DETECTION OF COMPLEMENT PROTEINS

Abstract

Methods for detecting and determining the level of complement proteins are disclosed, in particular using mass spectrometry. Also disclosed are methods of identifying subjects having or at risk of developing a complement-related disorder, and methods of treating the same.

Claims

1. A method for determining the level of at least one complement protein in a sample, the method comprising: digesting the protein(s) with endoproteinase GluC to obtain one or more peptides; and determining the level of the one or more peptides by mass spectrometry.

2.-3. (canceled)

4. A method according to claim 1, wherein the method comprises determining the concentration of the complement protein(s) in the sample.

5. (canceled)

6. A method according to claim 1, wherein the complement protein(s) is one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5.

7. (canceled)

8. A method according to claim 1, wherein the complement protein(s) is involved in the complement amplification loop and/or C3 convertase activity.

9. A method according to claim 1, wherein the complement protein(s) is a breakdown product of C3b.

10. A method according to claim 1, wherein the complement protein(s) is one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.

11. (canceled)

12. A method according to claim 1, wherein the complement protein is FI.

13. A method according to claim 1, wherein the sample has been obtained from a subject, optionally wherein the sample comprises, or is derived from, blood, lymph, plasma, serum, tissue, or cells.

14. A method according to claim 1, wherein the method comprises a step of obtaining the sample from a subject, optionally wherein the sample comprises, or is derived from, blood, lymph, plasma, serum, tissue, or cells.

15. (canceled)

16. A method according to claim 1, wherein the one or more peptides are selected from the group consisting of: TABLE-US-00011 (a) (SEQ ID NO: 20) VTYKCFE; (b) (SEQ ID NO: 21) NGWSPTPRCIRVSFTL; (c) (SEQ ID NO: 22) ATFCDFPKINHGILYDEE; (d) (SEQ ID NO: 23) RGWSTPPKCRSTISAE or (SEQ ID NO: 24) AMFCDFPKINHGILYDEE; (e) (SEQ ID NO: 25) VACHPGYGLPKAQTTVTCTE; (f) (SEQ ID NO: 26) YQCQSYYE; (g) (SEQ ID NO: 27) RGWSTPPICSFTKGE; (h) (SEQ ID NO: 28) GTAFVIFGIQDGE; (i) (SEQ ID NO: 29) LRRQHARASHLGLARSNLDE; (j) (SEQ ID NO: 30) LRRQHARASHLGLAR and/or (SEQ ID NO: 156) LRRQHARASHLGLA; (k) (SEQ ID NO: 31) LNLDVSLQLPSRSSKITHRIHWE; (l) (SEQ ID NO: 32) LNLDVSLQLPSR; (m) (SEQ ID NO: 33) RLGRE; (n) (SEQ ID NO: 34) SSKITHRIHWE; (o) (SEQ ID NO: 35) SASLLR and/or (SEQ ID NO: 157) SASLL; (p) (SEQ ID NO: 36) RLGR; (q) (SEQ ID NO: 37) HLIVTPSGCGE; and/or (r) any one or more of SEQ ID NO: 38 to 60.

17.-18. (canceled)

19. A method of treating a complement-related disorder in a subject, the method comprising: (a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; (b) determining the presence and/or level of the one or more peptides by mass spectrometry; (c) using the results of (b) to determine whether the subject has an increase in the level of one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, and/or FHR5; and/or a decrease in the level of one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5, as compared to reference value(s) obtained previously from the same subject or obtained from a subject without a complement-related disorder; and (d) treating the subject with a therapeutically effective amount of a complement-targeted therapeutic.

20.-21. (canceled)

22. A method of treating a complement-related disorder in a subject, the method comprising: (a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; (b) determining the presence and level of the one or more peptides by mass spectrometry, wherein the subject has an increase in the level of one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, and/or FHR5; and/or a decrease in the level of one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5, as compared to reference value(s) obtained previously from the same subject or obtained from a subject without a complement related disorder; and based on the results of (b), administering a therapeutically effective amount of a complement-targeted therapeutic.

