Method for the Diagnosis of Hereditary Angioedema

Abstract

The present invention is related to a method for differential diagnosis of hereditary angioedema, wherein the method comprises determining the level of C4 protein, C1-INH protein and C1q protein in a sample from a subject, wherein the sample is a dried blood spot sample and wherein the level is determined by mass spectrometry.

Claims

1. A method for differential diagnosis of hereditary angioedema, wherein the method comprises determining the level of C4 protein, C1-INH protein and C1q protein in a sample from a subject, wherein the sample is a dried blood spot sample and wherein the level is determined by mass spectrometry.

2. The method of claim 1, wherein determining the level of C4 protein comprises detecting and quantifying the level of a C4 fragment peptide, wherein determining the level of C1-INH protein comprises detecting and quantifying the level of a C1-INH fragment peptide, and wherein determining the level of C1q protein comprises detecting and quantifying the level of a C1q fragment peptide.

3. The method of claim 1, wherein if the sample tests negative for C4 protein and tests positive for C1-INH protein and C1q protein, the subject is suffering from hereditary angioedema type II.

4. The method of claim 1, wherein if the sample tests negative for C4 protein and C1-NH protein and tests positive for C1q protein, the subject is suffering from hereditary angioedema type I.

5. The method of claim 1, wherein the method is a method for differentiating between hereditary angioedema type I and hereditary angioedema type II.

6.-8. (canceled)

9. The method of claim 2, wherein the C4 fragment peptide, the C1-INH fragment peptide and/or the C1q fragment peptide is prepared from the sample by a protease digest or a peptidase digest.

10. The method of claim 9, wherein the protease is selected from the group comprising Arg-C, Asp-N, Asp-N(N-terminal Glu), BNPS or NCS/urea, Caspase-1, Caspase-10, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Chymotrypsin, Chymotrypsin (low specificity), Clostripain, CNBr, CNBr (methyl-Cys), CNBr (with acids), Enterokinase, Factor Xa, Formic acid, Glu-C (AmAc buffer, Glu-C (Phos buffer), Granzyme B, HRV3C protease, Hydroxylamine, Iodosobenzoic acid, Lys-C, Lys-N, Lys-N(Cys modified), Mild acid hydrolysis, NBS (long exposure), NBS (short exposure), NTCB, Pancreatic elastase, Pepsin A, Pepsin A (low specificity), Prolyl endopeptidase, Proteinase K, TEV protease, Thermolysin, Thrombin and trypsin.

11. (canceled)

12. The method claim 2, wherein the C4 fragment peptide is selected from the group consisting of TABLE-US-00027 Peptide Sequence(N-terminus.fwdarw.C-terminus) C4Alpha_[1006-1008] LPR C4Alpha_[1009-1026] GCGEQTMIYLAPTLAASR C4Alpha_[1009- GCGEQTMIYLAPTLAASR 1026]_Cys_CAM:1010 C4Alpha_[1027-1030] YLDK C4Alpha_[1031-1042] TEQWSTLPPETK C4Alpha_[1043-1051] DHAVDLIQK C4Alpha_[1052-1055] GYMR C4Alpha_[1062-1072] ADGSYAAWLSR C4Alpha_[1073-1084] GSSTWLTAFVLK C4Alpha_[1085-1099] VLSLAQEQVGGSPEK C4Alpha_[1100-1126] LQETSNWLLSQQQADGSFQDLSPVIHR C4Alpha_[1168-1174] VEASISK C4Alpha_[1175-1182] ASSFLGEK C4Alpha_[1183-1204] ASAGLLGAHAAAITAYALTLTK C4Alpha_[1211-1248] GVAHNNLMAMAQETGDNLYWGSVTGSQSNAVSP TPAPR C4Alpha_[1249-1278] NPSDPMPQAPALWIETTAYALLHLLLHEGK C4Alpha_[1279-1291] AEMADQAAAWLTR C4Alpha_[1292-1300] QGSFQGGFR C4Alpha_[1301-1325] STQDTVIALDALSAYWIASHTTEER C4Alpha_[1326-1336] GLNVTLSSTGR C4Alpha_[1337-1340] NGFK C4Alpha_[1341-1349] SHALQLNNR C4Alpha_[1350-1352] QIR C4Alpha_[1353-1365] GLEEELQFSLGSK C4Alpha_[1370-1375] VGGNSK C4Alpha_[1383-1390] TYNVLDMK C4Alpha_[1391-1404] NTTCQDLQIEVTVK C4Alpha_[1391- NTTCQDLQIEVTVK 1404]_Cys_CAM:1394 C4Alpha_[1405-1428] GHVEYTMEANEDYEDYEYDELPAK C4Alpha_[1429-1446] DDPDAPLQPVTPLQLFEG C4Alpha_[680-685] NVNFQK C4Alpha_[686-690] AINEK C4Alpha_[691-700] LGQYASPTAK C4Alpha_[702- CCQDGVTR 709]_Cys_CAM:702,703 C4Alpha_[710-714] LPMMR C4Alpha_[715- SCEQR 719]_Cys_CAM:716 C4Alpha_[723-729] VQQPDCR C4Alpha_[723- VQQPDCR 729]_Cys_CAM:728 C4Alpha_[730- EPFLSCCQFAESLR 743]_Cys_CAM:735,736 C4Alpha_[750-756] GQAGLQR C4Alpha_[757-775] ALEILQEEDLIDEDDIPVR C4Alpha_[776-785] SFFPENWLWR C4Alpha_[786-791] VETVDR C4Alpha_[792-815] FQILTLWLPDSLTTWEIHGLSLSK C4Alpha_[818-828] GLCVATPVQLR C4Alpha_[818- GLCVATPVQLR 828]_Cys_CAM:820 C4Alpha_[832-838] EFHLHLR C4Alpha_[846-861] FEQLELRPVLYNYLDK C4Alpha_[862-912] NLTVSVHVSPVEGLCLAGGGGLAQQVLVPAGSARP VAFSVVPTAATAVSLK C4Alpha_[862- NLTVSVHVSPVEGLCLAGGGGLAQQVLVPAGSARP 912]_Cys_CAM:876 VAFSVVPTAATAVSLK C4Alpha_[913-916] VVAR C4Alpha_[917-929] GSFEFPVGDAVSK C4Alpha_[936-941] EGAIHR C4Alpha_[942-954] EELVYELNPLDHR C4Alpha_[957-979] TLEIPGNSDPNMIPDGDFNSYVR C4Alpha_[980-1005] VTASDPLDTLGSEGALSPGGVASLLR C4Beta_[105-118] GPEVQLVAHSPWLK C4Beta_[119-123] DSLSR C4Beta_[124-137] TTNIQGINLLFSSR C4Beta_[139-155] GHLFLQTDQPIYNPGQR C4Beta_[158-159] YR C4Beta_[160-166] VFALDQK C4Beta_[167-185] MRPSTDTITVMVENSHGLR C4Beta_[190-214] EVYMPSSIFQDDFVIPDISEPGTWK C4Beta_[219-234] FSDGLESNSSTQFEVK C4Beta_[23-48] LLLFSPSVVHLGVPLSVGVQLQDVPR C4Beta_[236-244] YVLPNFEVK C4Beta_[245-269] ITPGKPYILTVPGHLDEMQLDIQAR C4Beta_[270-283] YIYGKPVQGVAYVR C4Beta_[284-292] FGLLDEDGK C4Beta_[294-297] TFFR C4Beta_[298-304] GLESQTK C4Beta_[305-316] LVNGQSHISLSK C4Beta_[326-337] LNMGITDLQGLR C4Beta_[338-373] LYVAAAIIESPGGEMEEAELTSWYFVSSPFSLDLSK C4Beta_[392-404] EMSGSPASGIPVK C4Beta_[405-459] VSATVSSPGSVPEVQDIQQNTDGSGQVSIPIIIPQTISE LQLSVSAGSPHPAIAR C4Beta_[460-484] LTVAAPPSGGPGFLSIERPDSRPPR C4Beta_[485-494] VGDTLNLNLR C4Beta_[49-53] GQVVK C4Beta_[495-512] AVGSGATFSHYYYMILSR C4Beta_[513-520] GQIVFMNR C4Beta_[521-523] EPK C4Beta_[525-559] TLTSVSVFVDHHLAPSFYFVAFYYHGDHPVANSLR C4Beta_[560-570] VDVQAGACEGK C4Beta_[560- VDVQAGACEGK 570]_Cys_CAM:567 C4Beta_[571-579] LELSVDGAK C4Beta_[580-582] QYR C4Beta_[583-588] NGESVK C4Beta_[589-614] LHLETDSLALVALGALDTALYAAGSK C4Beta_[60-63] NPSR C4Beta_[615-623] SHKPLNMGK C4Beta_[624-664] VFEAMNSYDLGCGPGGGDSALQVFQAAGLAFSDG DQWTLSR C4Beta_[624- VFEAMNSYDLGCGPGGGDSALQVFQAAGLAFSDG 664]_Cys_CAM:635 DQWTLSR C4Beta_[64-71] NNVPCSPK C4Beta_[64- NNVPCSPK 71]_Cys_CAM:68 C4Beta_[667-671] LSCPK C4Beta_[667- LSCPK 671]_Cys_CAM:669 C4Beta_[72-80] VDFTLSSER C4Beta_[81-92] DFALLSLQVPLK C4Beta_[93-95] DAK C4Beta_[96-104] SCGLHQLLR C4Beta_[96- SCGLHQLLR 104]_Cys_CAM:97 C4Gamma_[1458-1465] VVEEQESR C4Gamma_[1466-1474] VHYTVCIVVR C4Gamma_[1466- VHYTVCIVVR 1474]_Cys_CAM:1471] C4Gamma_[1475-1477] NGK C4Gamma_[1478-1498] VGLSGMAIADVTLLSGFHALR C4Gamma_[1499-1503] ADLEK C4Gamma_[1504-1510] LTSLSDR C4Gamma_[1511-1533] YVSHFETEGPHVLLYFDSVPTSR C4Gamma_[1534-1564] ECVGFEAVQEVPVGLVQPASATLYDYYNPER C4Gamma_[1534- ECVGFEAVQEVPVGLVQPASATLYDYYNPER 1564]_Cys_CAM:1535 C4Gamma_[1566-1575] CSVFYGAPSK C4Gamma_[1566- CSVFYGAPSK 1575]_Cys_CAM:1566 C4Gamma_[1578-1594] LLATLCSAEVCQCAEGK C4Gamma_[1578- LLATLCSAEVCQCAEGK 1594]_Cys_CAM:1583, 1588,1590 C4Gamma_[1595-1597] CPR C4Gamma_[1595- CPR 1597]_Cys_CAM:1595 C4Gamma_[1601-1604] ALER C4Gamma_[1616-1622] FACYYPR C4Gamma_[1616- FACYYPR 1622]_Cys_CAM:1618 C4Gamma_[1623-1630] VEYGFQVK C4Gamma_[1631-1633] VLR C4Gamma_[1638-1641] AAFR C4Gamma_[1642-1646] LFETK C4Gamma_[1656-1658] DVK C4Gamma_[1659-1665] AAANQMR C4Gamma_[1671-1674] ASCR C4Gamma_[1677-1681] LEPGK C4Gamma_[1682-1716] EYLIMGLDGATYDLEGHPQYLLDSNSWIEEMPSER C4Gamma_[1720-1722] STR C4Gamma_[1725-1744] AACAQLNDFLQEYGTQGCQV C4Gamma_[1725- AACAQLNDFLQEYGTQGCQV 1744]_Cys_CAM:1727, 1742

