Spatial omics-based machine learning (SOML) algorithms for early detection of hepatocellular carcinoma

Abstract

The present invention provides devices and methods for rapid diagnosis of hepatocellular carcinoma (HCC). The method includes the use of an affinity capture array for multiplexed analysis of the glycoprotein profile of a sample.

Description

Attomev Docket: 206085-0170-00WOSPATIAL OMICS-B ASED MACHINE LEARNING (SGML) ALGORITHMS FOR EARLY DETECTION OF HEPATOCELLULAR CARCINOMACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 679,400, filed on August 5, 2024, which is hereby incorporated by reference in its entirety.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under CA226052 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and a leading cause of cancer-related death in patients with cirrhosis (Singal et al., 2023, Nat Rev Clin Oncol, 20:864-884). Prognosis differs by tumor stage, with curative treatments facilitating long-term survival in patients with early-stage disease (Llovet et al., 2021, Nat Rev Dis Primers, 7:6). Ultrasound-based surveillance misses over one-third of HCC at an early-stage, particularly in patients with non-viral etiologies such as metabolic dysfunction associated steatotic liver disease (MASLD) (Tzartzeva et al., 2018, Gastroenterology, 154: 1706- 1718; Chong et al., 2022, Aliment Pharmacol Ther, 55:683-690). Alpha fetoprotein (AFP) is the only biomarker to complete all five phases of biomarker validation but has insufficient sensitivity for early-stage HCC detection(Singal et al., 2021, Gastroenterology, 160:2572-2584; Singal et al., 2023, Am J Gastroenterol, 78:207-216).

[0004] Accordingly, there is a need in the art for a means of performing rapid and sensitive detection of HCC. The present invention addresses this long felt, but unmet, need.SUMMARY OF THE INVENTION

[0005] In some embodiments, the present invention provides a method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining a glycoprotein profile of the serum sample; c) producing a patient healthAttomev Docket: 206085-0170-00WO profile comprising the glycoprotein profile; and d) detecting the presence of HCC in the subject when the combined health profile of the subject is greater than that of an average subject.

[0006] In some embodiments, step b) further comprises determining the serum alpha-fetoprotein (AFP) level of the subject. In some embodiments, the patient health profile comprises the serum AFP level of the subject, the glycoprotein profile of the subject, the age of the subject, and the sex of the subject.

[0007] In some embodiments, the glycoprotein profile comprises at least one glycoprotein selected from the group consisting of alpha-1 antitrypsin (AAT); alpha-l-acid glycoprotein (AGP); alpha-2-macroglobulin (A2M); angiotensin (ANGIO); apolipoprotein D (APOD); apolipoprotein H (APOH); ceruloplasmin (CERU); fetuin (FET); clusterin (CLUS); haptoglobin (HAPT); hemopexin (HEMO); immunoglobulin G (IGG); transferrin (TRAN); vitamin D binding protein (VDBP); histidine-proline rich glycoprotein (HPRG); a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13); attractin (ATRN); cluster of differentiation 109 (CD 109); cluster of differentiation 163 (CD 163); cluster of differentiation 42D (CD42D); colony stimulating factor 1 receptor (CSF1R); fibulin 1 (FBLN1); low density lipoprotein receptor-related protein 1 (LRP1); lumican (LUM); mannose receptor C-type 1 (MMR); plexin domain containing 2 (PLXDC2); serpin G1 (IC1), sex hormone-binding globulin (SHBG); and Susi, Von Willebrand Factor Type A (SVEP1).

[0008] In some embodiments, the glycoprotein profile comprises at least one glycan selected from the group consisting of: Glycan 933 (Hex3HexNAc2; [M+Na] m / z = 933.490), Glycan 1257 (Hex5HexNAc2; [M+Na] m / z = 1257.423), Glycan 1282 (Hex3dHexlHexNAc3;[M+Na] m / z = 1282.454), Glycan 1419 (Hex6HexNAc2; [M+Na] m / z = 1419.476), Glycan 1445 (Hex4dHexlHexNAc3; [M+Na] m / z = 1444.507), Glycan 1502 (Hex4HexNAc4;[M+Na] m / z = 1501.529), Glycan 1547 (Hex4dHexlHexNAc3; [M+SO4+2Na] m / z = 1546.539), Glycan 1591 (Hex4dHex2HexNAc3; [M+Na] m / z = 1590.558), Glycan 1607 (Hex5dHexlHexNAc3; [M+Na] / » z - 1606.560), Glycan 1611 (Hex4dHexNAc3NeuAcl;[M+2Na] m / z = 1611.604), Glycan 1664 (Hex5HexNAc4; [M+Na] m / z = 1663.581), Glycan 1689 (Hex3dHexlHexNAc5; [M+Na] m'z = 1688.613), Glycan 1736 (Hex4dHexlHexNAc3NeuAcl; [M+Na] m / z = 1735.516), Glycan 1744 (Hex8HexNAc2;[M+Na] m / z = 1743.538), Glycan 1810 (Hex5dHexlHexNAc4; [M+Na] m z = 1809.639),Attorney Docket: 206085-0170-00WOGlycan 1811 (Hex5dHexlHexNAc4; [M+H+Na] m / z = 1810.639), Glycan 1849 (Hex6dHexlHexNAc3S04; [M+Nal] m / z = 1848.722), Glycan 1851 (Hex4dHexlHexNAc5; [M+Na] m / z = 1850.666), Glycan 1854 (Hex5dHexNAc4NeuAclSO4; [M+Na2] m / z = 1853.674), Glycan 1855 (Hex5dHex2NAc3SO4; [M+Na2] m / z = 1854.675), Glycan 1860 ([M+] m / z = 1859.615), Glycan 1867 (Hex5HexNAc5; [M+Na] m / z = 1866.661), Glycan 1868 (Hex4HexNAc3NeuAcSO4; [M+Na3] m / z = 1867.75), Glycan 1876 (Hex5HexNAc5; [M+Na] m / z = 1875.611), Glycan 1890 (Hex5dHexlHexNAc4]SO4]; [M+Na] m / z = 1889.93), Glycan 1906 (Hex9HexNAc2; [M+Na] m / z = 1905.624), Glycan 1908 (Hex4HexNAc6; [M+Na] m / z = 1907.636), Glycan 1955 (Hex5HexNAc4]NeuAcl];[M+Na] m / z = 1954.676), Glycan 1956 (Hex5dHex2HexNAc4; [M+Na] m / z = 1955.693), Glycan 1973 (Hex6dHexlHexNAc3SO4; [M+Na3] m / z = 1972.693), Glycan 1994 (Hex6HexNAc3NeuAclSO4; [M+Na] m / z = 1993.655), Glycan 1995 (Hex6dHex2HexNAc3; [M+Na] m / z = 1994.662), Glycan 2013 (Hex5dHexlHexNAc5; [M+Na] m / z = 2012.719), Glycan 2016 (Hex6HexNAc3NeuAclSO4; [M+Na2] m / z = 2015.726), Glycan 2071 (Hex5HexNAc5SO42; [M+Na3] m / z = 2070.634), Glycan 2102 (Hex5dHex3HexNAc4; [M+Na] m / z = 2101.749), Glycan 2119 (Hex6dHex2HexNAc3NeuAclSO42; [M+Na3] m / z = 2118.609), Glycan 2120 (Hex4HexNAc5SO4; [M+Na3] m / z = 2119.605), Glycan 2123 (Hex5dHexlHexNAc4NeuAcl; [M+2Na] m / z = 2122.7173), Glycan 2134 (Hex4dHexlHexNAc6SO4; [M+Na] m / z = 2133.726), Glycan 2135 (Hex7HexNAc3(SO4)2]; [M+Na3] m / z = 2134.74), Glycan 2140 (Hex6dHexlHexNAc2NeuAcSO4]; [M+Na] m / z = 2139.709), Glycan 2142 (Hex4dHexlHexNAc5NeuAc; [M+Na] m / z = 2141.721), Glycan 2175 (Hex6dHexlHexNAc5; [M+Na] m / z = 2174.772), Glycan 2281 (Hex7HexNAc3SO42;[M+Na3] m / z = 2280.693), Glycan 2321 (Hex6dHex2HexNAc5; [M+Na] m / z = 2320.829), Glycan 2468 (Hex6dHex3HexNAc5; [M+H+Na] m / z =2467.892), and Glycan 2540 (Hex7dHexlHexNAc6+Nal; [M+H] m / z = 2539.904).

