INTESTINAL INFLAMMATORY DISEASE DESCRIPTION

The present invention relates to the medical, in particular diagnostic, sector.

More specifically, it relates to the sector of the diagnosis of intestinal inflammatory disease through MALDI-TOF mass spectrometry on faecal matrix.

BACKGROUND OF THE INVENTION

Inflammatory bowel (intestinal) disease (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC) typically affect young adults, although in 20-25% of cases the first appear may occur during paediatric age. The prevalence of such diseases is approximately 1% and their course is chronic, characterised by frequent stenosing and/or fistulising in CD (16% of patients) and higher risk of colorectal carcinoma, especially in UC. At the outset, clinical manifestations can, for a long time, be aspecific (abdominal pain, diarrhoea, anaemia, etc.) and common to functional pathologies that are very widespread in the population, such as irritable bowel syndrome (IBS). The treatment of IBD and of its complications is complex, requiring close clinical-instrumental monitoring of patients, medical therapies and often repeated surgeries.

The IBD diagnosis is essentially based on the result of instrumental and histological examinations.

Laboratory tests that are used for diagnostic framing and monitoring of the disease are aspecific inflammation indicators and include the C-reactive protein (CRP) and the determination with immunometric methods of the inflammatory protein Calprotectin in faeces. The latter, considered the best test now available for these diseases, with a specificity of approximately 80%, has sensibility that does not exceed 65%.

Hence, it would be desirable to have available more sensitive and specific biomarkers to support the non-invasive diagnosis of IBD and to direct the patient toward the execution of the instrumental examination necessary for diagnostic confirmation.

The inflammatory bowel process of IBD is characterised by an increased presence in the mucous membrane not only of inflammatory cells and proteins, but also of numerous proteases, such as metalloproteinases, which can catalyse the fragmentation of protein components in peptides; the latter can be carried directly and at high concentrations in faeces, which therefore represent a suitable matrix for their identification.

Therefore, peptidic biomarkers have been researched in the faecal sample.

However, the application of proteomics techniques to the faecal matrix for the identification of biomarkers has been limited by the complex and heterogeneous nature of the matrix, which consists of the commensal microbial component (approximately 20%, bacteria, fungi, viruses), of food residues (approximately 30%, vegetable fibres, fats, starches), of proteins (approximately 10%), of inorganic compounds (10-20%) and of variable quantities of water (50-75%).

The applications described so far relate to electrophoretic methods (SDS-PAGE and tryptic digestion of proteins) and mass spectrometry techniques with the use of instrumentations like Orbitrap, qTOF, QQQ.

In particular, analysis by mass spectrometry allows to search for unknown components in a biological sample and it has therefore been used for faeces analysis.

For example, in one study the analysis was carried out of the faecal proteome of Galphai2 ^/_ transgenic mice, which spontaneously develop chronic colitis, by bi- dimensional electrophoresis followed by LC-MS/MS mass spectrometry (Bergemalm D, Kruse R, Sapnara M, Halfvarson J, Hornquist EH. Elevated fecal peptidase D at onset of colitis in Galphai2 ^/_ mice, a mouse model of IBD. PLoS One. 2017;12(3):e0174275).

For example, in some studies the MALDI-TOF analysis of the faecal sample was used to analyse the microbiome or to detect the presence of occult blood.

For example, the works of Lin et al. (Lin SY, Shih SH, Wu DC, Lee YC, Wu Cl, Lo LH, Shiea J. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the detection of haemoglobins as the protein biomarkers for fecal occult blood. Rapid Commun Mass Spectrom. 2007;21 (20):3311-6) e di Wu Cl et al. (Wu Cl, Tsai CC, Lu CC, Wu PC, Wu DC, Lin SY, Shiea J. Diagnosis of occult blood in human faeces using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Clin Chim Acta. 2007;384(1-2):86-92) pertain to the use of MALDI- TOF analysis on the faecal sample in order to search for the alpha and beta chains of haemoglobin (mass range 6-20 kDa), with the objective of diagnosing the presence of faecal occult blood. The same type of analysis for the same purpose is described in the document US20030232446. In this type of analysis the faecal sample is subjected to sonication and then mixed directly with the MALDI matrix. Other studies use mass spectrometry on the faecal sample in order to study the intestinal microbiome. For example, the following works: Melnik AV, da Silva RR, Hyde ER, Aksenov AA, Vargas F, Bouslimani A, Protsyuk I, Jarmusch AK, Tripathi A, Alexandrov T, Knight R, Dorrestein PC. Coupling Targeted and Untargeted Mass Spectrometry for Metabolome-Microbiome-Wide Association Studies of Human Fecal Samples. Anal Chem. 2017;89(14):7549-7559; He Y, Li H, Lu X, Stratton CW, Tang YW. Mass spectrometry biotyper system identifies enteric bacterial pathogens directly from colonies grown on selective stool culture media. J Clin Microbiol. 2010 Nov;48(11):3888-92; Perry MJ, Centurioni DA, Davis SW, Hannett GE, Musser KA, Egan CT. Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection. Toxins (Basel). 2017 Mar 9;9(3). pii: E94. doi:10.3390/toxins9030094; Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI- TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front Microbiol. 2015 Aug 5;6:791.

The state of the art that uses this type of techniques for faecal analysis is centred on the clinical context of colorectal carcinoma and not on that of inflammatory bowel disease.

For example, the work Ang CS, Baker MS, Nice EC. Mass Spectrometry-Based Analysis for the Discovery and Validation of Potential Colorectal Cancer Stool Biomarkers. Methods Enzymol. 2017;586:247-274 describes methods for the analysis of faecal proteome, in order to identify biomarkers for colorectal cancer.

Hence, there remains in the state of the art the need for a methodology that allows to diagnose in a manner that is non-invasive but sufficiently sensitive and specifies the presence of an inflammatory bowel disease, in particular by means of the analysis of the faecal sample.