23. A method according to claim 22, wherein the method comprises obtaining a sample from a subject comprising at least one complement protein, optionally wherein the sample comprises or is derived from blood, lymph, plasma, serum, tissue, or cells.

24. A method according to claim 22, wherein the complement-related disorder is selected from macular degeneration, age related macular degeneration (AMD), geographic atrophy (‘dry’ (i.e. non-exudative) AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late/advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, Haemolytic Uremic Syndrome (HUS), atypical Haemolytic Uremic Syndrome (aHUS), DEAP HUS (Deficiency of FHR plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome), autoimmune uveitis, Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, Henoch-Schonlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosis (SLE), Sjogren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3 NF GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, neurodegeneration/neurodegenerative disease, dementia, multiple sclerosis (MS), cancer, stroke, Parkinson's disease, and/or Alzheimer's disease.

25-26. (canceled)

27. The method according to claim 22, wherein the subject has an increase in the level of one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, and/or FHR5 as compared to reference value(s) obtained previously from the same subject or obtained from a subject without a complement related disorder, and the complement targeted therapeutic is an agent that reduces the level of the one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, and/or FHR5 that are increased as compared to the reference value(s).

28. The method according to claim 22, wherein the subject has a decrease in the level of one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5 as compared to reference value(s) obtained previously from the same subject or obtained from a subject without a complement related disorder, and the complement targeted therapeutic is an agent that increases the level of the one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5 that are decreased as compared to the reference value(s).

29. The method according to claim 19, wherein the method comprises obtaining a sample from a subject comprising at least one complement protein, optionally wherein the sample comprises or is derived from blood, lymph, plasma, serum, tissue, or cells.

30. The method according to claim 19, wherein the subject has or is determined to have a complement-related disorder selected from macular degeneration, age related macular degeneration (AMD), geographic atrophy (‘dry’ (i.e. non-exudative) AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late/advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, Haemolytic Uremic Syndrome (HUS), atypical Haemolytic Uremic Syndrome (aHUS), DEAP HUS (Deficiency of FHR plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome), autoimmune uveitis, Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosis (SLE), Sjogren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3 NF GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, neurodegeneration/neurodegenerative disease, dementia, multiple sclerosis (MS), cancer, stroke, Parkinson's disease, and/or Alzheimer's disease.

31. The method according to claim 19, wherein the subject has an increase in the level of one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, and/or FHR5 as compared to reference value(s) obtained previously from the same subject or obtained from a subject without a complement related disorder, and the complement-targeted therapeutic is an agent that reduces the level of the one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, and/or FHR5 that are increased as compared to the reference value(s).

32. The method according to claim 19, wherein the subject has a decrease in the level of one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5 as compared to reference value(s) obtained previously from the same subject or obtained from a subject without a complement related disorder, and the complement-targeted therapeutic is an agent that increases the level of the one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and/or FHR5 that are decreased as compared to the reference value(s).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0311] FIG. 1. Schematic showing the C3 proteolytic cascade and the proteolytic events leading to the generation, breakdown and inactivation of C3b (modified from Maillard et al, J Am Soc Nephrol. 2015 Jul;26(7):1503-12). Proteoform-specific peptides for mass spectrometry are underlined.

[0312] FIG. 2. LC-SRM Trace showing detection of the heavy-labelled synthetic standard peptides of each individual RCA locus protein from a plasma sample.

[0313] FIG. 3. Linearity data for peptides derived from FH, FHL-1, and FHR1-5.

[0314] FIGS. 4A to 4D. Data confirming that C3 and C3 breakdown products in human plasma can be detected by MS with sufficient specificity and sensitivity. 4A: Total ion chromatograph from SRM-MS analysis showing specific and simultaneous detection of C3b fragment-specific peptides. 4B: Linearity data for seven of the ten peptides spiked into a plasma background. 4C: Coomassie-stained electrophoresis gel of C3 breakdown products obtained in vitro. 4D: MS quantification of key C3 fragments from the in vitro assay products shown in 4C.