[0009] In some embodiments, the glycoprotein profile comprises the level of at least one glycoform selected from the group consisting of angiotensin (ANGIO) conjugated to Glycan 2540 (ANGIO_2540), clusterin (CLUS) conjugated to Glycan 1607 (CLUS_1607), transferrin (TRAN) conjugated to Glycan 1607 (TRAN_1607), haptoglobin (HAPT) conjugated to Glycan 1689 (HAPT 1689), HAPT conjugated to Glycan 1851 (HAPT 1851),Attorney Docket: 206085-0170-00WO hemopexin (HEMO) conjugated to Glycan 2540 (HEMO_2540), and immunoglobulin G (IGG) conjugated to Glycan 1419 (IGG_1419), alpha-2-macroglobulin (A2M) conjugated to Glycan 1867 (A2M 1867), apolipoprotein D (APOD) conjugated to Glycan 2175 (APOD 2175), CLUS conjugated to Glycan 1689 (CLUS 1689), HAPT conjugated to Glycan 2321 (HAPT 2321), HEMO conjugated to Glycan 2175 (HEMO 2175), IGG conjugated to Glycan 1257 (IGG 1257), IGG conjugated to Glycan 1867 (IGG 1867), TRAN conjugated to Glycan 2175 (TRAN_2175), and vitamin D binding protein (VDBP) conjugated to Glycan 2175 (VDBP_2175), alpha-1 antitrypsin (AAT) conjugated to Glycan 2321 (AAT_2321), alpha-1 acid glycoprotein (AGP) conjugated to Glycan 2321 (AGP 2321), fetuin (FET) conjugated to Glycan 2175 (FET 2175), FET conjugated to Glycan 2540 (FET 2540), IGG conjugated to Glycan 1502 (IGG 1502), AGP conjugated to Glycan 2540 (AGP_2540), AGP conjugated to Glycan 1257 (AGP_1257), AGP conjugated to Glycan 1219 (AGP_1219), AGP conjugated to Glycan 1445 (AGP_1445), ANGIO conjugated to Glycan 1851 (ANGIO 1851), apolipoprotein H (APOH) conjugated to Glycogen 1851 (APOH 1851), APOH conjugated to Glycogen 1867 (APOH 1867), APOH conjugated to Glycogen 2157 (APOH 2157), ceruloplasmin (CERU) conjugated to Glycogen 1257 (CERU_1257), CERU conjugated to Glycogen 2175 (CERU_2175), CERU conjugated to Glycogen 2321 (CERU_2321), CLUS conjugated to Glycan 1257 (CLUSJ257), CLUS conjugated to Glycan 1851 (CLUS_1851), CLUS conjugated to Glycan 2013 (CLUS_2013), CLUS conjugated to Glycan 2540 (CLUS_2540), HEMO conjugated to Glycan 1664 (HEMO 1664), HEMO conjugated to Glycan 1851 (HEMO 1851), histidine-proline rich glycoprotein (HPRG) conjugated to Glycan 1689 (HPRG 1689), HPRG conjugated to Glycan 2321 (HPRG 2321), IGG conjugated to Glycan 1607 (IGG 1607), IGG conjugated to Glycan 1689 (IGG 1689), IGG conjugated to Glycan 1744 (IGG 1744), IGG conjugated to Glycan 1810 (IGG_1810), IGG conjugated to Glycan 2123 (IGG_2123), TRAN conjugated to Glycan 1419 (TRAN_1419), TRAN conjugated to glycan 1810 (TRAN_1810), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) conjugated to Glycan 2016 (ADAMTS13_2016), ADAMTS13 conjugated to Glycan 2119 (ADAMTS_2119), attractin (ATRN) conjugated to Glycan 1851 (ATRN 1851), ATRN conjugated to Glycan 1865 (ATRN_1854), ATRN conjugated to Glycan 1876 (ATRN-1876), cluster of differentiation 109 (CD 109) conjugated to Glycan 933Attorney Docket: 206085-0170-00WO(CD109_933), CD109 conjugated to Glycan 1851 (CD109 1851 ), CD109 conjugated to Glycan 1854 (CD109 1854), cluster of differentiation 163 (CD 163) conjugated to Glycan 1282 (CD163 1611), CD163 conjugated to Glycan 1591 (CD163 1591), CLDN163 conjugated to Glycan 1612 (CD163_1612), CLDN163 conjugated to Glycan 1849(CD163 1849), CLDN163 conjugated to Glycan 1868 (CD163 1868), CLDN163 conjugated to Glycan 1955 (CD163_1955), CLDN163 conjugated to Glycan 1995 (CD163_1955), CLDN163 conjugated to Glycan 2071 (CD163_2071), CLDN163 to conjugated Glycan 2102 (CD163_2102), CLDN163 conjugated to Glycan 2142 (CD163_2142), CLDN163 conjugated to Glycan 2468 (CD163_2468), cluster of differentiation 42D (CD42D) conjugated to Glycan 1994 (CD42D_1994), colony stimulating factor 1 receptor (CSF1R) conjugated to Glycan 1689 (CSF1R 1689), CSF1R conjugated to Glycan 1851 (CSF1R 1851), CSF1R conjugated to Glycan 1906 (CSF1R 1906), CSF1R conjugated to Glycan 1956 (CSF1R 1956), CSF1R conjugated to Glycan 2071 (CSF1R_2O71), CSF1R conjugated to Glycan 2102 (CSF1R_21O2), CSF1R conjugated to Glycan 2120 (CSF1R_212O), CSF1R conjugated to Glycan 2134 (CSF1R_2134), CSF1R conjugated to Glycan 2140 (CSF1R_214O), CSF1R conjugated to Glycan 2281 (CSF1R_2281), fibulin 1 (FBLN1) conjugated to Glycan 1282 (FBLN1 1282), low density lipoprotein receptor-related protein 1 (LRP1) conjugated to Glycan 933 (LRP1 933), LRP1 conjugated to Glycan 1851 (LRP1 1851), LRP1 conjugated to Glycan 1876 (LRP1_1876), lumican (LUM) conjugated to Glycan 1851 (LUM_1851), LUM conjugated to Glycan 1854 (LUM_1854), LUM conjugated to Glycan 1855 (LUM_1855), mannose receptor C-type 1 (MMR) conjugated to Glycan 1689 (MMR_1689), MMR conjugated to Glycan 1736 (MMR 1736), MMR conjugated to Glycan 1810 (MMR_1810), MMR conjugated to Glycan 1811 (MMR_1811), MMR conjugated to Glycan 1851 (MMR_1851), MMR conjugated to Glycan 1854 (MMR_1854), MMR conjugated to Glycan 1908 (MMR 1908), plexin domain containing 2 (PLXDC2) conjugated to Glycan 1547 (PLXDC2 1547), PLXDC2 conjugated to Glycan 1851 (PLXDC2_1851), PLXDC2 conjugated to Glycan 1860 (PLXDC2 1860), PLXDC2 conjugated to Glycan 1890 (PLXDC2 1890), PLXDC2 conjugated to Glycan 1973 (PLXDC2 1973), PLXDC2 conjugated to Glycan 2135 (PLXDC2_2135), Serpin G1 (IC1) conjugated to Glycan 1851 (IC 1 1851), IC1 conjugated to Glycan 1855 (IC 1 1855), IC1 conjugated to Glycan 1860 (IC 1 1860), sex hormone-binding globulin (SHBG) conjugated to Glycan 1736Attorney Docket: 206085-0170-00WO(SHBGJ736), SHBG conjugated to Glycan 1810 (SHBGJ810), SHBG conjugated to Glycan 1811 (SHBG 1811), SHBG conjugated to Glycan 1851 (SHBG 1851), SHBG conjugated to Glycan 1854 (SHBG 1854), Sushi, von Willebrand Factor Type A (SVEP1) conjugated to Glycan 1851 (SVEP1 1851), and SVEP1 conjugated to Glycan 2142 (SVEP1_2142).

[0010] In some embodiments, the glycoprotein profile comprises: i) the level of: ANGIO_2540, CLUS_1607, and TRAN 607, or ii) the level of: ANGIO_2540, CLUS_1607, and TRAN_1607, andA) the level of: HAPT 1689, HAPT 1851, HEMO_2540, and IGGJ419;B) the level of: A2M 1867, APOD 2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, and VDBP 2175;C) the level of: HAPT 1689, HAPT 1851, HEMO 2540, APOD 2175, HAPT_2321, IGG 1257, AAT_2321, AGP_2321, FET_2175, FET_2540, and IGG 1502;D) the level of: HAPT 1689, HAPT 1851, HEMO 2540, A2M 1867, HAPT_2321, IGG 1257, FET_2175, FET_2540, and AGP_2540;E) the level of: HEMO_2175 and IGGJ257; orF) the level of: HEMO_2540, A2M_1867, APOD_2175, HAPT 2321, HEMO_2175, IGG 1257, TRAN_2175, VDBP 2175, AGP 2321, FET_2175, IGG 1502, AGP 1257, AGP 1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS 1257, CLUS 1851, CLUS 2013, CLUS 2540, HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRAN 1419, and TRAN 1810.

[0011] In some embodiments, step c) comprises: i) providing a substrate comprising a plurality of antibody spots; ii) incubating the substrate in a blocking solution; iii) incubating the substrate with the serum sample from the subject; iv) treating the substrate with an enzymatic releasing solution; and v) scanning the substrate by mass spectrometry to detect and identify the presence of glycans.

[0012] In some embodiments, the substrate comprising a plurality of antibody spots comprises a plurality of spots against at least one antigen, wherein the antibodies of each spot bind a singleAttomev Docket: 206085-0170-00WO antigen. In some embodiments, the at least one antigen comprises at least one selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1. In some embodiments, the substrate comprises at least one antibody spot for each of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG.

[0013] In some embodiments, the substrate is selected from the group consisting of glass, polydimethylsiloxane (PDMS), epoxy silane-coated glass, and epoxysilane-coated PDMS.

[0014] In some embodiments, the blocking solution is bovine serum albumin (BSA).

[0015] In some embodiments, the enzymatic releasing solution comprises PNGase F.

[0016] In some embodiments, the mass spectrometry is selected from the group consisting of: matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EAST) mass spectrometry.

[0017] In some embodiments, the step of scanning the substrate is preceded by a step of spraying the substrate with a MALDI matrix material.

[0018] In some embodiments, the MALDI matrix solution is selected from the group consisting of: 2,5-dihydroxybenzoic acid, a-cyano-4-hydroxycinnamic acid, sinapinic acid, 1,5- diaminonaphthalene, and 9-aminoacridine.

[0019] In some embodiments, the present invention provides a device for rapid detection of hepatocellular carcinoma (HCC) comprising a solid substrate spotted with a plurality of antibodies, wherein each antibody spot comprises a plurality of antibodies that bind a single antigen, wherein the substrate comprises at least one antibody spot for at least one antigen selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1Attomev Docket: 206085-0170-00WO

[0020] In some embodiments, the substrate comprises at least one antibody spot for each of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG.

[0021] In some embodiments, the present invention provides a kit for detecting hepatocellular carcinoma comprising: a device of the present invention; at least one blocking solution; at least one enzymatic releasing solution; and at least one MALDI matrix material. In some embodiments, the blocking solution is bovine serum albumin. In some embodiments, the enzymatic releasing solution comprises PNGase F. In some embodiments, the MALDI matrix solution is a-cyano-4-hydroxy cinnamic acid.

[0022] In some embodiments, the present invention provides a method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; and d) determining that the subject has HCC when the level of the at least one glycan is altered compared to that in the serum of an individual without HCC.

[0023] In some embodiments, the at least one glycoprotein comprises at least one selected from the group consisting of: AAT, AGP, A2M, ANGIO, APOD, APOH, CERU, FET, CLUS, HAPT, HEMO, IGG, TRAN, VDBP, HPRG, ADAMTS13, ATRN, CD 109, CD 163, CD42D, CSF1R, FBLN1, LRP1, LUM, MMR, PLXDC2, IC1, SHBG, and SVEP1.

[0024] In some embodiments, the at least one glycan is selected from the group consisting of: Glycan 933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.