Also desired is a diagnostic method able not only to detect the presence of a chronic intestinal inflammatory disease but also to distinguish the two main forms of intestinal chronic inflammatory disease, i.e. Chron’s disease and ulcerative rectocolitis. The state of the art that uses this type of techniques centred on the clinical context of chronic inflammatory bowel disease is referred exclusively to serum and plasma matrices, not comparable in biochemical composition with the faecal matrix. For example the work by Nanni et al. (Nanni P, Parisi D, Roda G, Casale M, Belluzzi A, Roda E, Mayer L, Roda A. Serum protein profiling in patients with inflammatory bowel diseases using selective solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemometric data analysis. Rapid Commun Mass Spectrom. 2007;21 (24):4142-8) regards the use of MALDI-TOF mass spectrometry applied to the serum of patients with chronic inflammatory disease in a range of 2000-10000 m/z. The study demonstrates the significant effect of the different preparation of the sample (extraction by chromatography on reversed-phase chromatography or by anionic exchange or by copper affinity chromatography) on the proteomic profiles of the serum after MALDI-TOF mass spectrometry analysis. Similarly in two abstracts of studies presented at the Crohn’s & Colitis CongressTM 2018, MALDI-TOF mass spectrometry is used to analyse the plasma of patients suffering from Crohn’s disease (P112 El-Din SB, Ahmed E, Header D, Moez P, Ibrahim M. Study of the proteomic profile in patients with Crohn’s disease, its correlation with diagnosis and disease activity. Gastroenterology. 2018; 154, Issue 1 , Supplement, S58) or from ulcerative rectocolitis (P113 El-Din SB, AN E, Header D, Moez P, Ibrahim M. Study of the proteomic profile in patients with ulcerative, its correlation with diagnosis and disease activity. Gastroenterology. 2018; 154, Issue 1 , Supplement, S58) after extraction of the sample on an unspecified solid phase. The international patent application no. WO 2015/127455 refers to irritable bowel syndrome, not to chronic inflammatory bowel disease. In this document, the MALDI-TOF analysis of the faecal sample, conducted within the context of the irritable bowel syndrome, is carried out on intact samples of after acetone mediated protein precipitation. The claims of the document make no reference to MALDI-TOF proteomic analysis of the faecal sample in patients affected by chronic inflammatory bowel disease.

Use of mass spectrometry for peptidomic analysis, however, still presents considerable difficulties.

Of particular relevance, also to assure an effective clinical translation of the results, is the protocol for the extraction of the proteins/peptides from the faecal matrix, which must be simple and efficient. This protocol should be as fast as possible, it should limit ex vivo protein degradation processes and follow procedures that allow its subsequent automation for large scale application. The extraction protocol on the market or described in the literature are based on the use of extraction buffers for proteins with molecular weight generally greater than 10 kDa and assure analytical stability for subsequent immunometric analyses. The abundance of salts and detergents normally present in these buffers, however, makes them unsuitable for mass spectrometry analysis, due to possible interference on the ionisation processes. Faecal proteomics studies report different approaches to preparation of the sample, but none has been verified for possible use for peptidomic analysis. Moreover, no study verifies the stability of the sample, extremely significant for this type of analysis where the peptide fragments could be the result of pre-analytic ex vivo fragmentation, due to the protease action also of bacterial origin.

Hence, there is a need for a sample preparation process that allows an effective and reliable peptidomic analysis.

SUMMARY OF THE INVENTION

A diagnostic method based on MALDI-TOF/MS mass spectrometry has now been found that, carried out on a faecal sample of a subject, allows the diagnosis of a intestinal chronic inflammatory disease. In particular, a method has been discovered that allows to obtain a peptidomic profile that permits diagnosing the presence of a chronic inflammatory bowel disease with high sensitivity and specificity.

Moreover, a method for preparing the faecal sample has also been discovered, to be used for said MALDI-TOF/MS analysis that allows to identify through a single analysis peptidomic profiles able to distinguish healthy persons from ill ones and among the ill ones to distinguish those affected by Crohn’s disease of ulcerative rectocolitis.

Thus, an object of the present invention is a method for the diagnosis of a chronic inflammatory bowel disease from a faecal sample previously collected from a subject comprising the following steps:

a. preparation of the faecal extract by the following steps:

3 re-suspending a faecal sample in water, or in aqueous solution of polar solvents;

a ₂ . stirring and subsequently centrifuging using a centrifugal force above

15000 RCF (Relative Centrifugal Field), then collecting the supernatant; a ₃ . adding an apolar organic solvent in a ratio between faecal extract and solvent able to precipitate the peptide content, for example between 1 :10 and 10:1 (v/v);

a ₄ . stirring and letting rest;

a ₅ . centrifuging using a centrifugal force above 20000 RCF and collect the supernatant;

a ₆ . letting the organic solvent evaporate avoiding complete drying;

a ₇ . adding water or an aqueous solution of polar solvent, for example trifluoroacetic acid or formic acid; a ₈ . further diluting the obtained solution in a range between 1 :2 and 1 :10 with water or an aqueous solution of polar solvents;

b. desalinating the faecal extract;

c. mixing the desalinated sample with a MALDI matrix;

d. executing a mass spectrometry analysis of the type selected from: MALDI- TOF/MS, MALDI, TOF, MALDI-TOF, MALDI-TOF/MS-MS and MALDI-Q- TOF in positive mode in the range of mass-to-charge (m/z) of 1000-4000; e. analysing the obtained mass spectrum by identification of all peaks comprised in the range m/z indicated in the previous step presenting a signal-to-noise ratio (S/N) higher than 3;

f. diagnosing the presence of a intestinal chronic inflammatory disease in the subject from whom the sample was previously collected if the spectrum thus obtained and analysed comprises at least one peak at a value of m/z ± 0.6 selected from the following:

1002.3 ; 1006.4; 1019.5; 1029.5; 1030.5; 1035.4; 1048.5; 1049.4; 1051.8; 1055.4;

1056.6 ; 1059.5; 1062.4; 1068.6; 1070.5; 1071.4; 1073.5; 1085.4; 1087.5; 1093.5;

1096.4 ; 1098.5; 1099.4; 1108.5; 1113.4; 1117.5; 1121.5; 1127.5; 1132.5; 1134.5;

1137.5; 1139.5; 1148.6; 1160.4; 1161.5; 1166.5; 1169.6; 1172.4; 1177.4; 1179.5;

1186.7 ; 1189.4; 1192.5; 1195.5; 1198.6; 1200.6; 1202.7; 1203.5; 1208.5; 1210.5; 1216.8; 1224.5; 1230.8; 1234.8; 1239.6; 1247.5; 1248.7; 1249.5; 1250.7 ; 1255.6;

1256.8 ; 1257.7; 1263.6; 1267.6; 1270.7; 1272.8; 1274.6; 1286.6; 1288.7; 1293.6;

1294.5; 1296.8; 1298.5; 1303.5; 1311.7; 1312.5; 1313.4; 1316.5; 1317.7 ; 1322.8;

1323.5 ; 1329.5; 1336.7; 1339.8; 1342.5; 1345.5; 1355.8; 1358.6; 1360.6; 1362.5;

1366.7; 1367.8; 1368.6; 1370.6; 1376.7; 1377.7; 1378.6; 1381.6; 1385.6 ; 1389.8; 1393.8; 1405.6; 1407.5; 1409.6; 1419.6; 1421.5; 1424.7; 1425.6; 1428.6; 1429.6; 1431.6; 1432.6; 1444.7; 1447.6; 1450.1 ; 1463.7; 1465.7; 1466.7; 1472.7 ; 1478.5;