13. The method of claim 8, wherein the C4 fragment is selected from the group consisting of C4Beta[571-579], C4Alpha[680-685], C4Alpha[786-791], C4Beta[294-297] and C4Gamma[1638-1641].

14. The method of claim 2, wherein the C1-INH fragment peptide is selected from the group consisting of TABLE-US-00028 Peptide Sequence(N-terminus.fwdarw.C-terminus) SerpinG1_[202-211] DFTCVHQALK SerpinG1_[202- DFTCVHQALK 211]_Cys_CAM:205 SerpinG1_[212-216] GFTTK SerpinG1_[217-233] GVTSVSQIFHSPDLAIR SerpinG1_[23-40] NPNATSSSSQDPESLQDR SerpinG1_[234-241] DTFVNASR SerpinG1_[242-249] TLYSSSPR SerpinG1_[250-268] VLSNNSDANLELINTWVAK SerpinG1_[269-273] NTNNK SerpinG1_[274-276] ISR SerpinG1_[277-286] LLDSLPSDTR SerpinG1_[301-306] TTFDPK SerpinG1_[310-316] MEPFHFK SerpinG1_[322-328] VPMMNSK SerpinG1_[330-341] YPVAHFIDQTLK SerpinG1_[344-364] VGQLQLSHNLSLVILVPQNLK SerpinG1_[367-380] LEDMEQALSPSVFK SerpinG1_[381-385] AIMEK SerpinG1_[386-390] LEMSK SerpinG1_[391-400] FQPTLLTLPR SerpinG1_[403-415] VTTSQDMLSIMEK SerpinG1_[41-44] GEGK SerpinG1_[416-466] LEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGV EAAAASAISVAR SerpinG1_[467-487] TLLVFEVQQPFLFVLWDQQHK SerpinG1_[488-494] FPVFMGR SerpinG1_[495-499] VYDPR SerpinG1_[53-77] MLFVEPILEVSSLPTTNSTTNSATK

15. The method of claim 10, wherein the C1-INH fragment peptide is selected from the group consisting of SerpinG1 [242-249] and SerpinG1 [391-400].

16. The method of claim 2, wherein the C1q fragment peptide is selected from the group consisting of TABLE-US-00029 Peptide Sequence(N-terminus.fwdarw.C-terminus) C1q-A_[104-110] GSPGNIK C1q-A_[111-121] DQPRPAFSAIR C1q-A_[123-150] NPPMGGNVVIFDTVITNQEEPYQNHSGR C1q-A_[151-180] FVCTVPGYYYFTFQVLSQWEICLSIVSSSR C1q-A_[151- FVCTVPGYYYFTFQVLSQWEICLSIVSSSR 180]_Cys_CAM:153,172 C1q-A_[186-195] SLGFCDTTNK C1q-A_[186- SLGFCDTTNK 195]_Cys_CAM:190 C1q-A_[196-219] GLFQVVSGGMVLQLQQGDQVWVEK C1q-A_[224-245] GHIYQGSEADSVFSGFLIFPSA C1q-A_[23-27] EDLCR C1q-A_[28-32] APDGK C1q-A_[34-41] GEAGRPGR C1q-A_[49-60] GEQGEPGAPGIR C1q-A_[82-94] VGYPGPSGPLGAR C1q-B_[118-121] ATQK C1q-B_[137-141] DQTIR C1q-B_[160-163] FTCK C1q-B_[164-177] VPGLYYFTYHASSR C1q-B_[178-186] GNLCVNLMR C1q-B_[178- GNLCVNLMR 186]_Cys_CAM:181 C1q-B_[194-215] VVTFCDYAYNTFQVTTGGMVLK C1q-B_[194- VVTFCDYAYNTFQVTTGGMVLK 215]_Cys_CAM:198 C1q-B_[216-229] LEQGENVFLQATDK C1q-B_[230-253] NSLLGMEGANSIFSGFLLFPDMEA Clq-B_[28-59] QLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIK C1q-B_[28- QLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIK 59]_Cys_CAM:31 C1q-B_[63-77] GLPGLAGDHGEFGEK C1q-B_[78-88] GDPGIPGNPGK C1q-B_[93-98] GPMGPK C1q-B_[99-110] GGPGAPGAPGPK C1q-C_[118-126] FQSVFTVTR C1q-C_[127-139] QTHQPPAPNSLIR C1q-C_[140-157] FNAVLTNPQGDYDTSTGK C1q-C_[162-184] VPGLYYFVYHASHTANLCVLLYR C1q-C_[162- VPGLYYFVYHASHTANLCVLLYR 184]_Cys_CAM:179 C1q-C_[189-198] VVTFCGHTSK C1q-C_[189- VVTFCGHTSK 198]_Cys_CAM:193 C1q-C_[199-210] TNQVNSGGVLLR C1q-C_[211-245] LQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD C1q-C_[29-47] NTGCYGIPGMPGLPGAPGK C1q-C_[29- NTGCYGIPGMPGLPGAPGK 47]_Cys_CAM:32 C1q-C_[48-57] DGYDGLPGPK C1q-C_[58-69] GEPGIPAIPGIR C1q-C_[76-86] GEPGLPGHPGK C1q-C_[87-113] NGPMGPPGMPGVPGPMGIPGEPGEEGR

17. The method of claim 12, wherein the C1q fragment peptide is selected from the group consisting of C1qBeta[178-186] and C1qBeta[63-77].