[0025] In some embodiments, step d) comprises determining that the subject has HCC when the level of the at least one glycan is increased in the serum of the subject compared to the serum ofAttomev Docket: 206085-0170-00WO an individual without HCC, and wherein the one glycan is selected from the group consisting of: Glycan 1257, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1607, Glycan 1664, Glycan 1689, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1867, Glycan 2013, Glycan 2123, Glycan 2175, Glycan 2321, and Glycan 2540.

[0026] In some embodiments, step d) comprises determining that the subject has HCC when the level of the at least one glycan is decreased in the serum of the subject compared to the serum of an individual without HCC, and wherein the one glycan is selected from the group consisting of: Glycan 933, Glycan 1736, Glycan 1860, Glycan 1876, Glycan 2071, Glycan 2102, and Glycan 2142.

[0027] In some embodiments, the present invention provides a method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; and d) determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein in the serum of the subject is altered compared to the serum of an individual without HCC.

[0028] In some embodiments, the at least one glycoprotein comprises at least one selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

[0029] In some embodiments, the at least one glycan is selected from the group consisting of: Glycan 933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.Attorney Docket: 206085-0170-00WO

[0030] In some embodiments, the at least one glycoform of the at least one protein comprises at least one selected from the group consisting of: ANGIO_2540, CLUS_1607, TRAN_1607, HAPT 1689, HAPT 1851, HEMO 2540, IGG 1419, A2M 1867, APOD 2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, VDBP 2175, AAT 2321, AGP 2321, FET 2175, FET_2540, IGG_1502, AGP_2540, AGP_1257, AGP_1219, AGP_1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU_1257, CERU_2175, CERU 2321, CLUS_1257, CLUS 1851, CLUS_2013, CLUS_2540, HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG_2123, TRANJ419, TRAN 1810, ADAMTS13_2016, ADAMTS_2119, ATRN 1851, ATRN_1854, ATRNJ876, CD109_933, CD109 1851, CD109_1854, CD163 1611, CD163 1591, CD163 1612, CD163 1849, CD163 1868, CD163_1955, CD163_1955, CD163_2071, CD163_2102, CD163_2142, CD163_2468, CD42D 1994, CSF1R 1689, CSF1R 1851, CSF1R 1906, CSF1R 1956, CSF1R 2071, CSF1R_21O2, CSF1R_212O, CSF1R 2134, CSF1R 2140, CSF1R 2281, FBLN1_1282, LRP1 933, LRP1 1851, LRP1 1876, LUM_1851, LUM 854, LUM_1855, MMR 1689, MMR 736, MMR 1810, MMR_1811, MMR 1851, MMR 1854, MMR_1908, PLXDC2_1547, PLXDC2 1851, PLXDC2 1860, PLXDC2 1890, PLXDC2 1973, PLXDC2 2135, IC 1_1851, IC1_1855, IC1 1860, SHBG 1736, SHBG 1810), SHBG 1811, SHBG 1851, SHBG 1854, SVEP1 1851, and SVEP1_2142.

[0031] In some embodiments, step d) comprises determining that the subject has HOC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HOC, wherein the at least one glycoform of the at least one glycoprotein is selected from the group consisting of: ADMTS13_2016, ATRN_1851, ATRNJ854, CD109 1851, CD109J854, CSF1R_1689, CSF1R 1851, FBLN1_1282, LRP1 1851, LUM_1851, LUMJ854, LUM 1855, MMR_1689, MMRJ810, MMR+1811, MMR 1851, MMR 1854, PLXDC2 1851, IC1 1851, IC1_1855, SHBG_1810, SHBG 1811, SHBG 1851, SHBG 1854, SVEP1 2142, ANGIO 2540, CLUS 1607, TRAN 1607, HAPT 1689, HAPT 1851, HEMO_2540, IGG 419, A2M 1867, APOD 2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, VDBP 2175, AAT_2321, AGP 2321, FET_2175, FET_2540, IGGJ502, AGP_2540, AGP_1257, AGP_1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257,Attomev Docket: 206085-0170-00WOCERU 2175, CERU 2321, CLUSJ257, CLUSJ 851, CLUS_2013, CLUS_2540,HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRAN 1419, and TRAN 1810.

[0032] In some embodiments, step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HCC, wherein the at least one glycoform of the at least one glycoprotein is selected from the group consisting of: ADAMTS13_2119, ATRN_1876, CD109_933, CD163J282, CD163 1591, CD163 1612, CD163_1849, CD163 1868, CD163 1955, CD163J995, CD163_2071, CD163_2102, CD163_2142, CD163_2468, CD42DJ994, CSF1R 1906, CSF1R_1956, CSF1R 2071, CSF1R_21O2, CSF1R 2120, CSF1R 2134, CSF1R 2140, CSF1R 2281, LRP1 933, LRP1 1876, MMR 736, MMR 1908, PLXDC2 1547, PLXDC2_1860, PLXDC2 1890, PLXDC2 1973, PLXDC2 2135, IC1 1860, SHBG 1736, and SVEP1 2142.BRIEF DESCRIPTION OF DRAWINGS

[0033] The following detailed description of embodiments provided herein will be better understood when read in conjunction with the appended drawings. It should be understood that embodiments provided in the present disclosure are not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0034] Figure 1, comprising Figure 1 A-Figure IL, depicts the results of example experiments demonstrating N-glycan heterogeneity in HCC tissue. Figures 1A-1J depict ten tissue sections with the pathology annotated cancer lesion highlighted showing tumor specific alterations in N- linked glycosylation. M / Z values are given below each image. Figures 1A-1E depict tissues with predominantly changes in fucosylation, while Figures IF- 1 J depict tissues with changes in branching but not fucosylation. Figure IK depicts a heat map showing N-glycan clusters in different patient groups. Figure IL depicts a correlation plot showing that N-glycan alteration occurs in clusters. For all panels, M / Z values are shown. Color scale is used to represent intensity of spectra, with blue being weakest and red being strongest.

[0035] Figure 2, comprising Figure 2A-Figure 2E, depicts the results of example experiments demonstrating glycans observed in tissue are also observed in matching serum. Figure 2A depicts the method of serum N-glycan analysis. Figures 2A-2D depict serum N-linked analysis from 14Attorney Docket: 206085-0170-00WO representative HCC patients for whom matched tissue N-glycan analysis was performed. Samples were analyzed in triplicate and sample numbers are shown either below or adjacent to spots. Figure 2B is for the N-glycan at 2320.829, Figure 2C for the N-glycan at 2393.848 and Figure 2D for the N-glycan at 2539.907. Figure 2E depicts a graph showing the relationship between positively correlated tissue and serum glycans. In all figures m / z values are included. Asterisk indicates those glycans that are fucosylated.

[0036] Figure 3, comprising Figure 3A-Figure 31, depicts the results of glycoproteomics for the analysis of fucosylated glycoproteins. Figure 3A depicts the proteomic workflow. Figure 3B depicts a volcano plot showing altered glycopeptides in HCC. Figures 3C-3E depict three glycopeptides that were found to be increased in HCC samples. Figure 3C is a glycopeptide from clutsterin, Figure 3D is a peptide from alpha-2 macroglobulin, and Figure 3E is a glycopeptide from hemopexin. Figure 3F depicts a workflow for the GlycoTyper, used to orthogonally confirm N-glycan alterations. Figure 3G depicts GlycoTyper analysis of clusterin, showing either changes in total fucosylation or on a specific N-glycan (m / z 2539.907), Figure 3H depicts GlycoTyper analysis of alpha-2-macroglobulin (A2M), showing either changes in total fucosylation or on a specific N-glycan (m / z 2172.772), Figure 31 depicts GlycoTyper analysis of hemopexin, showing either changes in total fucosylation or on a specific N-glycan (m / z 2539.907).

[0037] Figure 4, comprising Figure 4A- Figure 4L, depicts the GlycoTyper method for the serum analysis of specific glycoproteins. Figure 4A depicts slides spotted with 16 antibodies to the following: (1) anti-Alpha 1 Antitrypsin, (2) anti- Alpha IB-Glycopotein, (3) anti alphal-Acid Glycoprotein, (4) anti-Alpha-2-Macroglobulin, (5) anti-Angiotensinogen II / III, (6) anti- Apolipoprotein D, (7) anti-Apolipoprotein H (ApoH), (8) anti-Ceruloplasmin, (9) anti -Clusterin, 10) anti-Fetuin, 11) anti-Haptoglobin, 12) anti-Hemopexin, (13) anti histidine-proline rich glycoprotein; (14) anti-IgG, (15) anti-Transferrin; (16) anti-Vitamin D Binding Protein. Figures 4B-4D depict examples of GlycoTyper data for the 16 captured proteins for N-glycan at 2539.881 (Figures 4B & 4E), N-glycan at 2174.654 (Figures 4C & 4F) and N-glycan at 1809.639 (Figures 4D & 4G). Figures 4B-4D are from patients with HCC, while Figures 4E-4G are from patients with cirrhosis. Figure 4H depicts a bar group showing the mean (with SD) level of N- glycan at 2539.907 on haptoglobin. Figure 41 depicts a bar group showing the mean (with SD) level of N-glycan at 1663.581 on transferrin. Figures 4J-4K depict a similar analysis onAttomev Docket: 206085-0170-00WO angiotensinogen (Figure 4J) and hemopexin (Figure 4K). Figure 4L depicts ROC curves for Model G, AFP and AFP-L3 of discriminant ability to classify all or early-stage HCC from cirrhosis.

[0038] Figure 5 depicts the results of example experiments identifying the N-Glycans found in Tissue and Serum. 1) Each m / z peak that was detected and associated with a N-glycan is listed. Not all N-glycans were detected in a given tissue / serum sample. Analyses comparing tissue and serum utilized a consolidated peak list of only N-glycans detected in both sample types. 2) Observed m / z values 3) Error in ppm (particles per million) calculated as followed Error = ((Theoretical m / z - Observed m / z| / Theoretical m / z)* 106 4) Composition of N-glycans 5) Putative N-glycan structure by Glyco WorkBench.