1479.6; 1485.5; 1486.5; 1487.7; 1492.8; 1493.6; 1494.7; 1507.7; 1511.7; 1520.7;

1522.6; 1523.6; 1524.5; 1529.7; 1536.6; 1537.5; 1540.7; 1563.7; 1566.7; 1567.5; 1572.6; 1576.7; 1587.7; 1596.7; 1599.8; 1605.8; 1612.6; 1617.8; 1619.7 ; 1620.7;

1623.7; 1634.6; 1639.8; 1640.6; 1646.7; 1647.7; 1652.0; 1659.7; 1665.7; 1669.7;

1676.6; 1680.8; 1682.8; 1685.8; 1687.8; 1691.7; 1693.7; 1697.8; 1698.8; 1699.7;

1702.8; 1704.8; 1705.6; 1711.6; 1715.8; 1721.8; 1724.7; 1735.7; 1740.7; 1742.6;

1746.7; 1757.8; 1759.7; 1773.9; 1777.9; 1785.8; 1790.8; 1795.8; 1800.7; 1802.8;

1805.7; 1806.8; 1807.7; 1810.8; 1811.8; 1817.8; 1821.7; 1823.7; 1824.7; 1830.6;

1833.8; 1863.8; 1872.7; 1873.8; 1877.9; 1898.9; 1904.8; 1912.9; 1920.6; 1925.9; 1931.8; 1934.6; 1938.8; 1947.9; 1957.8; 1958.6; 1967.9; 1972.8; 1973.8;

1981.8; 1985.8; 1986.9; 1992.8; 1997.9; 2005.8; 2010.6; 2014.8; 2020.0; 2020.8;

2028.7; 2030.7; 2031.9; 2040.0; 2044.9; 2048.9; 2054.8; 2058.8; 2071.8; 2084.8;

2085.9; 2089.9; 2096.8; 2099.9; 2100.9; 2121.8; 2125.8; 2128.9; 2135.8; 2141.8;

2147.8; 2171.9; 2172.9; 2181.8; 2184.8; 2185.9; 2189.8; 2195.9; 2200.9; 2212.8;

2224.1 ; 2225.0; 2228.1 ; 2230.1 ; 2233.8; 2240.8; 2244.0; 2270.8; 2282.1 ; 2290.1 ;

2300.6; 2312.9; 2315.9; 2317.0; 2322.9; 2324.4; 2329.8; 2334.1 ; 2342.9; 2350.1 ; 2372.8; 2377.9; 2383.9; 2385.9; 2411.0; 2426.0; 2427.1 ; 2428.1 ; 2443.1 ; 2451.1 ;

2462.0; 2463.1 ; 2479.1 ; 2488.1 ; 2501.1 ; 2506.2; 2511.9; 2526.1 ; 2529.0; 2535.1 ;

2544.0; 2595.0; 2607.2; 2617.0; 2618.2; 2633.0; 2638.1 ; 2645.3; 2650.2; 2666.1 ;

2714.4; 2729.2; 2766.3; 2787.2; 2802.4; 2805.8; 2847.2; 2848.9; 2853.0; 2867.2;

2906.1 ; 2934.4; 2938.4; 2970.5; 2981.2; 3008.3; 3085.4; 3097.3; 3103.3; 3123.2; 3136.3; 3140.4; 3147.3; 3195.2; 3210.5; 3232.1 ; 3299.5; 3313.3; 3317.5; 3326.5; 3379.3; 3394.7; 3428.3; 3430.3; 3464.2; 3465.2; 3473.6; 3482.4; 3526.1 ; 3556.3;

3618.3; 3638.3; 3693.4; 3733.8; 3821.8; 3827.6; 3930.9; 3942.7; 3959.1 ; 3975.1. In particular, when the mass spectrum is obtained in such a way as to detect the isotopic forms of each peptide, the indicated value of m/z is that of the first isotope of the series, i.e. the peak with the lowest m/z peak of the series.

In a preferred embodiment of the invention, in the step f) the spectrum contains at least a peak at a value of m/z ± 0.6 selected from the following: 1035.4; 1099.4; 1121.5; 1263.6; 1267.6; 1286.6; 1293.6; 1294.5; 1323.5; 1329.5; 1366.7; 1367.8;

1376.7; 1405.6; 1419.6; 1428.6; 1466.7; 1639.8; 1693.7; 1697.8; 1724.7; 1773.9;

1805.7; 1810.8; 1863.8; 1957.8; 2020.0; 2096.8; 2171.9; 2185.9; 2212.8; 2228.1 ;

2282.1 ; 2315.9; 2342.9; 2529.0; 2934.4; 3821.8.

In an embodiment of the invention, the step f) can be replaced by the step f) in which the absence of a chronic inflammatory bowel disease in the subject from whom the sample was obtained if the spectrum does not contain any of the peaks per point f), and it contains only at least one peak at a value m/z ± 0.6 selected from the following: 1010.3; 1076.4; 1103.5; 1105.4; 1143.5; 1153.4; 1174.4; 1181.5; 1213.5; 1232.5; 1291.4; 1338.5; 1354.5; 1404.6; 1416.7; 1427.6; 1464.6; 1470.7;

1477.7; 1489.7; 1506.6; 1538.5; 1564.5; 1568.7; 1592.7; 1595.7; 1615.7; 1635.6;

1661.7; 1696.7; 1744.7; 1747.8; 1763.7; 1781.7; 1835.8; 1846.9; 1858.8; 1862.6;

1870.7; 1881.9; 1883.9; 1894.7; 1895.9; 1917.9; 1921.8; 1945.9; 2021.7; 2051.9;

2053.1 ; 2068.9; 2079.1 ; 2102.0; 2118.9; 2134.9; 2176.9; 2190.9; 2249.2; 2271.9; 2302.9; 2320.2; 2398.8; 2435.9; 2541.0; 2585.1 ; 2659.2; 2677.2; 2856.0; 2902.5;

2924.4; 3040.1 ; 3058.3; 3073.2; 3214.2; 3218.2; 3262.5; 3360.2; 3446.2; 3620.3;

3766.4.

In a preferred embodiment of the invention, in the step f) the spectrum contains at least one peak at a value of m/z ± 0.6 selected from the following: 1010.3; 1076.4; 1103.5; 1105.4; 1143.5; 1153.4; 1213.5; 1232.5; 1416.7; 1464.6; 1846.9; 2053.1 ; 2541.0; 3218.2.

