operated in multiple reaction monitoring (MRM) mode coupled with an M5 UPLC chromatography system (Sciex). Peptides were analyzed using a trap and elute system. Peptides were trapped on a Phenomenex Luna C18 column 20 x 0.3 mm, 5 pm, then chromatographically separated using a Phenomenex Kinetex C18 column 100 x 0.3 mm, 2.6 pm. The mobile phases consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The trap method was 10 minutes, starting with a hold time of 2.1 minutes at 2% MPB, followed by a linear gradient to 98% MPB from 2.1 to 6 minutes, held at 98% MPB from 6 to 7 minutes, a linear gradient to 2% MPB from 7 to 7.1 minutes, and a re-equilibration step of 2.9 minutes at 2% MPB (flow rate 60 pL / min). The trap column temperature was uncontrolled. The analytical method started 2.1 minutes after the start of the trap method. The run time for this method was 15 min starting with a hold time of 0.5 minutes at 2% MPB, followed by a linear gradient from 2% to 10% of MPB from 0.5 to 4 minutes, followed by a linear gradient to 30% MPB from 4 to 10.4 minutes, a linear gradient to 98% MPB from 10.4 to 10.5 min and held at 98% MPB for 2 minutes, a linear gradient to 1% of MPB from 12.5 to 12.6 min, and a re-equilibrium step of 2.4 min to the initial 2% MPB (flow rate 8 pL / min). The injection volume was 10 pL. The analytical column temperature was set to 40 °C. The complement peptides and isotopically-labeled internal standards were detected using MRM in positive mode, using an optiflow turbo spray ion source (Micro 1-50 pL probe) with the following parameters: curtain gas 35, collision gas high, ion spray voltage 4500 V, temperature 300, ion source gas 1 25, and ion source gas 2 30. The MS parameters including precursor mass (QI), ions monitored (Q3), retention time (RT), declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) for each peptide are listed in Table 2. The entrance potential for all peptides was 10 volts. Table 2. Signature peptides LC-MS / MS parameters for MRM method FL' / p2 Peptide Ql Q3 RT DP CE CXP B FL LSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIY 1018.5048 868, 1224, 1148, 1076, 1033 10.14 130 38.9 13 LSSLTETIEGVDAEDGHGPGEQQK-R*-KIVLDPSGSMNIY 1020.7561 868, 1227, 1151, 1079, 1036 10.14 110 46.9 13 pBa LSSLTETIEGVDAEDGHGPGEQQKR 664.0730 905, 840, 762, 705, 752 8.79 60 26.3 13 LSSLTETIEGVDAEDGHGPGEQQK-R* 666.3242 909, 845, 767, 755, 709 8.79 60 32.3 13 pBb KIVLDPSGSMNIY 718.8763 898, 1029, 1143,572, 628 9.87 70 26.5 13 FL' / p2 Peptide Ql Q3 RT DP CE CXP KIVLD-P*-SGSMNIY 721.8832 904, 1035, 1149,575, 631 9.87 80 39.5 13 C3 FL QKDAPDHQELNLDVSLQLPSRSSKITHRIHW 608.9893 754, 754, 716, 683, 753 9.33 60 22.4 30 Q-K*-DAPDHQELNLDVSLQLPSRSSKITHRIHW 610.3250 754, 754, 716, 683, 757 9.33 60 23.4 10 RSEETKENEGF 663.3020 502, 603, 731,860, 552 5.27 80 36.5 20 RSEET-K*-ENEGF 667.3091 502, 603, 739, 868, 556 5.27 80 47.5 20 pC3b TSSSGQQTAQRAELQCPQPAA 720.3430 258, 258, 867, 980, 654 7.4 60 25.6 30 TSSSGQQTAQ-R*-AELQCPQPAA 723.6791 258, 258, 872, 985, 657 7.4 60 22.6 13 pC3g SEETKENEGF 585.2515 953, 824, 346, 947, 1004 5.88 60 23.7 15 SEET-K*-ENEGF 589.2586 961,832, 346, 955, 1012 5.88 60 26.7 40 pC3d QKDAPDHQELNLDVSLQLPSR 601.5620 600, 359, 753, 802, 846 9.82 80 22.1 35 Q-K*-DAPDHQELNLDVSLQLPSR 603.5656 600, 359, 757, 806, 850 9.82 80 23.1 25 C4 FL AESLRKKSRDKGQAGLQRAL 553.8213 657,487, 714, 671, 519 5.51 80 28.7 20 AESLRKKSRD-K*-GQAGLQRAL 555.8248 657,487, 717, 674, 522 5.51 80 32.7 30 IASHTTEERGLNVTLSSTGRNGF 612.5623 825, 655, 705, 755, 812 8.45 40 22.6 10 IASHTTEE-R*-GLNVTLSSTGRNGF 615.0644 825, 660, 710, 760, 817 8.45 40 28.6 35 pC4b AESLRKKSRDKGQAGLQR 507.7910 544,473, 656,613, 522 3.63 80 26.4 20 AESLRKKSRD-K*-GQAGLQR 509.7945 544,473, 656,613, 522 3.63 80 30.4 25 pC4d IASHTTEERGLNVTLSSTGR 710.3697 973,861, 649, 620, 705 7.37 80 35.1 10 IASHTTEE-R*-GLNVTLSSTGR 713.7058 978, 866, 652, 623, 710 7.37 80 41.1 40 C2 FL GRKIQIQRSGH 427.2496 697, 584, 456,583, 563 4.05 40 24.5 20 GR-K*-IQIQRSGH 429.9210 697, 584, 456, 591, 567 4.05 40 26.5 30 FL' / p2 Peptide Ql Q3 RT DP CE CXP pC2b GRKIQIQRS 362.5561 631,503, 390, 342, 328 4.44 40 21.4 13 GR-K*-IQIQRS 365.2275 631,503, 390, 350, 330 4.44 40 22.4 25 C5 FL TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY 667.495 764, 804, 784, 740, 714 8.85 80 28.4 13 TECCVVASQL-R*- ANISHKDMQLGRLHMKTLLPVSKPEIRSY 668.925 764, 806, 786, 742, 715 8.85 80 36 10 pC5a TECCVVASQLRANISHKDMQLGR 535.466 675, 642, 619,612, 650 