[0039] Figure 6 depicts the glycoforms as part of the SOML algorithms. Column 1 is the feature used in model development. For simplicity, the protein and the m / z value rounded up to a whole integer was used. A plus mark within column indicates this feature was used in that model. Features in “bold” text indicate those features that play a heavy role in specific model. A-G are the specific models as described in Table 4.DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is based, in part, upon the discovery of novel combinations of biomarkers detected in subject samples that are unique to subjects with hepatocellular carcinoma (HCC). Accordingly, in some embodiments, the present invention provides methods for early detection of HCC based on alterations in the glycosylation of proteins in a biological sample.The method includes the determination of the glycoprotein profile in the sample, production of a patient health profile comprising a plurality of glycoproteins in the glycoprotein profile. The glycoprotein profile may be monitored by targeted analysis of proteins captured on a substrate by a plurality of antibodies.Definitions:

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.Attomev Docket: 206085-0170-00WO

[0042] As used herein, each of the following terms has the meaning associated with it in this section.

[0043] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0044] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0045] The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

[0046] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Natural antibodies are typically tetramers of immunoglobulin molecules. The term “antibody” as used herein encompasses antibody fragments, which refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Antibodies or antibody fragments as described herein may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fab, F(ab)2, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0047] The term “antigen” or “Ag” as used herein is defined as a molecule that binds to an antibody or a T cell receptor. Any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomicAttomev Docket: 206085-0170-00WODNA or RNA. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. The present disclosure provides, but is not limited to, the use of partial nucleotide sequences. Moreover, an antigen need not be encoded by a “gene” at all. An antigen can be generated, synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0048] The terms “biomarker” and “marker” are used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event and / or pathologic condition.

[0049] The phrase “body sample” or “biological sample” is used herein in its broadest sense. A sample may be of any biological tissue or fluid from which biomarkers of the present invention may be assayed. Examples of such samples include but are not limited to blood, saliva, buccal smear, feces, lymph, urine, gynecological fluids, biopsies, amniotic fluid and smears. Samples that are liquid in nature are referred to herein as “bodily fluids.” Body samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to aspirate bodily fluids. Methods for collecting various body samples are well known in the art. Frequently, a sample will be a “clinical sample,” i.e., a sample derived from a patient. Such samples include, but are not limited to, bodily fluids which may or may not contain cells, e.g., blood (e.g., whole blood, serum or plasma), urine, saliva, tissue or fine needle biopsy samples, and archival samples with known diagnosis, treatment and / or outcome history. Biological or body samples may also include sections of tissues such as frozen sections taken for histological purposes. The sample also encompasses any material derived by processing a biological or body sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample, proteins or nucleic acid molecules extracted from the sample. Processing of a biological or body sample may involve at least one of: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

[0050] As used herein, the term “carbohydrate” is intended to include any of a class of aldehyde or ketone derivatives of polyhydric alcohols. Therefore, carbohydrates include starches, celluloses, gums and saccharides. Although, for illustration, the term “saccharide” or “glycan” is used elsewhere herein, this is not intended to be limiting. It is intended that the methods providedAttorney Docket: 206085-0170-00WO herein can be directed to any carbohydrate, and the use of a specific carbohydrate is not meant to be limiting to that carbohydrate only.

[0051] As used herein, the term “cell-surface glycoprotein” refers to a glycoprotein, at least a portion of which is present on the exterior surface of a cell. In some embodiments, a cell-surface glycoprotein is a protein that is positioned on the cell-surface such that at least one of the glycan structures is present on the exterior surface of the cell.

[0052] In the context of the present invention, the term “control,” when used to characterize a subject, refers, by way of non-limiting examples, to a subject that is healthy, to a patient that otherwise has not been diagnosed with a disease. The term “control sample” refers to one, or more than one, sample that has been obtained from a healthy subject or from a non-disease tissue such as normal colon.

[0053] The term “control or reference standard” describes a material comprising none, or a normal, low, or high level of one of more of the marker (or biomarker) expression products of at least one the markers (or biomarkers) of the invention, such that the control or reference standard may serve as a comparator against which a sample can be compared.

[0054] “Differentially increased levels” refers to biomarker levels which are at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or more, and / or 0.5-fold, 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold higher or more, as compared with a control.

[0055] “Differentially decreased levels” refers to biomarker levels which are at least at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or less, and / or 0.9-fold, 0.8-fold, 0.6-fold, 0.4-fold, 0.2-fold, 0.1-fold or less, as compared with a control.

[0056] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.Attomev Docket: 206085-0170-00WO

[0057] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease, or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0058] As used herein, the terms “fragment” or “functional fragment” refer to a fragment of an antigen or a nucleic acid sequence encoding an antigen that, when administered to a subject, provides an increased immune response. Fragments are generally 10 or more amino acids or nucleic acids in length. “Fragment” may mean a polypeptide fragment of an antigen that is capable of eliciting an immune response in a subject. A fragment of an antigen may be 100% identical to the full length except missing at least one amino acid from the N and / or C terminal, in each case with or without signal peptides and / or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length antigen, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.

[0059] ‘ ‘Isolated” as used herein means (1) altered or removed from the natural state and / or (2) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting) and / or otherwise previously associated, and / or (3) designed, produced, prepared, and / or manufactured by the hand of man. In some embodiments, a nucleic acid or a peptide naturally present in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0060] As used herein “endogenous” refers to any material from or produced inside the organism, cell, tissue or system.Attomev Docket: 206085-0170-00WO

[0061] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0062] The “level” of at least one biomarkers means the absolute or relative amount or concentration of the biomarker in the sample. The term “level” also refers to the absolute or relative amount of glycosylation of the biomarker in the sample.

[0063] As is known in the art and used herein “glycans” are sugars (e.g., oligosaccharides and polysaccharides). Glycans can be monomers or polymers of sugar residues typically joined by glycosidic bonds also referred to herein as linkages. In some embodiments, the terms “glycan”, “oligosaccharide” and “polysaccharide” may be used to refer to the carbohydrate portion of a glycoconjugate (e.g., glycoprotein, glycolipid or glycoprotein). A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'- fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.). The term “glycan” includes homo and heteropolymers of sugar residues. The term “glycan” also encompasses a glycan component of a glycoconjugate (e.g., of a glycoprotein, glycolipid, glycoprotein, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from a glycoconjugate.

[0064] As used herein, the term “antibody array” refers to a tool used to identify glycans on proteins that interact with any of a number of different antibodies linked to the array substrate. In some embodiments, antibody arrays comprise a number of immobilized antibodies, referred to herein as “antibody spots”. In some embodiments, glycan arrays comprise at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 350, at least 1000 or at least 1500 antibody spots. In some embodiments, antibody arrays may be customized to present a desired set of antibody spots.

[0065] The term “glycoconjugate”, as used herein, encompasses all molecules in which at least one sugar moiety is covalently linked to at least one other moiety. The term specifically encompasses all biomolecules with covalently attached sugar moieties, including for example N- linked glycoproteins, O-linked glycoproteins, glycolipids, glycoproteins, etc.

[0066] The term “glycoform”, is used herein to refer to a particular form of a glycoconjugate. That is, when the same backbone moiety (e.g., polypeptide, lipid, etc.) that is part of a glycoconjugate has the potential to be linked to different glycans or sets of glycans, then eachAttomev Docket: 206085-0170-00WO different version of the glycoconjugate (i.e., where the backbone is linked to a particular set of glycans) is referred to as a “glycoform.”

[0067] The term “glycosidase” as used herein refers to an agent that cleaves a covalent bond between sequential sugars in a glycan or between the sugar and the backbone moiety (e.g. between sugar and peptide backbone of glycoprotein). In some embodiments, a glycosidase is an enzyme. In certain embodiments, a glycosidase is a protein (e.g., a protein enzyme) comprising at least one polypeptide chain. In certain embodiments, a glycosidase is a chemical cleavage agent.

[0068] A “glycoprotein preparation”, as that term is used herein, refers to a set of individual glycoprotein molecules, each of which comprises a polypeptide having a particular amino acid sequence (which amino acid sequence includes at least one glycosylation site) and at least one glycan covalently attached to the at least one glycosylation site. Individual molecules of a particular glycoprotein within a glycoprotein preparation typically have identical amino acid sequences but may differ in the occupancy of the at least one glycosylation site and / or in the identity of the glycans linked to the at least one glycosylation site. That is, a glycoprotein preparation may contain only a single glycoform of a particular glycoprotein, but more typically contains a plurality of glycoforms. Different preparations of the same glycoprotein may differ in the identity of glycoforms present (e.g., a glycoform that is present in one preparation may be absent from another) and / or in the relative amounts of different glycoforms.

[0069] The term “lectin” as used herein encompasses any amino acid and peptide bond-based compound having specific binding affinity to carbohydrates. Typically it relates to non-antibody polypeptides found in nature featuring specific carbohydrate binding. The term “lectin” includes functional fragments and derivatives thereof, the latter terms being defined in analogy to the same terms used in the context of antibodies.

[0070] “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject’s clinical parameters.

[0071] The term “N-glycan”, as used herein, refers to a polymer of sugars that has been released from a glycoconjugate but was formerly linked to the glycoconjugate via a nitrogen linkage. N-Attomev Docket: 206085-0170-00WO linked glycans are glycans that are linked to a glycoconjugate via a nitrogen linkage at asparagine residues within conserved protein structural motifs of N / X (any amino acid except proline) / S or T (serine or threonine). A diverse assortment of N-linked glycans exists, but is typically based on the common core pentasaccharide (Man)3(GlcNAc)(GlcNAc).

[0072] “Naturally-occurring” as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man is a naturally occurring sequence.

[0073] By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). The term “nucleic acid” typically refers to large polynucleotides.

[0074] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5’-direction.

[0075] The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand that are located 5’ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3’ to a reference point on the DNA are referred to as “downstream sequences.”

[0076] The term “O-glycan”, as used herein, refers to a polymer of sugars that has been released from a glycoconjugate but was formerly linked to the glycoconjugate via an oxygen linkage. O- linked glycans are glycans that are linked to a glycoconjugate via an oxygen linkage. O-linked glycans are typically attached to glycoproteins via N-acetyl-D-galactosamine (GalNAc) or via N- acetyl-D-glucosamine (GlcNAc) to the hydroxyl group of L-serine (Ser) or L-threonine (Thr). Some O-linked glycans also have modifications such as acetylation and sulfation. In someAttomev Docket: 206085-0170-00WO instances O-linked glycans are attached to glycoproteins via fucose or mannose to the hydroxyl group ofL-serine (Ser) or L-threonine (Thr).