18. The method of claim 9, wherein the cut-off value for C4 fragment peptide C4Beta[571-579] is 500 ng/ml; the cut-off value for C4 fragment peptide C4Alpha[680-685] is 260 ng/ml; the cut-off value for C4 fragment peptide C4Alpha[786-791] is 100 ng/ml; the cut-off value for C4 fragment peptide C4Beta[294-297] is 201 ng/ml; and the cut-off value for C4 fragment peptide C4Gamma[1638-1641] is 920 ng/ml.

19. The method of claim 11, wherein the cut-off value for C1-INH fragment peptide SerpinG1[242-249] is 835 ng/ml, and the cut-off value for C1-INH fragment peptide SerpinG1[391-400] is 392 ng/ml.

20. The method of claim 13, wherein the cut-off value for C1q fragment peptide C1qBeta[178-186] is 800 ng/ml, and the cut-off value for C1q fragment peptide and C1qBeta[63-77] is 1690 ng/ml.

21. (canceled)

22. The method of claim 1, wherein mass spectrometry is selected from the group comprising SELDI MS, MALDI MS, ESI MS, DESI MS and ion mobility MS.

23. The method of claim 1, wherein mass spectrometry uses an analyzer selected from the group comprising Triple Quad, ToF, QToF, ion trap, orbitrap, ion mobility and any combination thereof.

24. The method of claim 1, wherein spectrometric analysis comprises or uses MS/MS, MRM, SRM or any combination thereof.

25. A kit suitable for use in a method for differential diagnosis of hereditary angioedema, wherein the kit comprises at least one element selected from the group comprising an interaction partner of one biomarker, one biomarker, instructions of use for the kit, and one or more container, wherein the biomarker is selected from the group comprising C4 protein, a fragment peptide of C4 protein, C1-INH protein, a fragment peptide of C1-INH protein, C1q protein and a fragment peptide of C1q.

Description

[0080] The present invention is now further illustrated by the following figures and examples from which further features, embodiments and advantages may be taken.

[0081] More specifically,

[0082] FIGS. 1 to 9 are boxplots indicating levels of the indicated peptide; the y-axis demonstrates the logarithmised levels of said indicated peptide in ng/ml as determined from a dried blood spot on a filter card as described in the Example part; the x-axis depicts groups of subjects which have been grouped as described in the Example part. The boxplot represents the 25th and 75th percentile of each group of subjects by the bottom and top of the box, respectively; the band near the middle of the box represents the 50th percentile (i.e. the median) of each group; the whiskers represent one standard deviation above and below the mean of the data; any data not included between the whiskers is shown as an outlier with a small circle or star. The horizontal line represents the cut-off level of expressed as ng/ml for the indicated peptide.

[0083] FIG. 1 is a boxplot of peptide fragment C4Beta_[571-579] of protein C4 beta illustrating a cut-off of 500 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from HAE type 1 and HAE type 2.

[0084] FIG. 2 is a boxplot of peptide fragment SerpinG1_[242-249] of protein C1-INH illustrating a cut-off of 835 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from HAE type 1.

[0085] FIG. 3 is a boxplot of peptide fragment C1q Beta_[178-186] of protein C1q beta illustrating a cut-off of 800 ng/ml. This peptide fragment may be used as a control.

[0086] FIG. 4 is a boxplot of peptide fragment C4Alpha_[680-685] of protein C4alpha illustrating a cut-off of 260 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from either HAE type 1 or HAE type 2.

[0087] FIG. 5 is a boxplot of peptide fragment C4Alpha_[786-791] of protein C4alpha illustrating a cut-off of 100 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from either HAE type 1 or HAE type 2.

[0088] FIG. 6 is a boxplot of peptide fragment C4Beta_[294-297] of protein C4beta illustrating a cut-off of 201 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from either HAE type 1 or HAE type 2.

[0089] FIG. 7 is a boxplot of peptide fragment C4Gamma_[1638-1641] of protein C4gamma illustrating a cut-off of 920 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from either HAE type 1 or HAE type 2.

[0090] FIG. 8 is a boxplot of peptide fragment SerpinG1_[391-400] of protein C1-INH illustrating a cut-off of 392 ng/ml. Such cut-off allows to distinguish between healthy controls and patients suffering from HAE type 1.

[0091] FIG. 9 is a boxplot of peptide fragment C1q-Beta_63-77] of protein C1q beta illustrating a cut-off of 1690 ng/ml. This peptide fragment may be used as a control.

EXAMPLES

[0092] In the Examples described in the following a dried blood spot (abbr. DBS) on a filter card was used as a sample from a subject.

Example 1: Method for HAE Diagnostic Based on Fragmentation of C3, C1q, C4 and C1-INH into Peptides and Mass Spectrometry Thereof

[0093] To quantify the content/levels of C3, C1q, C4 and C1-INH in dried blood spots (DBS) extract a protocol as described was used. After extraction of blood components, the DBS extract was subjected in situ to reaction with dithiothreitol (DDT) to reduce the disulfide bridges in the proteins and to iodacetamide (IAA) to alkylate the free SH groups. The reaction mixture was then digested in its entirety with trypsin. The tryptic mixture containing peptide fragments of the proteins to be analyzed was injected in LC/IM-high resolution mass spectrometry. For all proteins, peptides without post-transactional modifications could be identified in blood matrix (see Table 1 C3, C1q, C4 and C1-INH peptides identified in tryptic mixture obtained after total tryptic digestion of DBS extract, selected peptides with +H adducts). For all peptides fragmentation spectra were obtained and, based on the experimental fragmentation pattern, transitions to be used in multiple reaction monitoring mass spectrometry.

[0094] The tryptic peptides could be measured next by LC/MRM-MS. Below are example of tryptic peptides from C3, C1q, C4 and C1-INH detected and quantified using LC/MRM-MS.

[0095] Equipment

[0096] For detecting the tryptic peptides of the proteins to be quantified in a biological sample of a donor, the following equipment was used:

TABLE-US-00012 Equipment Model Provider DBS Puncher 1296-071 Delfia Perkin Elmer Pipettes single and multichannel Eppendorf Vortexer Mixer UZUSIO VTX-300L LMS co. LTD Sonicator SW12H Sonoswiss Incubator Titramax 1000 Heidolph Centrifuge Benchtop Eppendorf UPLC Acuity iclass Waters IM-qToF Vion Waters TQ TQS-micro Waters Data mining tool Progenesis Nonlinear

[0097] Reagents

[0098] For detecting the peptides of the proteins to be quantified in a sample from a subject the following reagents were used. To the extent that values depend on temperature (e.g., the pH value) such values were determined at a temperature of 25 C.

TABLE-US-00013 Name Supplier Purity 1,4-Dithiothreitol Roche >97% Acetonitrile, waterfreei (max. 0.003% H.sub.2O) VWR UPLC/ HiPerSolv CHROMANORM UHPLC grade AcroPrep Advance 96 well filter PALL platesfor aqueous filtration, 350 l, 1.0 m glass fibre Ammonia solution Merck 25% Ammoniumbicarbonate ACROS 98% Formic acid, ACS VWR >96% Iodoacetamide, IAA Sigma Aldrich >99% Kinetex colums EVO C18 VWR Leucine-Enkephalin waters Methanol HiPerSolv CHROMANORM VWR LC-MS grade Mirco-Platte 96-wells, PP, F-GREINER VWR (100 pieces) Natur SafeSeal vial 1.5 ml Sarstedt Taurocholic acid sodium salt hydrate Sigma Aldrich >95% Trypsin 20 g/vial Promega sequencing grade Verex Cap (pre-assembled), 8-425, Phenomenex Screw top, w/PTFE/Silicone septa, black Verex Insert, 5 mm Dia., 175 L, Clear Phenomenex 51, Conical Bottom, w/bottom spring Verex Vial, 8 mm Screw Top, 2 mL, Phenomenex Clear 33, w/Patch Water HiPerSolv CHROMANORM VWR LC-MS grade Pipette tips Sarstedt

[0099] Preparation of Stock Solution of Internal Standard

[0100] Internal Standard (IS 1) stock solution was used as internal standard and was prepared by dissolving 3 mg Leucine-Enkephaline (as provided by Waters, UK) in water to a concentration of 400 g/mL.