The analysis of the mass spectra obtained with the method indicated above allowed to observe the total absence of peptides in the investigated range in 40% of the samples of healthy subjects. In the remaining 60% of the samples of healthy subjects, the spectrum does not contain any of the peaks listed in point f), but it may contain at least one peak at a value m/z ± 0.6 selected from those indicated above in step f). In particular, some peaks have been identified, among those listed in step f), whose presence, in the absence of peaks listed in point f), is sufficient to diagnose the absence of intestinal chronic inflammatory disease. These peaks are the following: 1010.3; 1076.4; 1103.5; 1105.4; 1143.5; 1153.4; 1213.5; 1232.5; 1416.7; 1464.6; 1846.9; 2053.1 ; 2541.0; 3218.2.

In the samples of the subjects affected by chronic inflammatory bowel disease, the spectrum contains numerous peptides.

In particular, 359 peaks were identified, corresponding to as many peptides, associated exclusively with the presence of chronic intestinal inflammatory disease and absent in healthy subjects. The comprehensive analysis of the mass spectra considering all 359 peptides therefore allows to attain a sensitivity of 83% and a specificity of 100%. More in particular, 38/359 diagnostic peptides were selected, detected only in patients with IBD. Their individual or variously combined detection allows the diagnosis of IBD with a sensitivity of 82% and a specificity of 100%. The research and identification of this narrow number of peaks allows to simplify the analysis of the result without altering sensitivity and diagnostic specificity.

Hence, this method has higher sensitivity and higher diagnostic specificity with respect to other faecal biomarkers, in particular faecal calprotectin.

Moreover, a method of this type has low analytical cost, lower than that of faecal calprotectin.

In addition, it has been verified experimentally that the method allows to obtain the same result regardless of the MALDI-TOF/MS instrumentation used, thus assuring its reproducibility. Analytical reproducibility was verified by repeatedly analysing the same sample in the same analytical session (intra-assay reproducibility) or in analytical sessions carried out on different days (inter-assay reproducibility), obtaining the same peptidome profile in both cases.

Lastly, it was found that the mass spectra obtained from faecal samples prepared according to the step a) of the aforesaid method and preserved at approximately +4°C are reproducible over time, confirming that this preparation allows to obtain samples that can be preserved before analysis. This is certainly an advantage within the context of the laboratory clinical analysis.

Said chronic intestinal inflammatory disease is preferably selected from ulcerative rectocolitis and Crohn’s disease. In some patients with chronic intestinal inflammatory disease, the clinical, radiological, endoscopic, histological characteristics and the biochemical indicators do not allow to distinguish between Crohn’s disease and ulcerative rectocolitis. These chronic intestinal inflammatory diseases are therefore defined as unclassified colitis.

In a preferred embodiment of the invention, in the step f) if the spectrum contains at least one peak at a value of m/z ± 0.6 selected from the following: 1002.3; 1006.4; 1029.5; 1030.5; 1035.4; 1055.4;1099.4; 1137.5; 1139.5; 1179.5; 1200.6; 1303.5; 1317.7; 1322.8; 1323.5; 1362.5; 1366.7; 1465.7; 1477.7; 1485.5; 1492.8; 1522.6; 1538.5; 1693.7; 1740.7; 1746.7; 2021.7; 2100.9; 2121.8; 2172.9; 2244; 2372.8; 2411.0; 3465.2, then Crohn’s disease is diagnosed.

In a preferred embodiment of the invention, if the spectrum contains at least one peak at a value of m/z ± 0.6 selected from the following: 1019.5; 1049.4; 1070.5; 1085.4; 1093.5; 1127.5; 1177.4; 1186.7; 1224.5; 1296.8; 1342.5; 1425.6; 1431.6; 1479.6; 1536.6; 1537.5; 1676.6; 1759.7; 1807.7; 1895.9; 2044.9; 2084.8; 2089.9; 2315.9; 2938.4, then ulcerative rectocolitis is diagnosed.

The specific diagnosis of Crohn’s disease or ulcerative rectocolitis achieved by identifying the aforesaid peaks allows to obtain a correct classification of patients in 72% of cases.

The method of the invention thus advantageously allows not only to distinguish healthy subjects from IBD patients but also to identify, among the latter, the subjects affected by Crohn’s disease and the subjects affected by ulcerative rectocolitis.

Another object of the present invention is an automation apparatus adapted to carry out the entire pre-analytical and analytical process, to implement the aforesaid method.

DESCRIPTION OF THE INVENTION

Figures

Figure 1. Detail of the MALDI/TOF/MS spectrum of a faecal sample obtained in positive reflectron mode illustrating in the m/z 1210-1280 range the isotopic forms of the four peptides detected. For each peptide, the only value of m/z considered is the one corresponding to the arrow.

Figure 2. MALDI-TOF/MS spectrum of a faecal sample almost completely lacking detectable peptides.

Figure 3. MALDI-TOF/MS spectrum of a faecal sample characterised by a limited number of detectable peptides.

Figure 4. MALDI-TOF/MS spectrum of a faecal sample characterised by a high number of detectable peptides. Figure 5. MALDI-TOF/MS spectrum of a faecal sample characterised by a high number of detectable peptides.

Figure 6. MALDI-TOF/MS spectrum of a faecal sample obtained from a healthy control subject (n 100). The spectrum does not include any of the 38 diagnostic peptides. The only reported peptide (m/z 1464.6) was detected both in the healthy subjects (6/34) and in patients with IBD (50/133).

Figure 7. MALDI-TOF/MS spectrum of a faecal sample obtained from a healthy control subject (n 34). The spectrum does not include any of the 38 diagnostic peptides. The only reported peptide (m/z 1076.4) was detected both in the healthy subjects (2/34) and in patients with IBD (2/133).

Figure 8. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 61). The detected diagnostic peptide is highlighted (m/z 1294.6). Figure 9. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 184). The detected diagnostic peptide is highlighted (m/z 1724.7). Figure 10. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 35). The 2 diagnostic peptides detected are highlighted (m/z 1810.7 and 2212.7).

Figure 11. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 52). The 3 diagnostic peptides detected are highlighted (m/z 1773.7, 2171.9 e 2281.8).

Figure 12. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 131). The 3 diagnostic peptides detected are highlighted (m/z 1810.7 1957.7 and 2185.9).

Figure 13. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 2). The 4 diagnostic peptides detected are highlighted (m/z 1121.5; 1286.6; 1639.8 and 1863.8).

Figure 14. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 78). The 4 diagnostic peptides detected are highlighted (m/z 1323.6; 1724.7; 1810.8 and 2185.9).

Figure 15. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD (n 147). The 4 diagnostic peptides detected are highlighted (m/z 1035.4; 1810.7; 1957.7 and 2185.9).