7.64 40 22.8 20 TECCVVASQL-R*-ANISHKDMQLGR 537.468 679, 646, 622, 614, 650 7.64 40 24.8 13 pC5b LHMKTLLPVSKPEIRSY 503.789 764, 588, 393,251, 419 7.78 60 21.2 13 LHM-K*-TLLPVSKPEIRSY 505.793 764, 588, 393,251, 423 7.78 60 27.2 13 'FL, full-length 2p, processed Aqueous humor sample preparation method: Procured healthy volunteer human aqueous humor samples (10 pL) were heat inactivated for 10 min, reduced using DTT, alkylated using IAA, and then digested overnight using chymotrypsin (1:10 enzyme: total protein ratio) at 25 °C. After digestion samples were deglycosylated using PNGase F at 37 °C for one hour. Isotopically labeled standards were spiked-in after digestion at 0.1 ng / pL (described in Table 2). Sample desalting was performed using SPEC Pt C18 stage tips and the elutes were dried down. Elutes were reconstituted using 40 pL of 0.1% formic acid + 0.005% DDM. Calibration curves were prepared from 500 ng / mL down to 0.122 ng / mL. The sample preparation method is schematically shown in Figure 5. Data analysis / quantification: For the DDA-MS data analysis, files were searched using Thermo Proteome Discoverer 3.0.0.757. For the targeted MRM method, complement protein / fragment levels were calculated using Skyline to first extract chromatographic peak areas for each peptide proteoform which was then normalized to the sum of the peak areas of all forms of that peptide. The lower limit of quantification (LLOQ) was defined as the lowest concentration of analyte that could be measured with a 10 signal / noise ratio peak area. The peptide concentration was calculated using the area under the curve and calibration curve for each peptide. Method reproducibility and robustness: Data-dependent acquisition (Thermo Q Exactive HF) was utilized to select chymotryptic signature peptides that would allow tracking of both full-length proteins and active cleavage products. After signature peptides were selected, an MRM multiplexed method was developed that 5 enabled the chromatographic separation of all peptides as shown in Figure 6. 13-point calibration curves ranging from 0.1-500 ng / mL were used to assess linearity and determine the LLOQ for all peptides in Table 3. For all peptides, the linearity was R2>0.99 and LLOQ (ng / mL) <3.9 ng / mL. Table 3. LLOQ of peptide standards in buffer FL7p2 Peptide LLOQ R2 B FL LSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIY 3.91 0.9967 pBa LSSLTETIEGVDAEDGHGPGEQQKR 0.49 0.9982 pBb KIVLDPSGSMNIY 0.49 0.9907 C3 FL RSEETKENEGF 0.24 0.9989 pC3g SEETKENEGF 0.49 0.9998 pC3b TSSSGQQTAQRAELQCPQPAA 0.24 0.9997 C4 FL AESLRKKSRDKGQAGLQRAL 0.24 0.9997 pC4b AESLRKKSRDKGQAGLQR 0.12 0.9996 FL ELNPLDHRGRTLEIPGNSDPNMIPDGDFNSY 0.49 0.9945 pC4c ELNPLDHRGR 0.49 0.9998 FL SCPKEKTTRKKRNVNF 0.98 0.9999 C5 FL TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY 3.91 0.9986 FL RANISHKDMQLGRL n / a n / a pC5a TECCVVASQLRANISHKDMQLGR 0.122 0.9971 pC5a RANISHKDMQLGR pC5b LHMKTLLPVSKPEIRSY 0.122 0.9956 10            ’FL, full-length 2p, processed In relation to C5, Table 3 contains sequence of signature peptide for the fulllength protein (TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY) identified in silico. Additional enzymatic cleavages (PNGase F) results in a shorter overlapping 15   peptide (RANISHKDMQLGRL) than was originally identified in silico. Results DDA mode was utilized to confirm the generation of chymotryptic signature peptides for each complement protein and fragment, enabling tracking of both full length proteins and active cleavage products. The results showed that not all in silica predicted peptides were generated in vitro and occasionally additional cleavage was generated. For instance, the predicted C5 overlapping peptide (TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY) compared to the observed peptide (RANISHKDMQLGRL), revealed an additional cleavage between Leu (L) and Arg (R). This untargeted analysis also confirmed the N-glycosylation site in the C5 overlapping peptide (RANISHKDMQLGRL), leading to developing a deglycosylation step in the sample preparation protocol to ensure detection. To detect the C5 overlapping peptide the DDA data was analyzed to identify alternative chymotryptic peptides that could be used to determine total amounts of C5. Through this analysis, 8 chymotryptic peptides (5 in the alpha chain and 3 in the beta chain) were identified that could report total amounts of C5. The presence of these peptides