[0101] Storing of Samples and Solutions

[0102] Control samples and study samples (dried blood spots) were stored at RT. Internal Standard working solutions were stored at room temperature until use.

[0103] Sample Preparation for Analysis

[0104] 1 punch of 3.2 mm was cut from the filter card with dried blood spots and subjected the following protocol:

[0105] First, for extraction 100 L 1 M NH.sub.4HCO.sub.3 were added to the punches, whereby the material was sonicated for 10 min. at 60 C., incubated for 30 min on a shaker (at 700 rpm) at 37 C.

[0106] Second, to the solution 125 l 1 M DTT was added and the reaction mixture was incubated for 3 h at 37 C. on a shaker (700 rpm).

[0107] Third, 375 l 1 M IAA was added and the solution was incubated for 1.5 hours on a shaker (700 rpm) in the dark.

[0108] Fourth, 10 l 0.5 g/l trypsin was added and the solution was incubated for 3 to 16 hours on a shaker (700 rpm) in the dark.

[0109] The thus obtained solution containing a digest of blood extract was transferred to a PTFE (polytetrafluoroethylene) filter plate (Acroprep, Pall, Germany) and then to a 96 well plate by centrifugation at 3.500 rpm. Afterwards, 100 L of internal standard with a known concentration of 20 to 400 ng/mL was added.

[0110] Methods

[0111] A person skilled in the art will acknowledge that methods for detecting the fragment peptides of the proteins to be analyzed in a sample from a subject using mass spectrometric analysis may also employ other tryptic peptides, specific transitions and specific fragments which allow for specific detection of and/or quantification of HAE relevant peptide fragments and their isoforms in said sample from a subject.

[0112] LC/IM-QToF-MS analyses of the peptide fragments of the proteins to be analyzed from DBS extracts were performed using a Waters Acquity iclass UPLC (Waters, UK) coupled with Vion mass spectrometer (Waters, UK) as follows. [0113] 1. Chromatographic run was performed on a Kinetex EVO C18 column (Phenomenex, Germany). 10 L of the extract were injected onto the column and the compounds of the extract were eluted using a linear gradient from 0% A (50 mM formic acid in water) to 100% B (50 mM formic acid in acetonitrile:methanol vol. 1:1). [0114] 2. Internal standard was continuously injected at a concentration of 200 ng/mL in water and the signal was used to normalize the sample signal across the batch.

[0115] IM-QToF MS analyses were performed in positive ion mode using the following parameters: [0116] Analyzer mode: sensitivity [0117] MS mode: High definition MSE [0118] Capillary voltage: 1.2 kV [0119] Source temperature: 150 C. [0120] Desolvation temperature: 600 C. [0121] Desolvation gas; 1000 L/h [0122] Cone gag: 50 L/h [0123] Low Collision Energy: 6 eV [0124] High Collision Energy Ramp: 20-40 eV [0125] Scan mass: 50-1000 m/z [0126] Scan time: 0.5 s

[0127] LC/MRM-MS analyses of the peptide fragments of the proteins to be analyzed for DBS extracts were performed using a Waters Acquity iclass UPLC (Waters, UK) coupled with a TQ-S micro mass spectrometer (Waters, UK).

[0128] For the examples the following parameters were used in the quantification of the peptide fragments: [0129] 1. Chromatographic run was performed on a Kinetex EVO C18 column (Phenomenex, Germany). The 10 L extract were injected on the column and the compounds were eluted using a linear gradient from 0% A (50 mM formic acid in water) to 100% B (50 mM formic acid in acetonitrile:methanol vol. 1:1). [0130] 2. For the internal standard, MRM transition 556.24.fwdarw.119.97 was monitored. For each peptide, specific transition was used as shown in Example 2.

[0131] MRM-MS analyses were performed in positive ion mode using the following parameters: [0132] Capillary voltage: 1.2 kV [0133] Cone voltage: 20 V [0134] Source temperature: 150 C. [0135] Desolvation temperature: 600 C. [0136] Desolvation gas: 1000 L/h [0137] Cone gag: 50 L/h [0138] Collision Energy: 20 V [0139] Collision Cell Entrance: 30 eV [0140] Collision Cell Exit: 30 eV.

Example 2: Quantifying Peptide Fragments of Proteins C1q, C4, C1-INH and C3 in Dried Blood Spots of Healthy Donors

[0141] Using the methods outlined in Example 1, the different peptide fragments of proteins C1 q, C4, C1-INH and C3 were quantified using DBS from a total of 270 healthy subjects.

[0142] For Complement C1q the following tryptic peptides could be quantified, whereby the numbers in the brackets represents the position of the first amino acid and the last amino acid of the peptide in amino acid sequence of C1q (with the sequences being indicated with the N-terminus being at the left side and the C-terminus being at the right side). Such peptides are shown in Table 1:

TABLE-US-00014 TABLE1 Peptide Sequence(N-terminus.fwdarw.C-terminus) C1q-A_[104-110] GSPGNIK C1q-A_[111-121] DQPRPAFSAIR C1q-A_[123-150] NPPMGGNVVIFDTVITNQEEPYQNHSGR C1q-A_[151-180] FVCTVPGYYYFTFQVLSQWEICLSIVSSSR C1q-A_[151- FVCTVPGYYYFTFQVLSQWEICLSIVSSSR 180]_Cys_CAM:153,172 C1q-A_[186-195] SLGFCDTTNK C1q-A_[186- SLGFCDTTNK 195]_Cys_CAM:190 C1q-A_[196-219] GLFQVVSGGMVLQLQQGDQVWVEK C1q-A_[224-245] GHIYQGSEADSVFSGFLIFPSA C1q-A_[23-27] EDLCR C1q-A_[28-32] APDGK C1q-A_[34-41] GEAGRPGR C1q-A_[49-60] GEQGEPGAPGIR C1q-A_[82-94] VGYPGPSGPLGAR C1q-B_[118-121] ATQK C1q-B_[137-141] DQTIR C1q-B_[160-163] FTCK C1q-B_[164-177] VPGLYYFTYHASSR C1q-B_[178-186] GNLCVNLMR C1q-B_[178- GNLCVNLMR 186]_Cys_CAM:181 C1q-B_[194-215] VVTFCDYAYNTFQVTTGGMVLK C1q-B_[194- VVTFCDYAYNTFQVTTGGMVLK 215]_Cys_CAM:198 C1q-B_[216-229] LEQGENVFLQATDK C1q-B_[230-253] NSLLGMEGANSIFSGFLLFPDMEA Clq-B_[28-59] QLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIK C1q-B_[28- QLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIK 59]_Cys_CAM:31 C1q-B_[63-77] GLPGLAGDHGEFGEK C1q-B_[78-88] GDPGIPGNPGK C1q-B_[93-98] GPMGPK C1q-B_[99-110] GGPGAPGAPGPK C1q-C_[118-126] FQSVFTVTR C1q-C_[127-139] QTHQPPAPNSLIR C1q-C_[140-157] FNAVLTNPQGDYDTSTGK C1q-C_[162-184] VPGLYYFVYHASHTANLCVLLYR C1q-C_[162- VPGLYYFVYHASHTANLCVLLYR 184]_Cys_CAM:179 C1q-C_[189-198] VVTFCGHTSK C1q-C_[189- VVTFCGHTSK 198]_Cys_CAM:193 C1q-C_[199-210] TNQVNSGGVLLR C1q-C_[211-245] LQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD C1q-C_[29-47] NTGCYGIPGMPGLPGAPGK C1q-C_[29- NTGCYGIPGMPGLPGAPGK 47]_Cys_CAM:32 C1q-C_[48-57] DGYDGLPGPK C1q-C_[58-69] GEPGIPAIPGIR C1q-C_[76-86] GEPGLPGHPGK C1q-C_[87-113] NGPMGPPGMPGVPGPMGIPGEPGEEGR

[0143] All of the Complement C1q tryptic peptides can be used to differentiate between healthy subjects and hereditary angioedema patients. Two of said peptides, namely C1q-B_[178-186] with MRM transition 510.26.fwdarw.254.58 and C1q-B_[63-77] with MRM transition 495.25.fwdarw.774.5 were used as representative examples (see Example 3).