Figure 16. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by Crohn’s disease (n 19). The peptides that allow to define the presence of chronic inflammatory bowel disease (IBD diagnosis: 1121.5; 1428.6; 1810.7 and 2019.8) and the peptide that allows to classify the chronic inflammatory bowel disease detected as Crohn’s disease (m/z 2410.9).

Figure 17. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by Crohn’s disease (n 73). The peptides that allow to define the presence of chronic inflammatory bowel disease (IBD diagnosis: m/z 1810.8) and to classify the chronic inflammatory bowel disease detected as Crohn’s disease (m/z 2100.9). Figure 18. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by ulcerative rectocolitis (n 36). The peptides that allow to define the presence of chronic inflammatory bowel disease (IBD diagnosis: m/z 2227.9) and to classify the chronic inflammatory bowel disease detected as ulcerative rectocolitis (m/z 1537.4).

Figure 19. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by ulcerative rectocolitis (n 63). The peptides that allow to define the presence of chronic inflammatory bowel disease (IBD diagnosis: m/z 1810.8; 2185.9 and 2315.9) and to classify the chronic inflammatory bowel disease detected as ulcerative rectocolitis (m/z 2084.9).

Figure 20. MALDI-TOF/MS spectrum of a faecal sample obtained from a healthy control subject. The mass spectrum does not indicate the presence of any peptide. Figure 21. MALDI-TOF/MS spectrum of a faecal sample obtained from a healthy control subject. In the mass spectrum are evident two peptides (m/s 1213.7 and 1470.9) detected both in the healthy subjects (7/34 and 1/34 respectively) and in the patients affected by IBD (74/133 and 4/133 respectively).

Figure 22. MALDI-TOF/MS spectrum of a faecal sample obtained from a healthy control subject. In the mass spectrum are evident two peptides (m/s 1213.7 and 2119.1) detected both in the healthy subjects (7/34 and 3/34 respectively) and in the patients affected by IBD (74/133 and 24/133 respectively).

Figure 23. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD. In the mass spectrum, the presence of a diagnostic peptide (m/z 1323.8) is detected.

Figure 24. MALDI-TOF/MS spectrum of a faecal sample obtained from a patient affected by IBD. In the mass spectrum, two diagnostic peptides are evident (m/z 1035.6 and 1323.7).

Figure 25. Intra-assay reproducibility. Individual aliquots of the same sample (P2) were analysed in quadruplicate in the same analytical session. All the peptides of the sample (m/z 1143.5; 1416.8; 1464.7; 1587.8; 1870.8; 2119.0) were detected in all spectra (reproducibility 100%). The peptide with m/z 1587.8 was present only in patients with IBD (8/133). The remaining peptides were instead detectable with the following representativity values, both in healthy subjects and in pathological subjects: m/z 1143.5 (2/34 and 53/133 respectively), m/z 1416.8 (2/34 and 17/133, respectively), m/z 1464.7 (6/34 e 50/133, respectively), m/z 1870.8 (3/34 and 24/133) and m/z 2119.0 (3/34 and 24/133).

Figure 26. Inter-assay reproducibility. Individual aliquots of the same sample (P4) were analysed in triplicate in different analytical sessions. All the peptides of the sample (m/z 1143.5; 1213.7; 1464.7; 1747.9; 1836.0; 1946.1 and 2119.0) were detected in all spectra (reproducibility 100%). All peptides were present both in healthy subjects and in patients with IBD, with following representativity values: m/z 1143.5 (2/34 and 53/133, respectively), m/z 1213.7 (7/34 and 74/133, respectively), m/z 1464.7 (6/34 and 50/133, respectively), m/z 1747.9 (2/34 and 15/133 respectively), m/z 1836.0 (6/34 and 33/133, respectively), m/z 1946.1 (4/34 and 46/133, respectively), m/z 2119.0 (3/34 and 24/133, respectively).

Figure 27. Classifying algorithm of chronic inflammatory bowel diseases.

Definitions

Within the meaning of the present invention, MALDI-TOF or MALDI-TOF/MS or MALDI-TOF/MS-MS or MALDI-Q-TOF means an analysis carried out by mass spectrometry instrumentations that couple the ionisation of the peptides present in the sample by laser energy adsorbed by the matrix to the detection of the ions by time of flight.

Within the meaning of the present invention, mass spectrum means the detection by time of flight of all ions in the range of mass/charge (m/z) between 1000 and 4000 present in the sample where the ionisation of the peptides by laser energy adsorbed by the matrix.

Within the meaning of the present invention, peak means a single ion of the mass spectrum with a signal/noise ratio above 3. The peak also comprises the isotopic forms.

Within the meaning of the present invention, intestinal chronic inflammatory diseases means the diseases that share a chronic bowel inflammatory process that can affect any segment of the mucous membrane of the gastro-enteric tract (from the mouth to the anus), for example in Crohn’s disease, or be located preferentially at the mucous membrane of the colon-rectum, for example in ulcerative rectocolitis. Detailed Description of the Invention

The method of the present invention comprises the steps as defined above and in the claims.

A more detailed description of the method of the invention and of some preferred embodiment thereof is provided below.

a) Preparation of the faecal extract

In this step, the faecal sample, previously obtained from a subject, is resuspended in water in a weight/volume ratio (w/v) preferably between 10:1 and 10000:1 , more preferably in a faecal sample/water ratio of 1000:1 (w/v) (for example 100 mg of faeces in 100 pl_ of water).

Alternatively, it is possible to resuspend the faecal sample in aqueous solutions containing polar solvents, for example trifluoroacetic acid (TFA) or formic acid (FA), at a concentration preferably between 0.01 % and 10%.

To facilitate the extraction process, the resuspended material is agitated, also by using automatic agitation systems, typically for a few minutes.

The faecal solution is then centrifuged for a time generally between 10 and 30 minutes, using a centrifugal force greater than 15000 RCF. If desired, the supernatant, which represents the extract of the faecal sample, can be preserved frozen at a temperature preferably between -15°C and -80°C.

The subsequent analytical step comprises precipitation using organic solvents, for example acetonitrile (ACN), of the proteins with high molecular weight present in the faecal extract. The faecal extract can be used fresh immediately after its preparation or after unfreezing, if it was preserved frozen. Samples subjected to freezing are preferably subjected to further mixing, also with the use of automatic agitation systems, for at least 5-30 seconds and centrifuged typically for approximately 5-25 minutes, using a centrifugal force greater than 15000 RCF, before recovering the supernatant. Subsequently, to the extract of the faecal extract is added the organic solvent in a faecal extract: solvent ratio preferably between 1 :10 and 10:1 , more preferably in a ratio of 1 :1 (v/v). The organic solvent can be selected from apolar solvents able to precipitate the protein content, for example acetonitrile, trichloroacetic acid (TCA) and acetone. Mixes of different apolar solvents can also be used. In one embodiment, to a quantity of extract of the faecal extract between 20 and 1000 pl_ is added pure acetonitrile maintaining a ratio of 1 :1 (v/v) between faecal extract and acetonitrile.