was tested in 10 aqueous humor samples and determined that the peptides VYLEVVSKHF (beta chain) and SGVTLDPRGIY (alpha chain) were the most readily detected across samples. These peptides were included in the MRM method. Example complement activation assay: To measure identified peptides in biologically relevant samples, complement activator reagents that activate both the classical and alternative pathways (HAGG and zymosan, respectively) were used on aqueous humor collected from n=3 healthy volunteers. After inducing the alternative complement activation pathway using zymosan, a -44% decrease in Factor B and an increase of -322% in fragment Ba were observed when comparing baseline to 60 minutes post-treatment (fragment Ba / factor B ratios of 0.14 vs 1.1 respectively), see Figure 7A. A -43% decrease in C3 and an increase of -90% in C3g were observed when comparing baseline to 60 minutes (C3g / C3 ratios of 0.93 vs 3.1 respectively). No C4 activation was observed in this alternative pathway activation. When the classical complement pathway was activated using HAGG, a -47% decrease in C4 and an increase of -88% in C4c were observed when comparing baseline to 60 minutes post-treatment (C4c / C4 ratios 0.11 vs 0.38 respectively), see Figure 7B. A -69% decrease in C3, and an increase of-170% in C3g (C3g / C3 ratios of 0.93 vs 7.9 respectively) were observed when comparing baseline to 60 minutes post-treatment, see Figure 7B. Brief summary of the example: The present example provides of a multiplexed LC-MS / MS method to track complement activation by measuring three complement proteins and their active fragments simultaneously. Data-dependent acquisition (Thermo Q Exactive HF) was utilized to select chymotryptic signature peptides that allowed tracking of both full-length proteins and active cleavage products. After signature peptides were selected, an MRM multiplexed method was developed (QTRAP Sciex 6500+). 13-point calibration curves ranging from 0.1-500 ng / mL were used to assess linearity and determine the LOD and LLOQ. For all analytes, the linearity was R2>0.99 and LLOQ <39 ng / mL. Inter- and intra-day reproducibility, repeatability, and stability were also assessed. Quantifiable levels of CFB, FBa, C3, C3g, C4, and C4c were detected in aqueous humor samples (n=5) with concentrations ranging from 107-1 ng / mL. After inducing the alternative complement activation pathway in aqueous humor samples (n=3) using zymosan, a -44% decrease in CFB and an increase of -322% in FBa was observed when comparing t=0 vs t=60 min (FBa / CFB ratios of 0.14 vs 1.1 respectively). A -43% decrease in C3, and an increase of -90% in C3g were observed when comparing t=0 vs t=60 min (C3g / C3 ratios of 0.93 vs 3.1 respectively). As expected, no C4 activation was observed in this activation. Similarly, when activating the classical complement pathway in aqueous humor samples (n=3) using HAGG, a -47% decrease in C4 and an increase of -88% in C4c were observed (C4c / C4 ratios 0.11 vs 0.38 respectively). A -69% decrease in C3 and an increase of -170% in C3g (C3g / C3 ratios of 0.93 vs 7.9 respectively) were also observed when comparing t=0 vs t=60 min. Collectively, these results demonstrate this multiplexed method enables the measurement of complement activation in human aqueous humor samples. This assay the assay can be used, e.g., to diagnose and / monitor treatment of ocular diseases like age-related macular degeneration and geographic atrophy and to aid clinical development in these and other ocular indications. OTHER EMBODIMENTS It is to be understood that while the present application has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present application, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A peptide selected from any one of the following sequences:IIGGSDADIK Active-Cis LLEVPEGR Total Cis LQVIFK Total Cis AARLPVAPLRK Total Cis YQSVFTVAR Total Clq LEQGENVFLQATDK Total Clq VVTFCGHTSK Total Clq LSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIY B LSSLTETIEGVDAEDGHGPGEQQKR Ba KIVLDPSGSMNIY Bb QKDAPDHQELNLDVSLQLPSRSSKITHRIHW C3 RSEETKENEGF C3 TSSSGQQTAQRAELQCPQPAA C3b SEETKENEGF C3g QKDAPDHQELNLDVSLQLPSR C3d AESLRKKSRDKGQAGLQRAL C4 IASHTTEERGLNVTLSSTGRNGF C4 AESLRKKSRDKGQAGLQR C4b IASHTTEERGLNVTLSSTGR C4d GRKIQIQRSGH C2 GRKIQIQRS C2b TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY C5 TECCVVASQLRANISHKDMQLGR C5a LHMKTLLPVSKPEIRSY C5bor a salt thereof.