EXAMPLES

Example 1: Generation of Peptides from Complement Proteins for Mass Spectrometry

[0315] GluC digestion was performed on FH, FHL-1, FHR1-5, FI, C3, C3b and C3b breakdown products to achieve distinct peptides for mass spectrometry. GluC digestion is described in Example 2.2.

[0316] Peptides that can be used to detect each protein or protein fragment are set out in Tables 1-4 below.

TABLE-US-00003 TABLE 1 Distinct FH family peptides after GluC digestion. SEQ ID Protein Peptide Sequence Mass No: Factor H VTYKCFE  888.4051 20 FHL1 NGWSPTPRCIRVSFTL 1832.9355 21 FHR1 ATFCDFPKINHGILYDEE 2110.9669 22 FHR2 RGWSTPPKCRSTISAE 1774.8784 23 and AMFCDFPKINHGILYDEE 2140.9598 24 FHR3 VACHPGYGLPKAQTTVTCTE 2074.9816 25 FHR4 YQCQSYYE 1082.4015 26 FHR5 RGWSTPPICSFTKGE 1664.7980 27

[0317] The series of proteolytic events leading to the generation, breakdown and inactivation of C3 are shown in FIG. 1. Proteoform-specific peptides produced by GluC digestion are underlined in FIG. 1 and are shown in Table 2. Table 3 shows how each protein can be detected individually using the peptides in Table 2.

TABLE-US-00004 TABLE 2 Peptide sequences for MS resulting from GluC digestion of C3, C3b and breakdown products. SEQ Contained in ID Peptide Peptide sequence C3b products No: C3.1 GTAFVIFGIQDGE C3 + C3b + 28 iC3b + C3c C3.2 LRRQHARASHLGLAR C3 only 29 SNLDE C3.3 LRRQHARASHLGLAR C3a only 30 C3.4 LNLDVSLQLPSRSSKI C3 + C3b 31 THRIHWE C3.5 LNLDVSLQLPSR iC3b + C3dg + C3d 32 C3.6 RLGRE C3 + C3b + iC3b 33 C3.7 SSKITHRIHWE C3f 34 C3.8 SASLLR C3f 35 C3.9 RLGR C3c 36 C3.10 HLIVTPSGCGE C3d 37

TABLE-US-00005 TABLE 3 Methodology for determining concentration of all C3/C3b breakdown products using GluC digestion peptides of Table 2. Protein or fragment Peptide(s) C3 (total) C3.1 C3 only C3.2 C3a C3.3 C3b C3.4 − C3.2 iC3b C3.6 − C3.4 C3f C3.7 or C3.8 C3c C3.9 C3dg C3.5 − C3.10 − iC3b [C3.6 − C3.4] C3d C3.10