[0144] For complement C3 the following tryptic peptides could be quantified, whereby the numbers in the brackets represents the position of the first amino acid and the last amino acid of the peptide in amino acid sequence of C3 (with the sequences being indicated with the N-terminus being at the left side and the C-terminus being at the right side). Such peptides are shown in Table 2:

TABLE-US-00015 TABLE2 Peptide Sequence(N-terminus.fwdarw.C-terminus) C3Beta_[105-119] FVTVQATFGTQVVEK C3Beta_[120-136] VVLVSLQSGYLFIQTDK C3Beta_[137-148] TIYTPGSTVLYR C3Beta_[149-155] IFTVNHK C3Beta_[156-161] LLPVGR C3Beta_[162-176] TVMVNIENPEGIPVK C3Beta_[177-205] QDSLSSQNQLGVLPLSWDIPELVNMGQWK C3Beta_[208-225] AYYENSPQQVFSTEFEVK C3Beta_[226-241] EYVLPSFEVIVEPTEK C3Beta_[23-35] SPMYSIITPNILR C3Beta_[242-249] FYYIYNEK C3Beta_[250-258] GLEVTITAR C3Beta_[259-263] FLYGK C3Beta_[265-281] VEGTAFVIFGIQDGEQR C3Beta_[291-304] IPIEDGSGEVVLSR C3Beta_[306-315] VLLDGVQNPR C3Beta_[316-322] AEDLVGK C3Beta_[323-343] SLYVSATVILHSGSDMVQAER C3Beta_[344-359] SGIPIVTSPYQIHFTK C3Beta_[363-386] YFKPGMPFDLMVFVTNPDGSPAYR C3Beta_[36-65] LESEETMVLEAHDAQGDVPVTVTVHDFPGK C3Beta_[387-408] VPVAVQGEDTVQSLTQGDGVAK C3Beta_[409-425] LSINTHPSQKPLSITVR C3Beta_[429-439] QELSEAEQATR C3Beta_[440-462] TMQALPYSTVGNSNNYLHLSVLR C3Beta_[463-478] TELRPGETLNVNFLLR C3Beta_[479-481] MDR C3Beta_[482-486] AHEAK C3Beta_[489-497] YYTYLIMNK C3Beta_[500-502] LLK C3Beta_[503-505] AGR C3Beta_[506-508] QVR C3Beta_[509-530] EPGQDLVVLPLSITTDFIPSFR C3Beta_[531-544] LVAYYTLIGASGQR C3Beta_[545-556] EVVADSVWVDVK C3Beta_[557-566] DSCVGSLVVK C3Beta_[557- DSCVGSLVVK 566]_Cys_CAM:559 C3Beta_[574-584] QPVPGQQMTLK C3Beta_[585-592] IEGDHGAR C3Beta_[616-622] IVVDVVEK C3Beta_[623-633] ADIGCTPGSGK C3Beta_[634-657] DYAGVFSDAGLTFTSSSGQQTAQR C3Beta_[658-667] AELQCPQPAA C3Beta_[658- AELQCPQPAA 667]_Cys_CAM:662 C3Beta_[67-73] LVLSSEK C3Beta_[74-94] TVLTPATNHMGNVTFTIPANR C3Beta_[95-97] EFK C3Beta_[98-100] SEK C3cAlpha1_[749-764] SNLDEDIIAEENIVSR C3cAlpha1_[765-779] SEFPESWLWNVEDLK C3cAlpha1_[780-783] EPPK C3cAlpha1_[784-789] NGISTK C3cAlpha1_[797-812] DSITTWEILAVSMSDK C3cAlpha1_[814-834] GICVADPFEVTVMQDFFIDLR C3cAlpha1_[814- GICVADPFEVTVMQDFFIDLR 834]_Cys_CAM:816 C3cAlpha1_[835-841] LPYSVVR C3cAlpha1_[842-848] NEQVEIR C3cAlpha1_[849-855] AVLYNYR C3cAlpha1_[856-861] QNQELK C3cAlpha1_[864-879] VELLHNPAFCSLATTK C3cAlpha1_[864- VELLHNPAFCSLATTK 879]_Cys_CAM:873 C3cAlpha1_[905-913] TGLQEVEVK C3cAlpha1_[914-926] AAVYHHFISDGVR C3cAlpha1_[938-940] MNK C3cAlpha1_[941-945] TVAVR C3cAlpha1_[946-951] TLDPER C3cAlpha1_[952-954] LGR C3cAlpha2_[1321-1325] SEETK C3cAlpha2_[1326-1337] ENEGFTVTAEGK C3cAlpha2_[1338-1351] GQGTLSVVTMYHAK C3cAlpha2_[1354-1360] DQLTCNK C3cAlpha2_[1354- DQLTCNK 1360]_Cys_CAM:1358 C3cAlpha2_[1361-1364] FDLK C3cAlpha2_[1365-1375] VTIKPAPETEK C3cAlpha2_[1376-1381] RPQDAK C3cAlpha2_[1382-1391] NTMILEICTR C3cAlpha2_[1382- NTMILEICTR 1391]_Cys_CAM:1389 C3cAlpha2_[1394-1419] GDQDATMSILDISMMTGFAPDTDDLK C3cAlpha2_[1420-1427] QLANGVDR C3cAlpha2_[1428-1431] YISK C3cAlpha2_[1432-1436] YELDK C3cAlpha2_[1437-1441] AFSDR C3cAlpha2_[1442-1450] NTLIIYLDK C3cAlpha2_[1451-1462] VSHSEDDCLAFK C3cAlpha2_[1451- VSHSEDDCLAFK 1462]_Cys_CAM:1458 C3cAlpha2_[1463-1478] VHQYFNVELIQPGAVK C3cAlpha2_[1479-1491] VYAYYNLEESCTR C3cAlpha2_[1479- VYAYYNLEESCTR 1491]_Cys_CAM:1489 C3cAlpha2_[1492-1497] FYHPEK C3cAlpha2_[1502-1504] LNK C3cAlpha2_[1505-1507] LCR C3cAlpha2_[1505- LCR 1507]_Cys_CAM:1506 C3cAlpha2_[1527-1532] VTLEER C3cAlpha2_[1533-1535] LDK C3cAlpha2_[1536-1546] ACEPGVDYVYK C3cAlpha2_[1536- ACEPGVDYVYK 1546]_Cys_CAM:1537 C3cAlpha2_[1552-1570] VQLSNDFDEYIMAIEQTIK C3cAlpha2_[1571-1582] SGSDEVQVGQQR C3cAlpha2_[1583-1589] TFISPIK C3cAlpha2_[1592-1595] EALK C3cAlpha2_[1596-1599] LEEK C3cAlpha2_[1601-1624] HYLMWGLSSDFWGEKPNLSYIIGK C3cAlpha2_[1625-1644] DTWVEHWPEEDECQDEENQK C3cAlpha2_[1625- DTWVEHWPEEDECQDEENQK 1644]_Cys_CAM:1637

[0145] All of the Complement C3 tryptic peptides can be used in the assay. Two of said peptides, namely C3Beta_[489-497] with MRM transition 604.8.fwdarw.327.22 and C3cAlpha1_[814-834]_Cys_CAM: 816 with MRM transition 824.74.fwdarw.798.44 were used as illustrative examples (see Example 3).

[0146] For complement C4 the following tryptic peptides could be quantified, whereby the numbers in the brackets represents the position of the first amino acid and the last amino acid of the peptide in amino acid sequence of C4 (with the sequences being indicated with the N-terminus being at the left side and the C-terminus being at the right side). Such peptides are shown in Table 3.