[0077] The term “pre-cancerous” or “pre-neoplastic” and equivalents thereof shall be taken to mean any cellular proliferative disorder that is undergoing malignant transformation. Examples of such conditions include, in the context of colorectal cellular proliferative disorders, cellular proliferative disorders with a high degree of dysplasia and the following classes of adenomas: Level 1: penetration of malignant glands through the muscularis mucosa into the submucosa, within the polyp head; Level 2: the same submucosal invasion, but present at the junction of the head to the stalk; Level 3: invasion of the stalk; and Level 4: invasion of the stalk's base at the connection to the colonic wall. In some instances, pre-neoplastic is used to describe a normal tissue that will form tumors.

[0078] As used herein, “predisposition” refers to the property of being susceptible to a cellular proliferative disorder. A subject having a predisposition to a cellular proliferative disorder has no cellular proliferative disorder, but is a subject having an increased likelihood of having a cellular proliferative disorder.

[0079] A “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0080] The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T ”

[0081] As used herein, the term “providing a prognosis” refers to providing a prediction of the probable course and outcome of colorectal cancer, including prediction of severity, duration, chances of recovery, etc. The methods can also be used to devise a suitable therapeutic plan, e.g., by indicating whether or not the condition is still at an early stage or if the condition has advanced to a stage where aggressive therapy would be ineffective.

[0082] A “reference level” of a biomarker means a level of the biomarker, for example level of a type of glycan that is indicative of a particular disease state, phenotype, or lack thereof, as wellAttorney Docket: 206085-0170-00WO as combinations of disease states, phenotypes, or lack thereof. A “positive” reference level of a biomarker means a level that is indicative of a particular disease state or phenotype. A “negative” reference level of a biomarker means a level that is indicative of a lack of a particular disease state or phenotype.

[0083] As used herein, the term “saccharide” refers to a polymer comprising at least one monosaccharide group. Saccharides, therefore, include mono-, di-, tri- and polysaccharides (or glycans). Glycans can be branched or branched. Glycans can be found covalently linked to nonsaccharide moieties, such as lipids or proteins (as a glycoconjugate). These covalent conjugates include glycoproteins, glycopeptides, peptidoglycans, glycoproteins, glycolipids and lipopolysaccharides. The use of any one of these terms also is not intended to be limiting as the description is provided for illustrative purposes. In addition to the glycans being found as part of a glycoconjugate, the glycans can also be in free form (i.e., separate from and not associated with another moiety).

[0084] By the term “specifically binds,” as used herein, is meant a molecule, such as an antibody, which recognizes and binds to another molecule or feature, but does not substantially recognize or bind other molecules or features in a sample.

[0085] “ Standard control value” as used herein refers to a predetermined glycan level. The standard control value is suitable for the use of a method of the present invention, in order for comparing the amount of glycan of interest that is present in a sample. An established sample serving as a standard control provides an average amount of glycan of interest that is typical for an average, healthy person of reasonably matched background, e.g., gender, age, ethnicity, and medical history. A standard control value may vary depending on the biomarker of interest and the nature of the sample.

[0086] The terms “subject,” “patient,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some non-limiting embodiments, the patient, subject or individual is a mammal, bird, poultry, cattle, pig, horse, sheep, ferret, primate, dog, cat, guinea pig, rabbit, bat, or human.

[0087] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, prevention, or eradication of at least one sign or symptom of a disease or disorder.Attomev Docket: 206085-0170-00WO

[0088] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, at least one of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0089] Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Description

[0090] The present invention is based in part on novel methods that allow for the early detection of hepatocellular carcinoma (HCC). The methods relate analyzing the glycoprotein profile of a sample, and comparing the levels of various glycoproteins to a control or reference value. In some embodiments, the glycoprotein profile of a sample is analyzed by capture of specific proteins in the sample using an antibody array, treatment of the captured proteins with an enzymatic releasing solution which releases the glycans on the captured proteins, and analysis of the specific glycans released from the captured proteins on a spot-by-spot basis by mass spectrometry.

[0091] In various embodiments, glycoproteins are profiled using mass spectrometry. In another embodiment, glycoproteins are profiled using matrix-assisted laser desorption / ionization (MALDI). In another embodiment, proteins and cells are characterized using MALDI Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry. In another embodiment,Attomev Docket: 206085-0170-00WO proteins and cells are characterized using MALDI time of flight (MALDI-TOF) mass spectrometry.Affinity capture Arrays

[0092] The present invention provides in part antibody arrays that allow for the generation of structural glycan information for hundreds of individual glycoprotein targets. The antibody arrays utilize an efficient workflow that allows for the capture of specific proteins, treatment of captured protein with an enzymatic releasing solution that releases glycans from the captured proteins, and glycan analysis of the specific captured proteins on a spot-by-spot basis by mass spectrometry.

[0093] In various embodiments, the array is formed on a substrate. The substrate can be any substrate suitable for mass spectrometric analysis. Examples of suitable substrates include, but are not limited to, glass, plastic, polymer, or ceramic slides or multiwell plates, nitrocellulose, graphene, and gold. In some embodiments, the substrate can be functionalized or coated to enhance cell adherence. For example, the substrate surface can include an indium tin oxide coating, a thiol-reactive coating, a carboxy-reactive coating, an aldehyde-reactive coating, an azide-reactive coating, a gelatin coating, a collagen coating, a poly-l-lysine coating, a polyornithine coating, an extracellular matrix coating, a protein coating (such as cadherins, immunoglobulins, selectins, mucins, integrins, streptavidin, and the like), an epoxysilane coating, surface ionization, and the like.

[0094] In various embodiments, the array comprises a plurality of protein-binding (capture) molecules. In some embodiments, the capture molecules are antibodies or antibody fragments (e.g., an antibody array). In some embodiments, the capture molecules are aptamers (e.g., an aptamer array). In some embodiments, the capture molecules are proteins known to interact with a target protein. In some embodiments, the cell-binding molecules are derivatives of proteins or cleavage proteins that interact with a target protein. In some embodiments, the derivatives lack their native enzymatic activity. In some embodiments, the derivatives comprise the binding site of the target protein.

[0095] An affinity capture array (e.g., an antibody array) spotted on the substrate allows for capture and analysis of one to hundreds of different glycoproteins. Thus, in some embodiments, the affinity capture arrays of the invention can comprise one to hundreds of different captureAttomev Docket: 206085-0170-00WO molecules (e.g., antibodies), each specific for one protein target of interest. In some embodiments, the affinity capture array comprises a single capture molecule (e.g., antibody) that binds a single glycoprotein. In some embodiments, the capture array comprises a single capture molecule that binds a variety of proteins.

[0096] Spotting of capture molecules can be achieved through any suitable technique, including but not limited to inkjet printing, acoustic printing, fine print spotting, flow patterning on a functionalized substrate, contact printing (stamping), contact printing on a functionalized substrate, incubating on coated substrates (such as a nitrocellulose coating), or microprinting using epoxy-coated glass substrate or poly-amine glass substrate with printing needles or strips with very fine feature resolution.

[0097] In some embodiments, capture molecules are bound to a substrate. In some embodiments, capture molecules, (e.g., antibodies) are covalently bonded to a substrate. For example, an antibody may be directly conjugated to a nucleophilic or electrophilic group of a substrate. In some embodiments, capture molecules are covalently bonded to a substrate through at least one linker. For example, a linker may be conjugated to a nucleophilic or electrophilic group of a substrate and an antibody may be conjugated to a nucleophilic or electrophilic group of the linker. In some embodiments, capture molecules are bound to a substrate by non-covalent interactions between the antibodies and the substrate. For example, an antibody may be bound to a substrate through ionic, polar, and / or non-polar interactions. In some embodiments, capture molecules are bound to a substrate by non-covalent interactions between the antibodies and at least one compound covalently bonded to the substrate. For example, a linker may be covalently bonded to a substrate and an antibody may interact with the linker through ionic, polar, and / or non-polar interactions. In some embodiments, capture molecules are bound to a substrate by non- covalent interactions between the antibodies and at least one compound bound to the substrate by non-covalent interactions. For example, a linker may be bound to a substrate through ionic, polar, and / or non-polar interactions and an antibody may be bound to the linker through ionic, polar, and / or non-polar interactions. In some embodiments, capture molecules are bound to a substrate by a covalent bond between the capture molecules and at least one compound bound to the substrate by non-covalent interactions. For example, a linker may be bound to a substrate through ionic, polar, and / or non-polar interactions and an antibody may be conjugated to a nucleophilic or electrophilic group of the linker.Attomev Docket: 206085-0170-00WO

[0098] The affinity capture (e.g., antibody) arrays can be arranged in any desired grid or pattern. In certain embodiments, individual antibody spots are spaced laterally and longitudinally in an array of rows and / or columns. In some embodiments, individual cell-binding molecule spots are regularly spaced at about 1-200 pm in separation. In some embodiments, individual cell-binding molecule spots are regularly spaced at about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 p, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, or about 200 pm. In some embodiments, the affinity capture arrays comprise about 10-1,000,000 individual capture molecule spots. In another embodiment, the affinity capture arrays comprise about 500-500,000 individual capture molecule spots. In another embodiment, the affinity capture arrays comprise about 100-100,000 individual capture molecule spots. In some embodiments, the affinity capture arrays comprise capture molecule spots at a density of about 200 cell-binding molecule spots per cm2to about 20,000 antibody spots per cm2. In various embodiments, arraying capture molecule spots can be aided with the use of at least one grid, such as a well slide module.

[0099] In certain embodiments, each spot comprises a single specific capture molecule. In another embodiment, each spot comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different capture molecules (e.g., antibodies). In these embodiments, specific binding to a number of particular capture molecules within the feature can be determined by use of different detectable labels on a second set of capture agents, with each label corresponding to a particular capture molecule.

[0100] In various embodiments, antibody arrays can include antibodies, antibody fragments, or combinations thereof. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies. Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, includingAttomev Docket: 206085-0170-00WO full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species.

[0101] In various embodiments, the antibody arrays can include antibodies, antibody fragments, or combinations thereof. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies. Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species.