TABLE-US-00006 TABLE 4 Alternative peptides for C3.1 resulting from GluC digestion, to measure total C3 content. SED ID Peptide Mass Position Peptide sequence No. C3.1.1 6957.8210 463-523 AKIRYYTYLIMNKGRLLKAGRQVREPGQDLV 61 VLPLSITTDFIPSFRLVAYYTLIGASGQRE C3.1.2 5778.9734 321-372 RSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMV 62 FVTNPDGSPAYRVPVAVQGE C3.1.3 5650.1364  97-146 KVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFT 63 VNHKLLPVGRTVMVNIE C3.1.4 3904.1017 373-408 DTVQSLTQGDGVAKLSINTHPSQKPLSITVRT 64 KKQE C3.1.5 3861.1628 565-599 GDHGARVVLVAVDKGVFVLNKKNKLTQSKI 65 WDVVE C3.1.6 3807.7645 600-637 KADIGCTPGSGKDYAGVFSDAGLTFTSSSG 66 QQTAQRAE C3.1.7 3263.6357 414-442 QATRTMQALPYSTVGNSNNYLHLSVLRTE 67 C3.1.8 2845.4609 24-50 AHDAQGDVPVTVTVHDFPGKKLVLSSE 68 C3.1.9 2819.4705 150-175 GIPVKQDSLSSQNQLGVLPLSWDIPE 69 C3.1.10 2691.3425 524-549 VVADSVWVDVKDSCVGSLVVKSGQSE 70 C3.1.11 2618.3261 296-320 DLVGKSLYVSATVILHSGSDMVQAE 71 C3.1.12 2511.2904 51-73 KTVLTPATNHMGNVTFTIPANRE 72 C3.1.13 2108.1378 78-96 KGRNKFVTVQATFGTQVVE 73 C3.1.14 1992.1480 278-295 VVLSRKVLLDGVQNPRAE 74 C3.1.15 1827.9414 448-462 TLNVNFLLRMDRAHE 75 C3.1.16 1769.8923 176-189 LVNMGQWKIRAYYE 76 C3.1.17 1745.9386  1-15 SPMYSIITPNILRLE 77 C3.1.18 1738.9036 550-564 DRQPVPGQQMTLKIE 78 C3.1.19 1623.9348 231-244 VTITARFLYGKKVE 79 C3.1.20 1352.6612 245-257 GTAFVIFGIQDGE 80 C3.1.21 1138.5335 219-226 KFYYIYNE 81 C3.1.22 1135.5146 190-199 NSPQQVFSTE 82 C3.1.23 954.5862 265-272 SLKRIPIE 83 C3.1.24 853.4221 205-211 YVLPSFE 84 C3.1.25 841.4657 258-264 QRISLPE 85 C3.1.26 826.4007 638-645 LQCPQPAA 86 C3.1.27 785.4171 212-218 VIVEPTE 87 C3.1.28 591.2938 19-23 TMVLE 88 C3.1.29 570.3125 443-447 LRPGE 89 C3.1.30 509.2485 74-77 FKSE 90

[0318] GluC digestion of Factor I (FI) produced the candidate peptides in Table 5 for MS analysis. SEQ ID NO:45 to 56 and 155 contain 8-21 amino acids and are a good length for MS analysis.

TABLE-US-00007 TABLE 5 Peptide sequences resulting from GluC digestion of Fl. SEQ Peptide Mass Position Peptide sequence ID NO: 1 3996.7183 510-548 CAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGVVS 38 WGE 2 3994.8853 57-92 GTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLE 39 3 3467.7211 555-583 FPGVYTKVANYFDWISYHVGRPFISQYNV 40 4 3397.6804 150-180 ANVACLDLGFQQGADTQRRFKLSDLSINSTE 41 5 3388.6605 446-475 LPRSIPACVPWSPYLFQPNDTCIVSGWGRE 42 6 3155.5773 31-56 KKCLAKKYTHLSCDKVFCQPWQRCIE 43 7 2991.2861 254-281 LCCKACQGKGFHCKSGVCIPSQYQCNGE 44 8 2531.2455 129-149 VKLVDQDKTMFICKSSWSMRE 45 9 2068.0320 489-505 VKLISNCSKFYGNRFYE 46 10 1805.8309  93-109 CLHPGTKFLNNGTCTAE 47 11 1698.7969 420-434 NYNAGTYQNDIALIE 48 12 1605.7867 110-124 GKFSVSLKHGNTDSE 49 13 1297.5729 291-303 VGCAGFASVTQEE 50 14 1168.272 291-302 VGCAGFASVTQE 155 15 1295.6082 435-445 MKKDGNKKDCE 51 16 1191.6156 411-419 YVDRIIFHE 52 17 1166.5557 181-190 CLHVHCRGLE 53 18 1121.5738 480-488 RVFSLQWGE 54 19 978.4448 306-314 ILTADMDAE 55 20 955.4731 19-26 KVTYTSQE 56 21 736.3182 282-288 VDCITGE 57 22 647.2817 549-554 NCGKPE 58 23 520.2613 191-195 TSLAE 59 24 505.2252 476-479 KDNE 60

Example 2: Mass Spectrometry

2.1 Preparation of Stable Isotopic Standards (SIS) Spiking Solution

[0319] High purity heavy-labelled synthetic standards, with S-carboxymethylated (CAM) cysteine residues, were obtained (Cambridge Research Biochemicals, Cambridge, UK) and diluted to 1 μg/μL with 50:50 acetonitrile:water +0.1% formic acid (Table 6).