TABLE-US-00016 TABLE3 Peptide Sequence(N-terminus.fwdarw.C-terminus) C4Alpha_[1006-1008] LPR C4Alpha_[1009-1026] GCGEQTMIYLAPTLAASR C4Alpha_[1009- GCGEQTMIYLAPTLAASR 1026]_Cys_CAM:1010 C4Alpha_[1027-1030] YLDK C4Alpha_[1031-1042] TEQWSTLPPETK C4Alpha_[1043-1051] DHAVDLIQK C4Alpha_[1052-1055] GYMR C4Alpha_[1062-1072] ADGSYAAWLSR C4Alpha_[1073-1084] GSSTWLTAFVLK C4Alpha_[1085-1099] VLSLAQEQVGGSPEK C4Alpha_[1100-1126] LQETSNWLLSQQQADGSFQDLSPVIHR C4Alpha_[1168-1174] VEASISK C4Alpha_[1175-1182] ASSFLGEK C4Alpha_[1183-1204] ASAGLLGAHAAAITAYALTLTK C4Alpha_[1211-1248] GVAHNNLMAMAQETGDNLYWGSVTGSQSNA VSPTPAPR C4Alpha_[1249-1278] NPSDPMPQAPALWIETTAYALLHLLLHEGK C4Alpha_[1279-1291] AEMADQAAAWLTR C4Alpha_[1292-1300] QGSFQGGFR C4Alpha_[1301-1325] STQDTVIALDALSAYWIASHTTEER C4Alpha_[1326-1336] GLNVTLSSTGR C4Alpha_[1337-1340] NGFK C4Alpha_[1341-1349] SHALQLNNR C4Alpha_[1350-1352] QIR C4Alpha_[1353-1365] GLEEELQFSLGSK C4Alpha_[1370-1375] VGGNSK C4Alpha_[1383-1390] TYNVLDMK C4Alpha_[1391-1404] NTTCQDLQIEVTVK C4Alpha_[1391- NTTCQDLQIEVTVK 1404]_Cys_CAM:1394 C4Alpha_[1405-1428] GHVEYTMEANEDYEDYEYDELPAK C4Alpha_[1429-1446] DDPDAPLQPVTPLQLFEG C4Alpha_[680-685] NVNFQK C4Alpha_[686-690] AINEK C4Alpha_[691-700] LGQYASPTAK C4Alpha_[702- CCQDGVTR 709]_Cys_CAM:702,703 C4Alpha_[710-714] LPMMR C4Alpha_[715- SCEQR 719]_Cys_CAM:716 C4Alpha_[723-729] VQQPDCR C4Alpha_[723- VQQPDCR 729]_Cys_CAM:728 C4Alpha_[730- EPFLSCCQFAESLR 743]_Cys_CAM:735,736 C4Alpha_[750-756] GQAGLQR C4Alpha_[757-775] ALEILQEEDLIDEDDIPVR C4Alpha_[776-785] SFFPENWLWR C4Alpha_[786-791] VETVDR C4Alpha_[792-815] FQILTLWLPDSLTTWEIHGLSLSK C4Alpha_[818-828] GLCVATPVQLR C4Alpha_[818- GLCVATPVQLR 828]_Cys_CAM:820 C4Alpha_[832-838] EFHLHLR C4Alpha_[846-861] FEQLELRPVLYNYLDK C4Alpha_[862-912] NLTVSVHVSPVEGLCLAGGGGLAQQVLVPAGS ARPVAFSVVPTAATAVSLK C4Alpha_[862- NLTVSVHVSPVEGLCLAGGGGLAQQVLVPAGS 912]_Cys_CAM:876 ARPVAFSVVPTAATAVSLK C4Alpha_[913-916] VVAR C4Alpha_[917-929] GSFEFPVGDAVSK C4Alpha_[936-941] EGAIHR C4Alpha_[942-954] EELVYELNPLDHR C4Alpha_[957-979] TLEIPGNSDPNMIPDGDFNSYVR C4Alpha_[980-1005] VTASDPLDTLGSEGALSPGGVASLLR C4Beta_[105-118] GPEVQLVAHSPWLK C4Beta_[119-123] DSLSR C4Beta_[124-137] TTNIQGINLLFSSR C4Beta_[139-155] GHLFLQTDQPIYNPGQR C4Beta_[158-159] YR C4Beta_[160-166] VFALDQK C4Beta_[167-185] MRPSTDTITVMVENSHGLR C4Beta_[190-214] EVYMPSSIFQDDFVIPDISEPGTWK C4Beta_[219-234] FSDGLESNSSTQFEVK C4Beta_[23-48] LLLFSPSVVHLGVPLSVGVQLQDVPR C4Beta_[236-244] YVLPNFEVK C4Beta_[245-269] ITPGKPYILTVPGHLDEMQLDIQAR C4Beta_[270-283] YIYGKPVQGVAYVR C4Beta_[284-292] FGLLDEDGK C4Beta_[294-297] TFFR C4Beta_[298-304] GLESQTK C4Beta_[305-316] LVNGQSHISLSK C4Beta_[326-337] LNMGITDLQGLR C4Beta_[338-373] LYVAAAIIESPGGEMEEAELTSWYFVSSPFSLDL SK C4Beta_[392-404] EMSGSPASGIPVK C4Beta_[405-459] VSATVSSPGSVPEVQDIQQNTDGSGQVSIPIIIPQ TISELQLSVSAGSPHPAIAR C4Beta_[460-484] LTVAAPPSGGPGFLSIERPDSRPPR C4Beta_[485-494] VGDTLNLNLR C4Beta_[49-53] GQVVK C4Beta_[495-512] AVGSGATFSHYYYMILSR C4Beta_[513-520] GQIVFMNR C4Beta_[521-523] EPK C4Beta_[525-559] TLTSVSVFVDHHLAPSFYFVAFYYHGDHPVANS LR C4Beta_[560-570] VDVQAGACEGK C4Beta_[560- VDVQAGACEGK 570]_Cys_CAM:567 C4Beta_[571-579] LELSVDGAK C4Beta_[580-582] QYR C4Beta_[583-588] NGESVK C4Beta_[589-614] LHLETDSLALVALGALDTALYAAGSK C4Beta_[60-63] NPSR C4Beta_[615-623] SHKPLNMGK C4Beta_[624-664] VFEAMNSYDLGCGPGGGDSALQVFQAAGLAFS DGDQWTLSR C4Beta_[624- VFEAMNSYDLGCGPGGGDSALQVFQAAGLAFS 664]_Cys_CAM:635 DGDQWTLSR C4Beta_[64-71] NNVPCSPK C4Beta_[64-71]_Cys_CAM:68 NNVPCSPK C4Beta_[667-671] LSCPK C4Beta_[667- LSCPK 671]_Cys_CAM:669 C4Beta_[72-80] VDFTLSSER C4Beta_[81-92] DFALLSLQVPLK C4Beta_[93-95] DAK C4Beta_[96-104] SCGLHQLLR C4Beta_[96-104]_Cys_CAM:97 SCGLHQLLR C4Gamma_[1458-1465] VVEEQESR C4Gamma_[1466-1474] VHYTVCIVVR C4Gamma_[1466- VHYTVCIVVR 1474]_Cys_CAM:1471 C4Gamma_[1475-1477] NGK C4Gamma_[1478-1498] VGLSGMAIADVTLLSGFHALR C4Gamma_[1499-1503] ADLEK C4Gamma_[1504-1510] LTSLSDR C4Gamma_[1511-1533] YVSHFETEGPHVLLYFDSVPTSR C4Gamma_[1534-1564] ECVGFEAVQEVPVGLVQPASATLYDYYNPER C4Gamma_[1534- 1564]_Cys_CAM:1535 ECVGFEAVQEVPVGLVQPASATLYDYYNPER C4Gamma_[1566-1575] CSVFYGAPSK C4Gamma_[1566- CSVFYGAPSK 1575]_Cys_CAM:1566 C4Gamma_[1578-1594 LLATLCSAEVCQCAEGK C4Gamma_[1578- LLATLCSAEVCQCAEGK 1594]_Cys_CAM:1583,1588, 1590 C4Gamma_[1595-1597] CPR C4Gamma_[1595- 1597]_Cys_CAM:1595 CPR C4Gamma_[1601-1604] ALER C4Gamma_[1616-1622] FACYYPR C4Gamma_[1616- FACYYPR 1622]_Cys_CAM:1618 C4Gamma_[1623-1630] VEYGFQVK C4Gamma_[1631-1633] VLR C4Gamma_[1638-1641] AAFR C4Gamma_[1642-1646] LFETK C4Gamma_[1656-1658] DVK C4Gamma_[1659-1665] AAANQMR C4Gamma_[1671-1674] ASCR C4Gamma_[1677-1681] LEPGK C4Gamma_[1682-1716] EYLIMGLDGATYDLEGHPQYLLDSNSWIEEMPS ER C4Gamma_[1720-1722] STR C4Gamma_[1725-1744] AACAQLNDFLQEYGTQGCQV C4Gamma_[1725- AACAQLNDFLQEYGTQGCQV 1744]_Cys_CAM:1727,1742