[0102] While the methods describe the use of antibody arrays, it should be understood that any suitable capture molecule having an affinity to a cell-surface molecule of interest can be used to cells as would be understood by those having skill in the art. For example, antibodies can be replaced or supplemented with at least one antigen, aptamers, affibodies, proteins, peptides, nucleic acids, carbon nanotubes, and fragments thereof. The capture molecules are also not limited to an array pattern and can be provided in any shape or form desired.Glycan Analysis

[0103] In various aspects, the present invention provides a method of isolating and analyzing glycoproteins from a sample. In some embodiments, the method comprises the steps of a) providing an affinity capture array according to the present invention; b) incubating the affinity capture array with a sample containing at least one glycoprotein to be analyzed; c) rinsing the affinity capture array to remove unbound proteins, d) spraying the affinity capture array with an enzymatic releasing solution to release glycans from the captured glycoproteins, and e) scanning the single-cell capture array by mass spectrometry.

[0104] In some embodiments, the sample of step b) is any liquid comprising at least one glycoprotein of interest. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a biofluid. Examples of biofluids include, but are notAttorney Docket: 206085-0170-00WO limited to, serum, plasma, whole blood, bronchial lavage, urine, cerebrospinal fluid (CSF), synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, otic fluid, gastric fluid, ascitic fluid, nasal mucus, and breast milk. In some embodiments, the sample comprises at least one additive. In some embodiments, the additive is an aqueous additive. In some embodiments, the additive is a non-aqueous additive. Examples of suitable additives include, but are not limited to, a diluent, a solvent, a base, an acid, a buffer, a salt, or any combination thereof. In some embodiments, the sample is a fluid derived from a solid tissue.

[0105] In some embodiments, step b) comprises incubating the affinity capture array with the sample for between about 1 minute and about 24 hours. In some embodiments, the affinity capture array is incubated with the sample for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0106] In some embodiments, step c) comprises rinsing away unbound proteins and glycoproteins off the array. In some embodiments, the step of rinsing unbound proteins and glycoproteins off the array comprises at least one wash with phosphate-buffered saline (PBS). InAttorney Docket: 206085-0170-00WO some embodiments, the step of rinsing unbound proteins and glycoproteins off the array comprises at least one water wash. In some embodiments, the step of rinsing unbound proteins and glycoproteins off the array comprises at least one PBS wash and at least one water wash.

[0107] In some embodiments, step d) comprises spraying the affinity capture array with an enzymatic releasing solution to release glycans from the captured glycoproteins. Examples of enzymatic releasing agents include, but are not limited to, trypsin, Endoglycosidase H (Endo H), Endoglycosidase F (EndoF), N-Glycanase F (PNGase F), PNGase A, O-glycanase, and / or at least one protease (e.g., trypsin, or LysC), or chemically (e.g., using anhydrous hydrazine (N) or reductive or non-reductive beta-elimination (O)).

[0108] In some embodiments, step d) further comprises incubating the affinity capture array after it has been sprayed with the enzymatic releasing solution. In some embodiments, the affinity capture array is incubated for between about 1 minute and about 24 hours. In some embodiments, the affinity capture array is incubated for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.Attomev Docket: 206085-0170-00WO

[0109] In some embodiments, step e) comprises analyzing the substrate by mass spectrometry. Examples of mass spectrometric methods include fast atom bombardment mass spectrometry (FAB-MS), liquid chromatography mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS / MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), and matrix-assisted laser desorption / ionization tandem mass spectrometry (MALDI-MS / MS).

[0110] Mass spectrometry imaging is a powerful tool that has been used to correlate various peptides, proteins, lipids and metabolites with their underlying histopathology in tissue sections. One significant advantage is that matrix-assisted laser desorption / ionization (MALDI) imaging combined with tandem mass spectrometry reveals detailed structural information about the molecules and fragments in a sample. A wide range of molecular weights can be detected by mass spectrometry imaging. Also, the high mass resolution allows distinguishing two peaks with close molecular weights, which subsequently improves the detection specificity. In addition, tens or even hundreds of single molecules and fragments can be detected at femtomole levels in one single image, allowing detection of low concentrations of molecules. Therefore, MALDI imaging facilitates high-throughput analysis of single cells. MALDI imaging can also be used for performing quantitative assays. Another significant advantage of MALDI imaging is that it has the capability of detecting an unknown compound without any prior knowledge of the analytes. Therefore, this technique is particularly suitable for biomarker discovery research.

[0111] MALDI is a soft ionization mass spectrometric technique that is suitable for use in the analysis of biomolecules, such as proteins, peptides, nucleic acids, sugars, and the like, which tend to be fragile and fragment when ionized by conventional ionization methods. This makes the coupling of MALDI and the single-cell capture arrays particularly amenable to glycomic metabolomic analysis.

[0112] Generally, MALDI comprises a two-step process. In the first step, desorption is triggered by an ultraviolet (UV) laser beam. The matrix material absorbs the UV laser radiation, which leads to the ablation of an upper layer of the matrix material, thereby producing a hot plume. The hot plume contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters, and nanodroplets. In the second step, the analyte molecules are ionized, e.g., protonated or deprotonated, in the hot plume.Attomev Docket: 206085-0170-00WO

[0113] The matrix material comprises a crystallized molecule capable of absorbing the UV laser radiation. Common matrix materials include, but are not limited to, a-cyano-4- hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid / 2-hydroxy-5- methoxybenzoic acid, 2,4,6-trihydroxyacetophenone, 6-aza-2 -thiothymine, 3-hydroxypicolinic acid, 3 -aminoquinoline, anthranilic acid, 5-chloro-2-mercaptobenzothiazole, 2,5- dihydroxyacetophenone, ferulic acid, and 2-(4-hydroxyphenylazo) benzoic acid. A solution of the matrix material is made in highly purified water and an organic solvent, such as acetonitrile or ethanol. In some embodiments, a small amount of trifluoroacetic acid (TFA) also can be added to the solution.

[0114] The matrix solution can then be mixed with the analyte, e.g., a protein sample. This solution is then deposited onto a MALDI plate, wherein the solvents vaporize leaving only the recrystallized matrix comprising the analyte molecules embedded in the MALDI crystals.

[0115] The glycans detectable by the invention include straight chain and branched oligosaccharides as well as naturally occurring and synthetic glycans. For example, the glycan can be a glycoaminoacid, a glycopeptide, a glycolipid, a glycosaminoglycan (GAG), a glycoprotein, a whole cell, a cellular component, a glycoconjugate, a glycomimetic, a glycophospholipid anchor, glycosylphosphatidylinositol (GPI)-linked glycoconjugates, bacterial lipopolysaccharides and endotoxins. The glycans can also include N-glycans, -glycans, glycolipids and glycoproteins.

[0116] In some instances, the glycans detectable by the invention include two or more sugar units. Any type of sugar unit can be present in the glycans of the invention, including, for example, allose, altrose, arabinose, glucose, galactose, gulose, fucose, fructose, idose, lyxose, mannose, ribose, talose, xylose, or other sugar units. Such sugar units can have a variety of modifications and substituents. For example, sugar units can have a variety of substituents in place of the hydroxy, carboxylate, and methylenehydroxy substituents. Thus, lower alkyl moieties can replace any of the hydrogen atoms from the hydroxy, carboxylic acid and methylenehydroxy substituents of the sugar units in the glycans of the invention. For example, amino acetyl can replace any of the hydroxy or hydrogen atoms from the hydroxy, carboxylic acid and methylenehydroxy substituents of the sugar units in the glycans of the invention.

[0117] In some embodiments, the methods of the present invention can include determining the glycoprofile of a glycoprotein. The properties of a glycoprotein can beAttomev Docket: 206085-0170-00WO determined by analyzing the glycans of the intact glycoprotein. Properties of the glycans which can be determined include: the mass of part or all of the saccharide structure, the charges of the chemical units of the saccharide, identities of the chemical units of the saccharide, confirmations of the chemical units of the saccharide, total charge of the saccharide, total number of sulfates of the saccharide, total number of acetates, total number of phosphates, presence and number of carboxylates, presence and number of aldehydes or ketones, dye-binding of the saccharide, compositional ratios of substituents of the saccharide, compositional ratios of anionic to neutral sugars, presence of uronic acid, enzymatic sensitivity, linkages between chemical units of the saccharide, charge, branch points, number of branches, number of chemical units in each branch, core structure of a branched or unbranched saccharide, the hydrophobicity and / or charge / charge density of each branch, absence or presence of GlcNAc and / or fucose in the core of a branched saccharide, number of mannose in an extended core of a branched saccharide, presence or absence or sialic acid on a branched chain of a saccharide, the presence or absence of galactose on a branched chain of a saccharide.

[0118] The property of the glycan that is detected by this method can also be any structural property of a glycan or unit. For instance, the property of the glycan can be the molecular mass or length of the glycan. In other embodiments the property can be the compositional ratios of substituents or units, type of basic building block of a polysaccharide, hydrophobicity, enzymatic sensitivity, hydrophilicity, secondary structure and conformation (i.e., position of helices), spatial distribution of substituents, linkages between chemical units, number of branch points, core structure of a branched polysaccharide, ratio of one set of modifications to another set of modifications (i.e., relative amounts of sulfation, acetylation or phosphorylation at the position for each), and binding sites for proteins.Methods of Biomarker Identification

[0119] Alterations in glycoprotein levels have been associated with a number of diseases. In various embodiments, the present invention provides methods of identifying biomarkers associated with a disease or disorder.

[0120] In some embodiments, a biomarker is a unique glycoprotein or glycoform that is present only in subjects with or without the disease or disorder. In some embodiments, aAttomev Docket: 206085-0170-00WO biomarker is a glycoprotein or glycoform present at an increased or decreased level in a subject with the disease or disorder relative to a normal healthy control subject.

[0121] In some embodiments, the method comprises the steps of: a) obtaining a plurality of biofluid samples from a number of subjects with a disease or disorder of interest and a plurality of healthy control subjects; b) contacting each sample with an affinity capture array of the present invention; c) scanning the arrays by mass spectrometry to detect and identify the presence of glycoproteins and form glycoprotein profiles for each subject; and d) selecting at least one glycoprotein or glycoform that are differentially expressed between subjects with the disease or disorder and healthy control subjects as biomarkers.

[0122] In some embodiments, step d) comprises performing differential analysis on the samples to identify glycoprotein or glycoform that are differentially expressed between disease and control subjects.