[0320] A mixed SIS solution was prepared by firstly diluting stock solution of FHL-1, FHR1, FHR2, FHR3, FHR4 and FHR5 by tenfold (no dilution of CFH stock was required), then adding the appropriate amounts of each individual diluted solution to a final volume of 200 μL in 0.1% TFA. This was then stored at −80° C. in 5 μL aliquots for further dilution immediately prior to spiking.

[0321] Spiking solution was prepared immediately prior to sample addition by adding 195 μL 50:50 acetonitrile:water to a 5 μL aliquot of the mixed SIS solution. 2 μL of this was carefully added to each digested sample prior to drying down.

TABLE-US-00008 TABLE 6 Stock solutions of stable isotopic standards (SIS) at 1 μg/MI. The residues in bold were chosen to carry a stable-heavy isotype to enable quantitation. Lower case ‘c’ denotes a S-carboxymethylated (CAM) cysteine residue. Residue in bold type contained an isotopically heavy amino acid, where K(+8), R(+10), F(+10) and Y(+10). Net Solvent Conc, Peptide MW, Purity, Content, Volume, pmol/ Protein Sequence g % % μL μL CFH VTYKcFE  953.4  98.6 72.6 716 1050 FHL-1 NGWSPTPRc 1900  97.4 78.9 768  526.3 IRVSFTL FHR1 ATFcDFPKI 2178 100.0 80.7 807  459.1 NHGILYDEE FHR2 RGWSTPPKE 1845.9 100.0 55.5 555  541.7 RSTISAE FHR3 VAcHPGYGL 2207.1  98.4 75.0 738  453.1 PKAQTTVT CTE FHR4 YQcQSYYE 1149.7  97.6 84.1 821  869.8 FHR5 RGWSTPPI 1739.8  95.6 74.0 707  574.8 CSFTKGE

2.2 Preparation of Samples for Analysis by LC-MS/MS

[0322] Frozen plasma samples were allowed to thaw to room temperature before being vortexed hard for 5 minutes to dissolve any soluble material, then centrifuged at 13,300 g for 30 min to settle any insoluble material.

[0323] To a 5 μL plasma aliquot (equivalent to approximately 350 μL protein), 90 μL of 50 mM ammonium bicarbonate (pH 7.8), 2 μL of ProteaseMAX™ (Promega, Southampton, UK) solution (1% w/v in 50 mM ammonium bicarbonate) and 1 μL of 500 mM dithiothreitol prepared in 50 mM ammonium bicarbonate was added. This was vortexed briefly to mix, then given a pulse spin before incubating at 56° C. for 25 min.

[0324] After cooling to room temperature, 3 μL 500 mM iodoacetamide (prepared in 50 mM ammonium bicarbonate) was added. This was vortexed briefly to mix, then given a pulse spin before incubating at room temperature and in the dark for 15 min.

[0325] A further 1 μL of ProteaseMAX solution (1% w/v in 50 mM ammonium bicarbonate) and 5 μL of 1 μg/uL endoproteinase GluC (Roche, Mannheim, Germany) were added. The mixture was vortexed briefly, then given a pulse spin before incubating for 16 hours at 25° C. with slight shaking (400 rpm).

[0326] To the digested peptide mix obtained, 6 μL 10% v/v trifluoroacetic acid (TFA) and 2 μL of SIS spiking solution were added, vortexed briefly to mix, then pulse spin. The solution was placed into an evaporator and dried. Finally the peptides were reconstituted in 50 μL 0.1% TFA and vortexed to dissolve any residue before centrifuging at 13,300 g for 30 min to settle any insoluble/particulate material. Approximately 48 μL (taking care to leave behind any precipitated material) was transferred to a LC autosampler vial for subsequent analysis by LC-MS/MS.