[0147] All of the Complement C4 tryptic peptides can be used to differentiate between healthy subjects and hereditary angioedema patients. for of the peptides, namely C4Alpha_[680-685] with MRM transition 375.2.fwdarw.536.28, C4Alpha_[786-791] with MRM transition 359.69.fwdarw.490.26, C4Beta_[294-297] with MRM transition 285.66.fwdarw.322.19, C4Beta_[571-579] with MRM transition 466.26.fwdarw.243.13, and C4 gamma_[1638-1641] with MRM transition 232.64.fwdarw.322.19] were used as representative examples (see Example 3).

[0148] For complement C1-INH (also referred to as SerpinG1), the following tryptic peptides could be quantified, whereby the numbers in the brackets represents the position of the first amino acid and the last amino acid of the peptide in amino acid sequence of C1-INH (with the sequences being indicated with the N-terminus being at the left side and the C-terminus being at the right side). Such peptides are shown in Table 4.

TABLE-US-00017 TABLE4 Peptide Sequence(N-terminus.fwdarw.C-terminus) SerpinG1_[202-211 DFTCVHQALK SerpinG1_[202- DFTCVHQALK 211]_Cys_CAM:205 SerpinG1_[212-216] GFTTK SerpinG1_[217-233] GVTSVSQIFHSPDLAIR SerpinG1_[23-40] NPNATSSSSQDPESLQDR SerpinG1_[234-241] DTFVNASR SerpinG1_[242-249] TLYSSSPR SerpinG1_[250-268] VLSNNSDANLELINTWVAK SerpinG1_[269-273] NTNNK SerpinG1_[274-276] ISR SerpinG1_[277-286] LLDSLPSDTR SerpinG1_[301-306] TTFDPK SerpinG1_[310-316] MEPFHFK SerpinG1_[322-328] VPMMNSK SerpinG1_[330-341] YPVAHFIDQTLK SerpinG1_[344-364] VGQLQLSHNLSLVILVPQNLK SerpinG1_[367-380] LEDMEQALSPSVFK SerpinG1_[381-385] AIIVIEK SerpinG1_[386-390] LEMSK SerpinG1_[391-400] FQPTLLTLPR SerpinG1_[403-415] VTTSQDMLSIMEK SerpinG1_[41-44] GEGK SerpinG1_[416-466] LEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTET GVEAAAASAISVAR SerpinG1_[467-487] TLLVFEVQQPFLFVLWDQQHK SerpinG1_[488-494] FPVFMGR SerpinG1_[495-499] VYDPR SerpinG1_[53-77] MLFVEPILEVSSLPTTNSTTNSATK

[0149] All the complement C1-INH tryptic peptides can be used to differentiate between healthy subjects and hereditary angioedema patients. Two peptides, namely SerpinG1_[242-249] with MRM transition 455.74.fwdarw.696.33 and SerpinG1 [391-400] with MRM transition 593.35.fwdarw.455.79], were used as representative example (see Example 3).

Example 3: Quantifying Tryptic Peptide Fragments of the Proteins C3, C1q, C4 and C1-INH in DBS Extract from Healthy Subjects and Hereditary Angioedema Patients with Known Pathogenic Variants in Serping1 Gene

[0150] HAE Patients

[0151] All patients with hereditary angioedema disease type 1/2 or of whom it was strongly assumed that they were suffering from hereditary angioedema disease type 1/2 sent to the participating centers were included into the study. SerpinG1 mutations were confirmed in all the patients taken in consideration for this study using techniques such as next generation sequencing, Sanger sequencing and/or multiplexed ligation dependent probe amplification.

[0152] Protein C4

[0153] Using the methods outlined in Example 1, the content of peptide fragments C4Alpha_[680-685], C4Alpha_[786-791], C4Beta_[294-297], C4Beta_[571-579] and C4 gamma_[1638-1641] of protein C4 was quantified in DBS from a total of 270 healthy subjects. Similarly, the content of peptide fragments C4Alpha_[680-685], C4Alpha_[786-791], C4Beta_[294-297], C4Beta_[571-579] and C4 gamma_[1638-1641] of the protein C4 was quantified in DBS from a total of 135 previously genetically diagnosed hereditary angioedema patients.

[0154] For this assay, pure synthetic peptides were obtained and used to obtain a standard curve used to quantify the peptides originating from blood samples. The linearity of the standard curve is reflected in R.sup.2 values in the following Table 5.

TABLE-US-00018 TABLE5 Parent ion Molecular charge Retention R.sup.2 Protein Sequence weight state Transition time Linearity C4Beta LELSVDGAK 930.50 2+ 466.26/243.13 3.5 0.998109 [571-579] C4Alpha NVNFQK 748.39 2+ 375.2/536.28 2.4 0.999882 [680-685] C4Alpha VETVDR 717.37 2+ 359.69/490.26 2 0.999694 [786-791] C4Beta TFFR 569.30 2+ 285.66/322.19 3.1 0.987227 [294-297] C4Gamma AAFR 463.25 2+ 232.64/322.19 2 0.999615 [1638-1641]

[0155] As shown in Table 6 below, peptides C4Alpha[680-685], C4Alpha_[786-791], C4Beta_[294-297], C4Beta_[571-579] and C4Gamma_[1638-1641] of protein C4 were reduced in a statistically significant manner in hereditary angioedema patients compared to healthy subjects (p<0,0001). The values for the various peptides are in ng/ml.

TABLE-US-00019 TABLE 6 HAE HAE Controls Type 1 Type 2 Peptide Number of values (N) 270 118 17 C4Beta_[571- Minimum 500 6.25 25 579] 25% Percentile 1075 6.25 56.25 Median 1572 37.5 165.6 75% Percentile 2408 228.1 298.4 Maximum 3400 475 375 Mean 1760 114.3 173 Std. Deviation 770.2 131.3 122.5 Std. Error of Mean 47.22 12.3 30.62 Lower 95% CI of mean 1667 89.94 107.8 Upper 95% CI of mean 1853 138.7 238.3 C4Alpha_[680- Minimum 268.8 0 0 685] 25% Percentile 462.5 0 6.25 Median 687.5 12.5 43.75 75% Percentile 943.8 50 100 Maximum 2131 237.5 137.5 Mean 723.5 36.74 52.21 Std. Deviation 327.7 50.34 47.18 Std. Error of Mean 19.98 4.694 11.44 Lower 95% CI of mean 684.1 27.44 27.95 Upper 95% CI of mean 762.8 46.04 76.47 C4Alpha_[786- Minimum 100 0 0 791] 25% Percentile 350 0 7.813 Median 531.3 12.5 18.75 75% Percentile 775 31.25 31.25 Maximum 1444 75 43.75 Mean 590.9 16.39 19.14 Std. Deviation 329.7 19.96 13 Std. Error of Mean 20.49 1.87 3.251 Lower 95% CI of mean 550.5 12.69 12.21 Upper 95% CI of mean 631.2 20.1 26.07 C4Beta_[294- Minimum 218.8 0 25 297] 25% Percentile 437.5 0 43.75 Median 634.4 18.75 75 75% Percentile 831.3 62.5 162.5 Maximum 1488 200 187.5 Mean 657.5 35.84 95.31 Std. Deviation 259 49.89 58.96 Std. Error of Mean 15.76 4.613 14.74 Lower 95% CI of mean 626.4 26.71 63.89 Upper 95% CI of mean 688.5 44.98 126.7 C4Gamma_ Minimum 975 0 218.8 [1638-1641] 25% Percentile 1980 0 309.4 Median 2659 181.3 418.8 75% Percentile 3381 318.8 715.6 Maximum 5550 912.5 962.5 Mean 2704 187.7 500.8 Std. Deviation 969.8 214.9 233 Std. Error of Mean 59.02 19.7 58.25 Lower 95% CI of mean 2588 148.6 376.6 Upper 95% CI of mean 2820 226.7 624.9

[0156] Protein C1-INH

[0157] Using the methods outlined in Example 1, the content of peptide fragments SerpinG1_[242-249] and SerpinG1_[391-400] of protein C1-INH were quantified in DBS from a total of 270 healthy subjects. Similarly, the content of peptide fragments SerpinG_[242-249] and SerpinG1_[391-400] of protein C1-INH was quantified in DBS from a total of 135 previously genetically diagnosed hereditary angioedema patients.