[0123] In some embodiments, step d) comprises inputting the glycoprotein profiles formed in step c) into a machine learning algorithm to select at least one biomarker that are differentially expressed between disease and control subjects. In some embodiments, the machine learning algorithm produces a diagnostic model comprising at least one biomarker and at least one additional input. Additional inputs include, but are not limited to, age, sex, race, ethnicity, and other molecules in a sample from a subject. Examples of other molecules include, but are not limited to, nucleic acids (e.g., DNA, RNA, free nucleic acids, modified nucleic acids, etc.), proteins (e.g., alpha fetoprotein (AFP), alanine transaminase (ALT), aspartate aminotransferase (AST), etc.), peptides (e.g., insulin, etc.), lipids (e.g., cholesterol, triglycerides, acyl fatty acids, etc.), carbohydrates (e.g., sucrose, glycogen, etc.), metabolites (e.g., bilirubin, creatinine, etc.), and combinations thereof.

[0124] In some embodiments, the method comprises: a) obtaining a plurality of biofluid samples from a number of subjects with a disease or disorder of interest and a plurality of subjects without the disease or disorder; b) contacting each sample with an affinity capture array of the present invention; c) scanning the arrays by mass spectrometry to detect and identify the presence of glycoproteins and form glycoprotein profiles for each subject; andAttomev Docket: 206085-0170-00WO d) selecting at least one glycoprotein or glycoform that are differentially expressed between subjects with the disease or disorder and the subjects without the disease or disorder as biomarkers.

[0125] In some embodiments, the subjects of step a) with the disease or disorder and the subjects without the disease or disorder may have at least one other disease in common. For example, the disease or disorder for which biomarkers are being identified may be HCC, but both subject populations have another liver disease or disorder, e.g., cirrhosis, hepatitis, nonalcoholic fatty liver disease (NAFLD), etc.

[0126] In some embodiments, step d) comprises performing differential analysis on the samples to identify glycoproteins or glycoforms that are differentially expressed between disease and control subjects.

[0127] In some embodiments, step d) comprises inputting the glycoprotein profiles formed in step c) into a machine learning algorithm to select at least one biomarker that are differentially expressed between disease and control subjects. In some embodiments, the machine learning algorithm produces a diagnostic model comprising at least one biomarker and at least one additional input. Additional inputs include, but are not limited to, age, sex, race, ethnicity, and other molecules in a sample from a subject. Examples of other molecules include, but are not limited to, nucleic acids (e.g., DNA, RNA, free nucleic acids, modified nucleic acids, etc.), proteins (e.g., alpha fetoprotein (AFP), alanine transaminase (ALT), aspartate aminotransferase (AST), etc ), peptides (e.g., insulin, etc ), lipids (e.g., cholesterol, triglycerides, acyl fatty acids, etc.), carbohydrates (e.g., sucrose, glycogen, etc.), metabolites (e.g., bilirubin, creatinine, etc.), and combinations thereof.

[0128] In some embodiments, the method comprises: a) obtaining a plurality of biofluid samples from a number of subjects with a disease or disorder of interest, a plurality of subjects without the disease or disorder, and a plurality of healthy control subjects; b) contacting each sample with an affinity capture array of the present invention; c) scanning the arrays by mass spectrometry to detect and identify the presence of glycoproteins and form glycoprotein profiles for each subject; andAttomev Docket: 206085-0170-00WO d) selecting at least one glycoprotein or glycoform that are differentially expressed between subjects with the disease or disorder and both the subjects without the disease or disorder and the healthy control subjects as biomarkers.

[0129] In some embodiments, the subjects of step a) with the disease or disorder and the subjects without the disease or disorder may have at least one other disease in common that the healthy control subjects do not have. For example, the disease or disorder for which biomarkers are being identified may be HCC, but both subject populations have another liver disease or disorder, e.g., cirrhosis, hepatitis, non-alcoholic fatty liver disease (NAFLD), etc.

[0130] In some embodiments, step d) comprises performing differential analysis on the samples to identify glycoproteins or glycoforms that are differentially expressed between disease and control subjects.

[0131] In some embodiments, step d) comprises inputting the glycoprotein profiles formed in step c) into a machine learning algorithm to select at least one biomarker that are differentially expressed between disease and control subjects. In some embodiments, the machine learning algorithm produces a diagnostic model comprising at least one biomarker and at least one additional input. Additional inputs include, but are not limited to, age, sex, race, ethnicity, and other molecules in a sample from a subject. Examples of other molecules include, but are not limited to, nucleic acids (e.g., DNA, RNA, free nucleic acids, modified nucleic acids, etc.), proteins (e.g., alpha fetoprotein (AFP), alanine transaminase (ALT), aspartate aminotransferase (AST), etc ), peptides (e.g., insulin, etc ), lipids (e.g., cholesterol, triglycerides, acyl fatty acids, etc.), carbohydrates (e.g., sucrose, glycogen, etc.), metabolites (e.g., bilirubin, creatinine, etc.), and combinations thereof.

[0132] In some embodiments, the biomarkers selected have a p-value less than 0.05.

[0133] In some embodiments, the method further comprises performing logistic regression analysis to determine the accuracy (area under the curve, AUC), specificity, and / or sensitivity of each glycoprotein or glycoform as a diagnostic biomarker for the disease or disorder. In some embodiments, the method further comprises performing logistic regression analysis to determine the accuracy (area under the curve, AUC), specificity, and / or sensitivity of a diagnostic model produced by a machine learning algorithm for the disease or disorder.

[0134] In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the fold-change between a subject with a disease or disorder and a control subject is at leastAttomev Docket: 206085-0170-00WO about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000.

[0135] In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the AUC is at least about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the AUC is 1.0.

[0136] In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the specificity is at least about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the specificity is 1.0.

[0137] In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the sensitivity is at least about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. In some embodiments, a glycoprotein or glycoform is selected as a biomarker if the sensitivity is 1.0.

[0138] In some embodiments, a diagnostic model is selected if the AUC is at least about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. In some embodiments, a diagnostic model is selected as a biomarker if the AUC is 1.0.

[0139] In some embodiments, a diagnostic model is selected as a biomarker if the specificity is at least about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. In some embodiments, a diagnostic model is selected as a biomarker if the specificity is 1.0.

[0140] In some embodiments, a diagnostic model is selected as a biomarker if the sensitivity is at least about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or aboutAttomev Docket: 206085-0170-00WO0.99. In some embodiments, a diagnostic model is selected as a biomarker if the sensitivity is 1.0.Method of Diagnosis

[0141] Alterations in glycosylation have been associated with a number of diseases. Generally, these changes are observed through glycan analysis of complex protein mixtures or through the analysis of a few specific individual proteins. In various embodiments, the present invention also provides methods for diagnosing a disease state or disorder state or the progression of a disease state or disorder state by detecting at least one specific glycan, glycoprotein, and / or glycoform whose presence or level (whether absolute or relative) may be correlated with a particular disease state (including susceptibility to a particular disease) and / or the change in the concentration of such glycans over time.

[0142] The detected glycans and detected changes in glycosylation can be used for detecting, treating, and / or preventing a variety of early stage diseases and / or cancers. In some embodiments, the presence of such glycans is indicative of the presence of cancer and can provide information on the prognosis of such a disease, for example, whether the disease is in remission or is becoming more aggressive. Patients with familial history of cancer, and hence a heightened risk of developing the disease, can be tested regularly to monitor their propensity for disease.

[0143] In some embodiments, the methods of the present invention provide a method of diagnosing a disease or condition in a subject comprising the steps of detecting the glycans present in a biological sample from a subject, establishing a glycan profile for the subject, comparing the glycan profile from the subject to glycan profile from a normal sample or diseased sample, and determining whether the subject has the disease or condition, wherein the glycans are detected using the presently disclosed methods described elsewhere herein.

[0144] In some embodiments, the methods of the present invention provide a method of diagnosing or detecting the presence of hepatocellular carcinoma (HCC). In some embodiments, the present invention provides a method of differentiating HCC from a different liver disease or disorder. In some embodiments, the present invention provides a method of determining that a subject with another liver disease or disorder also has HCC. Examples of other liver diseases and disorders that may also be present include, but are not limited to, hepatitis, cirrhosis, alcoholicAttomev Docket: 206085-0170-00WO fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic liver steatohepatitis (NASH), cholangiocarcinoma, and primary sclerosing cholangitis.

[0145] In some embodiments, the method comprises the steps of: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; and d) determining that the subject has HCC when the level of the at least one glycan in the serum of the subject is altered compared to the serum of an individual without HCC.

[0146] In some embodiments, the at least one glycoprotein comprises at least one selected from the group consisting of alpha-1 antitrypsin (AAT); alpha-l-acid glycoprotein (AGP); alpha-2-macroglobulin (A2M); angiotensin (ANGIO); apolipoprotein D (APOD); apolipoprotein H (APOH); ceruloplasmin (CERU); fetuin (FET); clusterin (CLUS); haptoglobin (HAPT); hemopexin (HEMO); immunoglobulin G (IGG); transferrin (TRAN); vitamin D binding protein (VDBP); histidine-proline rich glycoprotein (HPRG); a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13); attractin (ATRN); cluster of differentiation 109 (CD 109); cluster of differentiation 163 (CD 163); cluster of differentiation 42D (CD42D); colony stimulating factor 1 receptor (CSF1R); fibulin 1 (FBLN1); low density lipoprotein receptor-related protein 1 (LRP1); lumican (LUM); mannose receptor C-type 1 (MMR); plexin domain containing 2 (PLXDC2); serpin G1 (IC1), sex hormone-binding globulin (SHBG); and Susi, Von Willebrand Factor Type A (SVEP1).