The solution thus obtained is then briefly agitated, also using automatic agitation systems, and then allowed to rest preferably for approximately 20-60 minutes, during which the agitation procedure should be repeated at least three times.

Then centrifuge, typically for at least ten minutes, using a centrifugal force greater than 20000 RCF. The supernatant is then preferably transferred to a test tube suitable for the evaporation of solutions containing organic solvents.

Evaporation can be carried out with any method known in the art for the evaporation of organic solvents. For example, the flow of nitrogen or suitable evaporators can be used. Evaporators entailing the freezing of the sample are to be avoided. Evaporation is protracted for example until a residual volume corresponding approximately to a drop is present, avoiding full desiccation. Subsequently add a volume preferably between 5 and 500 mI_ of water or an aqueous solution containing polar solvents in a range of concentration preferably variable from 0.01 % to 10%, for example a 0.1 % solution of trifluoroacetic acid (TFA) or of formic acid. Before purification, the samples are further diluted with water or aqueous solutions containing polar solvents, for example TFA 0.1 %, preferably maintaining a v/v ratio from 1 :2 to 1 :10.

b) Purification of the faecal extract

The preparatory step of the MALDI-TOF/MS by purification of the faecal extract is then carried out.

Purification is effected with desalination, by any method known in the art.

Purification is preferably carried out on pipette tip of the Zip Tip type, or alternatively on solid phase using C18 or C8 column. Both these methods are known in the art. c) Mixing of the purified sample with a MALDI matrix

The sample-MALDI matrix solution is then prepared by mixing the purification sample with the MALDI matrix. Any MALDI matrix suitable to generate after desorption of charged ions of peptides and proteins can be used for the purpose. These matrices are known and commercially available. In a preferred embodiment, this matrix is alpha-cyano-4-hydroxycinnamic acid (CHCA).

A quantity variable between 0.2 and 5 pL of this solution can be deposited in a MALDI plate with hydrophobic characteristics or suitable to the determination of the peptides.

d) Mass spectrometry analysis

The mass spectrometry analysis is carried out in positive mode, with suitable instrumental conditions to allow the evaluation of the individual charges of the peptides in the mass/charge (m/z) range of 1000-4000.

Preferably, a mode is used that allows to highlight the isotopic forms, more preferably the reflectron mode.

The mass spectrometry analysis can be carried out with MALDI-TOF/MS, MALDI, TOF, MALDI-TOF, MALDI-TOF/MS-MS, or MALDI-Q-TOF technology. Preferably, a MALDI-TOF/MS analysis is carried out.

Any instrument allowing to perform a MALDI-TOF/MS, MALDI, TOF, MALDI-TOF, MALDI-TOF/MS-MS or MALDI-Q-TOF analysis in the mass/charge (m/z) range of 1000-4000 can be used in the present invention.

e) Analysis of the mass spectrum

The mass spectrum is typically analysed with appropriate software, generally a software supplied with the instrument or other known software for the analysis of these spectra. By way of example, the commercial software programmes FlexAnalysis (Bruker Daltonics, Bremen, Germany), Data Explorer (Applied Biosystems) and the mMass freeware by Martin Strohalm (freeware) can be used. With this analysis, the peptides in the m/z range from 1000 to 4000 are identified Peaks considered significantly different from analytical noise are those that have a signal-to-noise (S/N) ratio greater than 3. The analysis then entails the identification of all peaks with the described characteristics (S/N>3).

If the mass spectrum detects the isotopic forms of each peptide, the identified value of m/z corresponding to the selected peptide is that of the first isotope of the series, i.e. the peak with the lowest m/z of the series. Figure 1 shows and example of mass spectrum where the isotopes of the identified peptides are evident.

f) Diagnosis

The mass spectrum thus obtained and analysed allows the diagnosis of intestinal inflammatory diseases, if at least one peak is identified at the m/z values listed above. In particular, the method of the invention permits not only the diagnosis of an IBD but also the differential analysis between Chron’s Disease and ulcerative rectocolitis, if a peak is identified among those indicated above as diagnostic for these pathologies.

In an embodiment, the method of the invention also allows to diagnose the absence of a chronic intestinal inflammatory disease, if no peaks are identified among those indicated as diagnostic of IBD, but only peaks included among those also observed in healthy subjects are identified, i.e. the peaks listed above in step f), in particular the following ones: 1010.3; 1076.4; 1103.5; 1105.4; 1143.5; 1153.4; 1213.5; 1232.5; 1416.7; 1464.6; 1846.9; 2043.1 ; 2541.0; 3218.2. If from the analysis with the method of the invention only one or more peaks among those listed above emerge, and no peak among those indicated above as diagnostic of IBD, the absence of IBD can be diagnosed.

The method can be carried out on faecal samples coming from subjects suspected to have a intestinal chronic inflammatory disease, for example in patients with intestinal symptomatology characterised by persistent pain, diarrhoea, weight loss or patients with more aspecific abdominal symptomatology, for example in patients affected by irritable bowel syndrome, or in patients with extra-intestinal manifestations compatible with malabsorption, for example patients with iron- deficiency anaemia or patients with reduced bone mineralisation.

An object of the invention is also an automatable system, hereafter called “apparatus”, adapted to carry out the pre-analytical and analytical steps of the method of the invention. Said apparatus is then able to execute all the steps of the method, from sampling of the faeces to spectra analysis and the consequent computation of a diagnosis. Said apparatus comprises a device for the recognition of the sample by bar code, a device for sampling and preparation of the sample, an instrument for mass spectrometry analysis and a software or an IT application that contains an algorithm able to provide the classification of the result of the MALDI-TOF faecal analysis.

Said apparatus can comprise for example: a device for sampling and preparation of the sample connected to an instrument for mass spectrometry analysis and a software or an IT application that contains an algorithm able to provide the classification of the result of the MALDI-TOF faecal analysis. This algorithm will have as its input the MALDI-TOF peptidomic profile and it will output the classification of the result comprising the“Normal”,“IBD”,“Crohn’s Disease” and “Ulcerative rectocolitis” categories.