[0158] For this assay, pure synthetic peptides were obtained and used to obtain a standard curve used to quantify the peptides originating from blood samples. The linearity of the standard curve is reflected in R.sup.2 values in the following Table 7:

TABLE-US-00020 TABLE7 Parent ion Molecular charge Retention R.sup.2 Protein Sequence weight state Transition time Linearity SerpinG1 TLYSSSPR 909.46 2+ 455.74/696.33 2.6 0.999621 [242-249] SerpinG1 FQPTLLTLPR 1184.69 2+ 593.35/455.79 4.8 0.972887 [391-400]

[0159] As shown in Table 8 below, SerpinG_[242-249] and SerpinG1_[391-400] of protein C1-IHN were reduced in a statistically significant manner in hereditary angioedema patients type 1 in comparison to healthy subjects (p<0,0001). The values for the various peptides are in ng/ml.

TABLE-US-00021 TABLE 8 HAE HAE Number of Controls Type 1 Type 2 Peptide values (N) 270 118 17 SerpinG1_[242- Minimum 1113 0 837.5 249] 25% Percentile 2606 0 884.4 Median 3291 190.6 2463 75% Percentile 4081 381.3 6156 Maximum 6475 831.3 6394 Mean 3412 223.1 2956 Std. Deviation 1174 228.9 2319 Std. Error of Mean 71.43 21.07 562.4 Lower 95% CI of mean 3272 181.4 1764 Upper 95% CI of mean 3553 264.8 4148 SerpinG1_[391- Minimum 675 0 418.8 400] 25% Percentile 1566 0 640.6 Median 2169 81.25 1500 75% Percentile 2930 189.1 5406 Maximum 5069 393.8 6169 Mean 2322 107.4 2384 Std. Deviation 985.7 113.8 2284 Std. Error of Mean 59.99 10.47 553.9 Lower 95% CI of mean 2204 86.62 1210 Upper 95% CI of mean 2440 128.1 3558

[0160] For all samples included in the study, the HAE type 1 patients could be distinguished from healthy controls (non-HAE).

[0161] Protein C1q

[0162] Using the methods outlined in Example 1, the content of peptide fragments C1q-B_[178-186] and C1q-B_[63-77] of protein C1q were quantified in DBS from a total of 270 healthy subjects.

[0163] Similarly, the content of peptide fragments C1q-B_[178-186] and C1q-B_[63-77] of the protein C1q were determined in DBS from a total of 135 previously genetically diagnosed hereditary angioedema patients.

[0164] For this assay, pure synthetic peptides were obtained and used to obtain a standard curve used to quantify the peptides originating from blood samples. The linearity of the standard curve is reflected in R.sup.2 values in the following Table 9.

TABLE-US-00022 TABLE9 Parent ion Molecular charge Retention R.sup.2 Protein Sequence weight state Transition time Linearity C1qBeta GNLCVNLMR 1018.51 2+ 510.26/254.58 3.9 0.983744 [178-186] C1q-B GLPGLAGD 1482.7104 3+ 495.25/774.5 4.1 0.973842 [63-77] HGEFGEK

[0165] As shown in Table 10 below, peptide fragments C1q-B_[178-186] and C1q-B_[63-77] of protein C1q were not significantly reduced in a statistically significant manner in hereditary angioedema patients type 1 compared to healthy subjects. The values for the various peptides are in ng/ml.

TABLE-US-00023 TABLE 10 HAE HAE Controls Type 1 Type 2 Peptide Number of values (N) 270 118 17 C1q-B_[178-186] Minimum 1000 247 25% Percentile 2982 3857 Median 4790 4750 75% Percentile 6240 5355 Maximum 9365 5916 Mean 4698 4250 Std. Deviation 2062 1647 Std. Error of Mean 291.6 425.3 Lower 95% CI of mean 4112 3338 Upper 95% CI of mean 5284 5162 C1q-B_[63-77] Minimum 1783 552.6 25% Percentile 6262 5635 Median 8212 7196 75% Percentile 9666 8200 Maximum 11821 9729 Mean 7651 6684 Std. Deviation 2652 2183 Std. Error of Mean 364.2 563.7 Lower 95% CI of mean 6920 5475 Upper 95% CI of mean 8382 7893

[0166] Protein C3

[0167] Using the methods outlined in Example 1, the content of peptide fragment C3Beta_[250-258] of protein C3 was quantified in DBS from a total of 270 healthy subjects. Similarly, the content of peptide fragment C3Beta_[250-258] of protein C3 was quantified in DBS from a total of 135 previously genetically diagnosed hereditary angioedema patients.

[0168] For this assay, pure synthetic peptides were obtained and used to obtain a standard curve used to quantify the peptides originating from blood samples. The linearity of the standard curve is reflected in R.sup.2 values in the following Table 11.

TABLE-US-00024 TABLE11 Parent ion Molecular charge Retention R.sup.2 Protein Sequence weight state Transition time Linearity C3Beta GLEVTITAR 958.54 2+ 480.28/660.4 3.7 0.943337 [250-258]

[0169] As shown in Table 12 below, C3Beta_[250-258] of the protein C1q is not reduced in a statistically significant matter in hereditary angioedema patients type 1 compared to healthy subjects. The values for the various peptides are in ng/ml.

TABLE-US-00025 TABLE 12 HAE HAE Controls Type 1 Type 2 Peptide Number of values (N) 270 118 17 C3Beta_[250-258] Minimum 1 0.0 25% Percentile 3.785 0.0300 Median 6.73 2.840 75% Percentile 10.36 3.960 Maximum 15.78 8.710 Mean 7.07 2.339 Std. Deviation 3.923 2.347 Std. Error of Mean 0.2466 0.2020 Lower 95% CI of mean 6.584 1.940 Upper 95% CI of mean 7.555 2.739

Example 4: Determination of Biomarker Cut-Off Levels in DBS Extract from Healthy Subjects and Hereditary Angioedema Patients with Known Pathogenic Variants in Serping1 Gene

[0170] Based on the data and results of Example 3 and using synthetic peptides as calibration standards for SerpinG_[242-249], C4Alpha_[680-6851, C4Alpha_[786-791], C4Beta_[294-297], C4Beta_[571-579], C1q-Beta_[178-186], C4Gamma_[1638-1641], SerpinG1_[391-400] and C1q-Beta_63-77] a cut-off level was determined empirically for each peptide.

TABLE-US-00026 Peptide Cut-off C4Beta_[571 -579] 500 ng/mL SerpinG1_[242-249] 835 ng/mL C1q-Beta_[178-186] 800 ng/mL C4Alpha_[680-685] 260 ng/mL C4Alpha_[786-791] 100 ng/mL C4Beta_[294-297] 201 ng/mL C4Gamma_[1638-1641] 920 ng/mL SerpinG1_[391-400] 392 ng/mL C1q-Beta_[63-77] 1690 ng/mL

[0171] The results are shown in FIGS. 1 to 9.

[0172] For all samples included in the study, the HAE patients could be distinguished from the healthy controls (non-HAE, NC).

[0173] The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.