2. The peptide of claim 1, selected from any one of the following sequences:LLEVPEGR Cls-total LQVIFK Cls-total AARLPVAPLRK Cls-totalor a salt thereof.

3. The peptide of claim 1, having the following sequence:IIGGSDADIK Cls-activeor a salt thereof.

4. The peptide of claim 1, having the following sequence:LSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIY Bor a salt thereof.

5. The peptide of claim 1, selected from any one of the following sequences:LSSLTETIEGVDAEDGHGPGEQQKR Ba KIVLDPSGSMNIY Bbor a salt thereof.

6. The peptide of claim 1, having the following sequence:GRKIQIQRSGH C2or a salt thereof.

7. The peptide of claim 1, having the following sequence:GRKIQIQRS C2bor a salt thereof.

8. The peptide of claim 1, selected from any one of the following sequences:QKDAPDHQELNLDVSLQLPSRSSKITHRIHW C3 RSEETKENEGF C3or a salt thereof.

9. The peptide of claim 1, selected from any one of the following sequences:TSSSGQQTAQRAELQCPQPAA C3b SEETKENEGF C3g QKDAPDHQELNLDVSLQLPSR C3dor a salt thereof.

10. The peptide of claim 1, peptide selected from any one of the following sequences:AESLRKKSRDKGQAGLQRAL C4 IASHTTEERGLNVTLSSTGRNGF C4or a salt thereof.

11. The peptide of claim 1, selected from any one of the following sequences:AESLRKKSRDKGQAGLQR C4b IASHTTEERGLNVTLSSTGR C4dor a salt thereof.

12. The peptide of claim 1, selected from any one of the following sequences:TECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSY C5or a salt thereof.

13. The peptide of claim 1, selected from any one of the following sequences:TECCVVASQLRANISHKDMQLGR C5a LHMKTLLPVSKPEIRSY C5bor a salt thereof.