2.3 LC-SRM/MS Analysis of Plasma Digests

[0327] SRM analyses of plasma digests were performed on a 6495 triple quadrupole mass spectrometer with iFunnel-equipped electrospray ion source (Agilent, Santa Clara, CA, USA) coupled to an Infinity 1200 Series liquid chromatography system consisting of 1290 autosampler, 1260 Quat Pump VL pump and TCC column oven modules (Agilent, Santa Clara, CA, USA). Samples were injected directly (4 μL) onto a C18 column (250 mm×2.1 mm i.d., Thermo Scientific Acclaim 120, 3 μm particle size) that was maintained at a column temperature of 50° C. Compounds were developed using a gradient elution of increasing acetonitrile concentration with Buffer A consisting of Water +0.1% formic acid and Buffer B being Acetonitrile +0.1% formic acid. The flow rate was maintained at 250 μL/min with an initial composition of 5% Buffer B.

[0328] The following gradient elution profile was used to separate the peptides (time: % B): 0 min: 5% B; 2 min: 5% B; 3 min: 12% B; 12 min: 15% B; 15 min: 20% B; 30 min: 25% B; 31 min: 90% B; 39 min: 90% B; 40 min: 5% B; 49 min: 5% B.

[0329] Optimized SRM settings were determined using SIS solutions and are provided in Table 7.

TABLE-US-00009 TABLE 7 SRM transitions and optimal collision energies for FH family peptides (Quantitation ions in bold). Precursor Product Collision Peptide ion ions energy, Protein Sequence m/z m/z eV CFH VTYKcFE 473.7 583.3, 16, 16, (Light) 847.4, 16 746.3 CFH VTYKcFE 477.7 591.3, 16, 16, (Heavy) 855.4, 16 754.3 FHL-1 NGWSPTPR 631.2 723.9, 19, 19, CIRVSFTL 860.5, 19 (Light) 767.4 FHL-1 NGWSPTPR 634.3 728.9, 19, 19, cIRVSFTL 865.5, 19 (Heavy) 772.4 FHR1 ATFcDFPK 724.2 925.6, 20, 16, INHGILYD 1011.9, 20 EE (Light) 947.1 FHR1 ATFcDFPK 727.2 930.6, 20, 16, INHGILYD 1016.9, 20 EE (Heavy) 952.1 FHR2 RGWSTPPK 459.1, 539.2,  8, 26 ERSTISAE 611.8 798.9 (Light) FHR2 RGWSTPPK 462.6, 543.9,  8, 26 cRSTISAE 616.3 806.0 (Heavy) FHR3 VACHPGYG 730.7 1022.4, 16, 18 LPKAQTTV 971.7 TcTE (Light) FHR3 VACHPGYG 736.7 1031.4, 16, 18 LPKAQTTV 980.7 TCTE (Heavy) FHR4 YQcQSYYE 570.7 830.3, 11, 10, (Light) 993.1, 14 311.1 FHR4 YQcQSYYE 575.7 840.3, 11, 10, (Heavy) 1003.1, 14 311.1 FHR5 RGWSTPPI 575.2 828.4, 16, 15, CSFTKGE 895.5, 20 (Light) 588.3 FHR5 RGWSTPPI 581.2 836.4, 16, 15, CSFTKGE 905.5, 20 (Heavy) 598.3