Said apparatus comprises, in particular, a system, which may be automated, able to carry out the sampling and dilution of the faeces assuring a pre-set suitable weight/volume ratio. Said possibly automated system comprises at least a sampling test tube ready for use, i.e. already pre-filled with the solution suitable for the preparation of the faecal extract. Moreover, said test tube is preferably characterised by quantitative precision of the sampled material, rapidity and ease of use, which can also assure, for example, its utilisation by the patient. An object of the present invention is also a computer programme to carry out the steps e) and f) of the method of the invention. In particular, said programme is able to process the mass spectra obtained with the method of the invention and to provide a diagnostic classification. This classification can for example be based on the algorithm represented in figure 27. This programme can be designed by the person versed in the art on the basis of his/her general knowledge in the sector and of the method described herein. For example, the programme can comprise the following steps:

1. In case of absence of peptides, diagnosis of absence of intestinal chronic inflammatory disease.

2. In case of presence of peptides: if the spectrum comprises at least one value m/z among those listed in step f) and no value m/z among those listed in step f), diagnosis of absence of chronic intestinal inflammatory disease; if the spectrum comprises at least one value m/z between those listed in step f), diagnosis of chronic intestinal inflammatory disease.

3. In case of diagnosis of chronic intestinal inflammatory disease, if the spectrum comprises at least one m/z value among those indicated above as diagnostic of Crohn’s disease, classification of the chronic intestinal inflammatory disease as Crohn’s disease; if the spectrum comprises at least one m/z value among those indicated above as diagnostic of ulcerative rectocolitis, classification of the chronic intestinal inflammatory disease as ulcerative rectocolitis. A data support comprising said computer programme and a processor on which said computer programme is loaded are also objects of the present invention.

With the method of the invention, 5 faecal samples obtained from healthy subjects and 5 faecal samples obtained from subjects affected by chronic inflammatory bowel disease (IBD) were analysed in a preliminary exploratory step. Analysis of the mass spectra showed the nearly total absence of peptides in the samples of the healthy subjects and the presence of numerous peptides in the samples of the subjects affected by IBD.

With the goal of verifying the impact of preservation of the sample on the peptidomic profile, before extending the assessment of the method in a second and more extensive series of sample, the MALDI-TOF/MS analysis with the method described above was replicated in a series of 8 samples obtained from ill subjects. The faecal samples were analysed immediately after collection and after being preserved at different temperature (ambient and +4°C) for different time intervals (from 1 to 7 days) before analysis. The mass spectra obtained from the samples preserved at +4°C were found to be reproducible over time. On the basis of this result, 33 samples were collected from healthy control subjects and from 133 patients affected by IBD. The analysis of the results obtained from the entire series of analysed samples documented MALDI-TOF/MS spectra with variable peptidomic complexity. The detected spectra were spectra almost completely lacking peptidomic signals, spectra characterised by the presence of few peptides and spectra particularly enriched with peptides.

A total number of 438 peptides were detected. Sixty-nine peptides were detectable in the healthy subjects. 67/79 peptides detected in the controls were also detectable in the ill subjects, while 359 peptides were associated exclusively with the presence of IBD. These 359 peptides, individually or variously combined, were detectable in 111/133 patients with IBD. The comprehensive analysis of the mass spectra considering all 359 peptides therefore allows to attain a sensitivity of 83% and a specificity of 100%.

Since many peptides were mutually correlated, the analysis of the spectra was carried out again choosing the most representative peptides among the mutually correlated ones. From this analysis were selected 38 peptides in all, whose individual or variously combined presence allows the identification of patients with IBD with a sensitivity of 82% and a specificity of 100%.

The diagnostic system was further verified with the MALDI-TOF/MS analysis of an independent series of healthy control subjects (n=13) and of patients with IBD (n=25) using the same method for the preparation of the sample, but MALDI- TOF/MS instrumentation obtained from a different supplier. In no healthy subject analysed were the diagnostic peptides detected, but only the common peptides. The presence of the diagnostic peptides was confirmed in the patients.

Analytical reproducibility was verified by repeatedly analysing the same sample in the same analytical session (intra-assay reproducibility) or in analytical sessions carried out on different days (inter-assay reproducibility), obtaining the same peptidome profile in both cases.

The present invention will now be further illustrated by the following examples.

EXAMPLES

Materials and methods

The faecal samples were resuspended in H ₂ 0 with a 1000:1 (p/v) ratio. The resuspended material, after vortex agitation for a few minutes, was centrifuged for 30 minutes at 15000 RCF (Relative Centrifugal Field). The supernatant, collected with pipette, was frozen at -80°C for up to one month. After rapid unfreezing (37°C for 15 minutes), the samples were passed in the vortex for a few minutes, and then centrifuged for 15 minutes at 15000 RCF. 50 pL of the supernatant were mixed with 50 pL of acetonitrile (ACN) (1 :1 , v/v). The solution thus obtained was maintained at ambient temperature for 30 minutes, during which it was subjected at regular intervals, every 10 minutes, to agitation by passage in the vortex. The solution was then centrifuged for 15 minutes at 20000 RCF. The supernatant was transferred with a pipette to a test tube suitable for the evaporation of solutions containing organic solvents. Evaporation was carried out by nitrogen flow for such a duration as to avoid the complete desiccation of the sample and that on average was approximately 8-10 minutes. To the residual volume, approximately one drop, were added 30 pl_ of a 0.1 % solution of trifluoroacetic acid (TFA). The samples were further diluted 1 :5 with 0.1 % TFA immediately prior to purification by means of Zip Tip. The eluate thus obtained was mixed 1 :1 with the alpha-cyano-4- hydroxycinnamic acid (CHCA) MALDI matrix. A quantity of 1 mI_ of this solution was deposited in a MALDI plate.

The MALDI-TOF/MS analysis was carried out with two different MALDI instrumentations: Ultraflex II instrument (Bruker Daltonics, Bremen, Germany) abd 4800 Plus MALDI TOF/TOF Analyzer (AB Sciex) following the instrument specifications provided below.

4800 Plus MALDI TOF/TOF Analyzer: the MALDI-TOF/MS analysis was carried out in positive reflectron mode, with an electric field of 20 kV of acceleration and a grid of 16 kV and an extraction delay of 450 nanoseconds. The individual samples were analysed after collecting on average 1500 laser pulses of intensity equal to 3500 arbitrary units.

Ultraflex II instrument: the MALDI-TOF/MS was carried out in positive reflectron mode, with an electric field of IS1 25 kV, IS2 21.65 kV, reflectron potential 26.3 kV, extraction time 0 nanoseconds. The individual samples were analysed after collecting on average 1500 laser pulses of intensity equal to 50%.