14. A method of diagnosing a complement associated disorder in a subject, the method comprising:determining (i) a first amount of at least one peptide of claim 3 and (ii) a second amount of at least one peptide of claim 2 in a processed sample obtained by treating a sample obtained from the subject with a protease;wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder.

15. A method of diagnosing a complement associated disorder in a subject, the method comprising:determining (i) a first amount of at least one peptide of claim 5 and (ii) a second amount of at least one peptide of claim 4 in a processed sample obtained by treating a sample obtained from the subject with a protease;wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder.

16. A method of diagnosing a complement associated disorder in a subject, the method comprising:determining (i) a first amount of at least one peptide of claim 7 and (ii) a second amount of at least one peptide of claim 6 in a processed sample obtained by treating a sample obtained from the subject with a protease;wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder.

17. A method of diagnosing a complement associated disorder in a subject, the method comprising:determining (i) a first amount of at least one peptide of claim 9 and (ii) a second amount of at least one peptide of claim 8 in a processed sample obtained by treating a sample obtained from the subject with a protease;wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder.

18. A method of diagnosing a complement associated disorder in a subject, the method comprising:determining (i) a first amount of at least one peptide of claim 11 and (ii) a second amount of at least one peptide of claim 10 in a processed sample obtained by treating a sample obtained from the subject with a protease;wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder.

19. A method of diagnosing a complement associated disorder in a subject, the method comprising:determining (i) a first amount of at least one peptide of claim 13 and (ii) a second amount of at least one peptide of claim 12 in a processed sample obtained by treating a sample obtained from the subject with a protease;wherein an increase in the ratio of the first amount to the second amount in the processed sample compared to a reference ratio of the peptides is indicative of the subject having the complement-associated disorder.

20. The method of any one of claims 14-19, wherein the increase is about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 2 fold, about 3 fold, about 5 fold, about 7 fold, or about 10 fold.

21. The method of any one of claims 14-20, wherein the sample obtained from the subject is selected from blood, plasma, serum, cerebrospinal fluid (CSF), aqueous humor, vitreous humor, brain homogenate, and eye homogenate.

22. The method of any one of claims 14-21, wherein the protease is selected from trypsin, chymotrypsin, lysC, and gluC, or a combination thereof.

23. The method of any one of claims 14-22, wherein the determining is carried out using a mass spectrometry technique.

24. The method of any one of claims 14-23, wherein the determining is carried out using an immunoassay.

25. The method of any one of claims 14-23, wherein the reference amount is an amount of the corresponding peptide determined in a processed sample derived from a healthy subject.

26. The method of any one of claims 14-25, wherein the complement-associated disorder is ophthalmic disease or disorder.T1. The method of claim 26, wherein the ophthalmic disorder is age-related macular degeneration (AMD), geographic atrophy (GA), retinopathy, uveitis, glaucoma, Stargardt disease, retinal occlusive vasculitis, intraocular neovascular syndrome, corneal inflammation (e.g., acute or chronic), intraocular inflammation (e.g., acute or chronic), or choroidal neovascularization (CNV), retinal vein occlusion, dry eye syndrome, age-related maculopathy (e.g., early to intermediate dry AMD in which GA has not developed), posterior keratitis (e.g., unilateral posterior interstitial keratitis), or non-ulcerating inflammation of the corneal stroma.

28. The method of any one of claims 14-25, wherein the complement-associated disorder is neurodegenerative disorder.

29. The method of claim 28, wherein the neurodegenerative disorder is selected from Alzheimer’s disease, schizophrenia, frontotemporal dementia, Huntington’s disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome (GBS), Neuromyelitis Optica (NMO), Myasthenia gravis (MG), Multifocal Motor Neuropathy (MMN), Cold agglutinin disease (CAD), and Immune thrombocytopenic purpura (ITP).

30. The method of any one of claims 14-25, wherein the disorder is paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (MG), or neuromyelitis optical spectrum disorder (NMOSD).

31. The method of any one of claims 14-30, wherein the method further comprises a step of administering to the subject a therapeutic agent in an effective amount to treat the complement-associated disorder.

32. The method of claim 31, wherein the therapeutic agent is an inhibitor of complement selected from cinryze, berinert, ruconest, sutimlimab, pegcetacoplan,eculizumab, ravulizumab, avacincaptad, pozelimab, zilucoplan, iptacopan, danicopan, and avacopan.

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