TABLE-US-00010 TABLE 8 Peptides and transitions for quantitation of C3/C3b breakdown products. Se- Contained Peptide quence in . . . Transitions C3.1 GTAFVIF C3 + C3b + 569.0/483.2 GIQDGE iC3b + C3c 569.0/654.3 569.0/801.4 C3.2 LRRQHAR C3 only 384.2/431.6 ASHLGLA 384.2/510.4 RSNLDE 461.0/503.3 C3.3 LRRQHARA C3a only 291.2/304.3 SHLGLAR 291.2/395.8 436.3/598.7 C3.4 LNLDVSL C3 + C3b 546.8/578.7 QLPSRSS 683.2/797.1 KITHRIH 683.2/834.9 WE C3.5 LNLDVSL iC3b + C3dg + 677.9/1014.4 QLPSR C3d 677.9/359.1 677.9/800.2 C3.6 RLGRE C3 + C3b + 251.3/175.1 iC3b 251.3/232.1 251.3/345.2 C3.7 SSKITHRI C3f 349.3/354.2 HWE 349.3/436.4 349.3/489.8 C3.8 SASLLR C3f 323.7/159.1 323.7/288.2 323.7/488.3 C3.9 RLGR C3c 251.3/175.1 251.3/232.1 251.3/345.2 C3.10 HLIVTPSG C3d 585.3/251.1 CGE 585.3/394.5 585.3/404

[0330] In order to protect the source region from unwanted contaminants, a switching valve located between the column and source was diverted to the waste position at points in the chromatogram when the analyte peptides were not eluting. This allowed for six windows (two of the peptides, FHR-2 and FHL-1, eluted within the same window) of acquisition, of approximately one minute each, to be acquired with the column on-line to the mass spectrometer.

2.4 Results

FH Family Proteins

[0331] FIG. 2 shows a LC-SRM Trace showing detection of the heavy-labelled synthetic standards of each individual RCA locus protein from a plasma sample. This demonstrates that the method is feasible, specific and has the required sensitivity to distinguish between peptides from these seven proteins, in particular between FH and FHL-1.

[0332] FIG. 3 shows linearity data for FH, FHL-1, and FHR1-5. This demonstrates that the GluC digestion produces peptides that can be detected individually and specifically in native serum at endogenous levels. It also shows that the assay is capable of quantifying the level of each protein in the sample. Increasing amounts of protein increase the signal in a predictable manner, allowing determination of the levels, as well as the presence, of each of the proteins. Also demonstrated is that the assay is free from interference.

[0333] Lower limits of quantitation were defined as plasma concentrations of FH=25 nM, FHL-1=0.25 nM, FHR-1=2 nM, FHR-2=1 nM, FHR-3=1 nM, FHR-4=4 nM and FHR-5=3 nM.

C3 and C3 Breakdown Products

[0334] Synthetic versions of the peptides in Table 2 were synthesised to confirm and optimise their detection by MS to confirm that they could be quantified in a linear manner, and to demonstrate that they could be detected at endogenous levels in a serum or plasma sample. This is shown by FIGS. 4A to 4D.

[0335] FIG. 4A shows that all peptides in Table 2 can be detected individually in a plasma sample by SRM-MS using at least three transitions. The specificity of the assay for the peptides of interest is confirmed by the relative intensities of the transitions matching the relative intensity of the relevant product ions in an MS/MS scan. FIG. 4B confirms the specificity of the peptides, showing experiments in which the plasma sample was spiked with crude synthetic peptide which demonstrated the appropriate increase in signal.

[0336] C3b breakdown was further analysed in an in vitro assay. C3b was incubated along with FI and a fragment of cofactor CR1, selected over FH as CR1 drives the reaction to cleavage of iC3b to C3c+C3dg, whereas FH will only support cleavage of C3b to iC3b. Sequential samples were taken from the reaction and stopped by boiling.

[0337] FIG. 4C shows the time course of the C3b breakdown via gel electrophoresis. Analysis using MS and the peptides of Table 2 demonstrates that the formation of C3b fragments iC3b, C3f and C3c, and loss of intact C3b can be clearly detected over time (FIG. 4D). Not all peptides are shown since some (e.g. C3a) will not be present in the in vitro set-up, and others represent multiple products.

[0338] These data demonstrate that C3/C3b breakdown can be measured in a quantitative manner using GluC-derived peptides and MS. This enables the presence and levels of complement proteins to be detected in complement-related diseases such as AMD, as well as providing information as to successful treatment outcomes.

[0339] A single assay which can measure all FH family, C3 fragments and FI proteins allows for the simultaneous analysis of all key proteins in the complement amplification loop from just one sample and with efficient throughput.