The MALDI-TOF/MS mass spectra were analysed by the freeware mMass V5.5.0 for Windows, contemplating the peptides included in the m/z range from 1000 to 4000. Peaks considered significantly different from analytical noise were those that had a signal-to-noise (S/N) ratio greater than 3. In the presence of isotopic forms of each peptide, the identified value of m/z was the one corresponding to the first isotope of the series. With the method described, a preliminary series of 10 faecal samples was analysed (5 from healthy subjects and 5 from subjects affected by chronic intestinal inflammatory diseases). The analysis was then extended to a series of 33 healthy subjects (21 males, 12 females, age range 30-63 years) and 133 patients affected by chronic intestinal inflammatory diseases (78 males, 55 females, age range 17- 83). The 133 patients affected by chronic intestinal inflammatory disease comprised 79 subjects affected by Crohn’s disease, 47 affected by ulcerative rectocolitis and 7 by unclassified colitis. The diagnoses were always defined by an experienced team on the basis of the clinical-biochemical presentation and of the results of the radiological, endoscopic and histological investigations. The mass spectrometry analysis of a faecal sample obtained by a patient affected by ulcerative rectocolitis did not yield results that could be interpreted, due to the presence of interfering polymeric forms. The faecal samples obtained by an additional series of 8 patients affected by chronic intestinal inflammatory disease (3 males, 5 females, age range 18-70 years) were used for the verification of the impact of the preservation of the sample on the MALDI-TOF/MS result. Each sample, immediately after collection, was divided in seven aliquots. One aliquot was analysed with the method described within two hours from collection (reference sample). Three aliquots were preserved at ambient temperature, while the remaining three were preserved at +4°C for 1 , 4 and 7 days before the analysis. The faecal samples obtained from an additional series of 13 healthy subjects and 25 patients affected by chronic inflammatory bowel diseases were collected for the verification of instrumental reproducibility.

All enrolled subjects expressed their informed consent in writing.

Results

Example 1 - Preliminary exploration step With the method described above, 5 faecal samples obtained from healthy subjects and 5 faecal samples obtained from subjects affected by chronic inflammatory bowel disease (IBD) were analysed in a preliminary exploratory step.

Analysis of the mass spectra showed the nearly total absence of peptides in the samples of the healthy subjects and the presence of numerous peptides in the samples of the subjects affected by IBD.

Example 2 - Verification of the impact of preservation of the sample on the peptidomic profile

With the goal of verifying the impact of preservation of the sample on the peptidomic profile, before extending the assessment of the new method in a second and more extensive series of sample, the MALDI-TOF/MS analysis with the method described above was replicated in a series of 8 samples obtained from ill subjects.

The faecal samples were analysed immediately after collection and after being preserved at different temperature (ambient and +4°C) for different time intervals (from 1 to 7 days) before analysis.

The mass spectra obtained from the samples preserved at +4°C were found to be reproducible over time.

Example 3 - Validation of the method on a higher number of subjects

On the basis of the result obtained above, 33 samples were collected from healthy control subjects and from 133 patients affected by IBD.

The analysis of the results obtained from the entire series of analysed samples documented MALDI-TOF/MS spectra with variable peptidomic complexity.

The detected spectra were spectra almost completely lacking peptidomic signals (Figure 2), spectra characterised by the presence of few peptides (Figure 3) and spectra particularly enriched with peptides (Figures 4 and 5). A total number of 438 peptides were detected (Table 1). Sixty-nine peptides were detectable in the healthy subjects. 67/79 peptides detected in the controls were also detectable in the ill subjects (signal shared between healthy subjects and patients in column B of table 1), while 359 peptides were associated exclusively with the presence of IBD. These 359 peptides, individually or variously combined, were detectable in 111/133 patients with IBD. The comprehensive analysis of the mass spectra considering all 359 peptides therefore allows to attain a sensitivity of 83% and a specificity of 100%.

Table 1. The table shows the list of detected peptides (column A), their possible sharing between healthy and ill subjects (column B), the number of healthy subjects (column C), the number of ill subjects considered as a whole (column D) or individually on the basis of the diagnosis of Crohn’s disease (column E), of ulcerative rectocolitis (column F) or of unclassified colitis (column G) in which the individual peptides were detected individually.

Since many peptides were mutually correlated, the analysis of the spectra was carried out again choosing the most representative peptides among the mutually correlated ones. From this analysis were selected 38 peptides in all, shown in table 2, whose individual or variously combined presence allows the identification of patients with IBD with a sensitivity of 82% and a specificity of 100%.

Table 2. The table shows the list of the main 38 diagnostic peptides, detected only in patients with IBD. Their individual or variously combined detection allows the diagnosis of IBD with a sensitivity of 82% and a specificity of 100%.

Figures 6-15 show MALDI-TOF/MS spectra obtained from two healthy control subjects and from eight patients affected by IBD. In each of the spectra shown, only the peptides included among the 38 selected as diagnostic are indicated (see table 2); these have never been detected in the healthy control subjects (figures 6 and 7), while their presence in various combinations was detected in 82% of the patients with IBD. The mass spectra of the patients (Figures 8-15) highlight some examples of possible combinations of peptides useful for the diagnosis of chronic inflammatory diseases.

Diagnostic peptides were also identified for Crohn’s disease (table 3) and for ulcerative rectocolitis (table 4).

Table 3. Diagnostic peaks for Crohn’s disease.

Table 4. Diagnostic peaks for ulcerative rectocolitis.

Figures 16-19 show MALDI-TOF/MS spectra obtained from two patients affected by Crohn’s disease and from two patients affected by ulcerative rectocolitis. Indicated in each of the spectra shown are the peptides included among the 38 selected as diagnostic for IBD (see table 2), the peptides included among the 34 diagnostics for Crohn’s disease (see table 3) and the peptides included among the 25 diagnostics for ulcerative rectocolitis (see table 4).

Figure 27 shows the diagnostic flowchart based on the MALDI-TOF/MS analysis of the faecal sample.

Example 4 - instrumental and analytical reproducibility

The diagnostic system was further verified with the MALDI-TOF/MS analysis of an independent series of healthy control subjects (n=13) and of patients with IBD (n=25) using the same method for the preparation of the sample, but MALDI- TOF/MS instrumentation obtained from a different supplier

Figures 20-24 show the mass spectra obtained from three healthy subjects (figures 20-22) and from three subjects affected by IBD (figures 23-24).

In no healthy subject analysed were the diagnostic peptides detected (Table 2), but only the common peptides (Table 1), and these are indicated with the corresponding m/z in figures 20-22.

The presence of the diagnostic peptides was confirmed in the patients, as shown in figures 23-24.

Analytical reproducibility was verified by repeatedly analysing the same sample in the same analytical session (intra-assay reproducibility) or in analytical sessions carried out on different days (inter-assay reproducibility), obtaining the same peptidome profile in both cases (figures 25 and 26).