[0147] In some embodiments, the at least one glycan comprises at least one glycan selected from the group consisting of: Glycan 933 (Hex3HexNAc2; [M+Na] m / z = 933.490), Glycan 1257 (Hex5HexNAc2; [M+Na] m z = 1257.423), Glycan 1282 (Hex3dHexlHexNAc3; [M+Na] m / z = 1282.454), Glycan 1419 (Hex6HexNAc2; [M+Na] m / z = 1419.476), Glycan 1445 (Hex4dHexlHexNAc3; [M+Na] m z = 1444.507), Glycan 1502 (Hex4HexNAc4; [M+Na] m / z = 1501.529), Glycan 1547 (Hex4dHexlHexNAc3; [M+SO4+2Na] m / z = 1546.539), Glycan 1591 (Hex4dHex2HexNAc3; [M+Na] m / z = 1590.558), Glycan 1607 (Hex5dHexlHexNAc3; [M+Na] m z = 1606.560), Glycan 1611 (Hex4dHexNAc3NeuAcl; [M+2Na] m / z = 1611.604), Glycan 1664 (Hex5HexNAc4; [M+Na] m / z = 1663.581), Glycan 1689 (Hex3dHexlHexNAc5; [M+Na] m / z = 1688.613), Glycan 1736 (Hex4dHexlHexNAc3NeuAcl; [M+Na] m / z = 1735.516), Glycan 1744 (Hex8HexNAc2; [M+Na] m z = 1743.538), Glycan 1810 (Hex5dHexlHexNAc4;Attomev Docket: 206085-0170-00WO[M+Na] m / z = 1809.639), Glycan 1811 (Hex5dHexlHexNAc4; [M+H+Na] m / z = 1810.639), Glycan 1849 (Hex6dHexlHexNAc3S04; [M+Nal] m / z = 1848.722), Glycan 1851 (Hex4dHexlHexNAc5; [M+Na] m 'z = 1850.666), Glycan 1854 (Hex5dHexNAc4NeuAclSO4; [M+Na2] m / z = 1853.674), Glycan 1855 (Hex5dHex2NAc3SO4; [M+Na2] m / z = 1854.675), Glycan 1860 ([M+] m / z = 1859.615), Glycan 1867 (Hex5HexNAc5; [M+Na] m / z = 1866.661), Glycan 1868 (Hex4HexNAc3NeuAcSO4; [M+Na3] m / z = 1867.75), Glycan 1876 (Hex5HexNAc5; [M+Na] m / z = 1875.611), Glycan 1890 (Hex5dHexlHexNAc4]SO4]; [M+Na] m / z = 1889.93), Glycan 1906 (Hex9HexNAc2; [M+Na] m / z = 1905.624), Glycan 1908 (Hex4HexNAc6; [M+Na] m / z = 1907.636), Glycan 1955 (Hex5HexNAc4]NeuAcl]; [M+Na] m / z = 1954.676), Glycan 1956 (Hex5dHex2HexNAc4; [M+Na] m / z = 1955.693), Glycan 1973 (Hex6dHexlHexNAc3SO4; [M+Na3] m / z = 1972.693), Glycan 1994 (Hex6HexNAc3NeuAclSO4; [M+Na] m / z = 1993.655), Glycan 1995 (Hex6dHex2HexNAc3 ; [M+Na] m / z = 1994.662), Glycan 2013 (Hex5dHexlHexNAc5; [M+Na] m z = 2012.719), Glycan 2016 (Hex6HexNAc3NeuAclSO4; [M+Na2] m / z = 2015.726), Glycan 2071 (Hex5HexNAc5SO42; [M+Na3] m / z = 2070.634), Glycan 2102 (Hex5dHex3HexNAc4; [M+Na] m / z = 2101.749), Glycan 2119 (Hex6dHex2HexNAc3NeuAclSO42; [M+Na3] m / z = 2118.609), Glycan 2120 (Hex4HexNAc5SO4; [M+Na3] m / z = 2119.605), Glycan 2123 (Hex5dHexlHexNAc4NeuAcl; [M+2Na] m / z = 2122.7173), Glycan 2134 (Hex4dHexlHexNAc6SO4; [M+Na] m / z = 2133.726), Glycan 2135 (Hex7HexNAc3(SO4)2];[M+Na3] m / z = 2134.74), Glycan 2140 (Hex6dHexlHexNAc2NeuAcSO4]; [M+Na] m / z = 2139.709), Glycan 2142 (Hex4dHexlHexNAc5NeuAc; [M+Na] m / z = 2141.721), Glycan 2175 (Hex6dHexlHexNAc5; [M+Na] m 'z = 2174.772), Glycan 2281 (Hex7HexNAc3SO42; [M+Na3] m / z = 2280.693), Glycan 2321 (Hex6dHex2HexNAc5; [M+Na] m / z = 2320.829), Glycan 2468 (Hex6dHex3HexNAc5; [M+H+Na] m / z =2467.892), and Glycan 2540 (Hex7dHexlHexNAc6+Nal; [M+H] m / z = 2539.904).

[0148] In some embodiments, step d) comprises determining that the subject has HCC when the level of the at least one glycan is increased in the serum of the subject compared to the serum of an individual without HCC. In some embodiments, the at lest one glycan is selected from the group consisting of Glycan 1257, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1607, Glycan 1664, Glycan 1689, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1851,Attomev Docket: 206085-0170-00WOGlycan 1854, Glycan 1855, Glycan 1867, Glycan 2013, Glycan 2123, Glycan 2175, Glycan 2321, and Glycan 2540.

[0149] In some embodiments, step d) comprises determining that the subject has HCC when the level of at least one glycan is decreased in the serum of the subject compared to the serum of an individual without HCC. In some embodiments, the at least one glycan is selected from the group consisting of Glycan 933, Glycan 1736, Glycan 1860, Glycan 1876, Glycan 2071, Glycan 2102, and Glycan 2142.

[0150] In some embodiments, the method comprises the steps of: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; and d) determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein in the serum of the subject is altered compared to the serum of an individual without HCC.

[0151] In some embodiments, the at least one glycoprotein comprises at least one selected from the group consisting of AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; ADAMTS13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

[0152] In some embodiments, the at least one glycoform of the at least one glycoprotein comprises at least one selected from the group consisting of ANGIO conjugated to Glycan 2540 (ANGIO_2540), CLUS conjugated to Glycan 1607 (CLUS_1607), TRAN conjugated to Glycan 1607 (TRAN 1607), HAPT conjugated to Glycan 1689 (HAPT 1689), HAPT conjugated to Glycan 1851 (HAPT 1851), HEMO conjugated to Glycan 2540 (HEMO 2540), IGG conjugated to Glycan 1419 (IGG_1419), A2M conjugated to Glycan 1867 (A2M_1867), APOD conjugated to Glycan 2175 (APOD_2175), CLUS conjugated to Glycan 1689 (CLUS_1689), HAPT conjugated to Glycan 2321 (HAPT_2321), HEMO conjugated to Glycan 2175 (HEMO_2175), IGG conjugated to Glycan 1257 (IGG 1257), IGG conjugated to Glycan 1867 (IGG 1867), TRAN conjugated to Glycan 2175 (TRAN_2175), VDBP conjugated to Glycan 2175 (VDBP_2175), AAT conjugated to Glycan 2321 (AAT_2321), alpha-1 acid glycoprotein (AGP) conjugated to Glycan 2321 (AGP_2321), FET conjugated to Glycan 2175 (FET_2175), FET conjugated to Glycan 2540 (FET_2540), IGG conjugated to Glycan 1502 (IGG_1502), AGPAttorney Docket: 206085-0170-00WO conjugated to Glycan 2540 (AGP_2540), AGP conjugated to Glycan 1257 (AGP_1257), AGP conjugated to Glycan 1219 (AGP_1219), AGP conjugated to Glycan 1445 (AGP_1445), ANGIO conjugated to Glycan 1851 (ANGIO 1851), APOH conjugated to Glycogen 1851 (APOH 1851), APOH conjugated to Glycogen 1867 (APOH 1867), APOH conjugated to Glycogen 2157 (APOH_2157), CERU conjugated to Glycogen 1257 (CERU_1257), CERU conjugated to Glycogen 2175 (CERU_2175), CERU conjugated to Glycogen 2321 (CERU_2321), CLUS conjugated to Glycan 1257 (CLUS_1257), CLUS conjugated to Glycan 1851 (CLUS_1851), CLUS conjugated to Glycan 2013 (CLUS_2013), CLUS conjugated to Glycan 2540 (CLUS_2540), HEMO conjugated to Glycan 1664 (HEMO 664), HEMO conjugated to Glycan 1851 (HEMO 1851), histidine-proline rich glycoprotein (HPRG) conjugated to Glycan 1689 (HPRG 1689), HPRG conjugated to Glycan 2321 (HPRG 2321), IGG conjugated to Glycan 1607 (IGG 1607), IGG conjugated to Glycan 1689 (IGG 1689), IGG conjugated to Glycan 1744 (IGG_1744), IGG conjugated to Glycan 1810 (IGG_1810), IGG conjugated to Glycan 2123 (IGG_2123), TRAN conjugated to Glycan 1419 (TRAN_1419), TRAN conjugated to glycan 1810 (TRAN_1810), ADAMTS13 conjugated to Glycan 2016 (ADAMTS13_2016), ADAMTS13 conjugated to Glycan 2119 (ADAMTS 2119), ATRN conjugated to Glycan 1851 (ATRN_1851), ATRN conjugated to Glycan 1865 (ATRN_1854), ATRN conjugated to Glycan 1876 (ATRN_1876), CD109 conjugated to Glycan 933(CD109 933), CD109 conjugated to Glycan 1851 (CD109 1851), CD109 conjugated to Glycan 1854 (CD109_1854), CD 163 conjugated to Glycan 1282 (CD163 1611), CD 163 conjugated to Glycan 1591 (CD163 1591), CLDN163 conjugated to Glycan 1612 (CD163 1612), CLDN163 conjugated to Glycan 1849 (CD163 1849), CLDN163 conjugated to Glycan 1868(CD163 1868), CLDN163 conjugated to Glycan 1955 (CD163 1955), CLDN163 conjugated to Glycan 1995 (CD163_1955), CLDN163 conjugated to Glycan 2071 (CD163_2071), CLDN163 to conjugated Glycan 2102 (CD163_2102), CLDN163 conjugated to Glycan 2142 (CD163_2142), CLDN163 conjugated to Glycan 2468 (CD163_2468), CD42D conjugated to Glycan 1994 (CD42D 1994), CSF1R conjugated to Glycan 1689 (CSF1R 1689), CSF1R conjugated to Glycan 1851 (CSF1R 1851), CSF1R conjugated to Glycan 1906 (CSF1R 1906), CSF1R conjugated to Glycan 1956 (CSF1R 1956), CSF1R conjugated to Glycan 2071 (CSF1R_2O71), CSF1R conjugated to Glycan 2102 (CSF1R_21O2), CSF1R conjugated to Glycan 2120 (CSF1R_212O), CSF1R conjugated to Glycan 2134 (CSF1R_2134), CSF1RAttomev Docket: 206085-0170-00WO conjugated to Glycan 2140 (CSF1R_214O), CSF1R conjugated to Glycan 2281 (CSF1R_2281), FBLN1 conjugated to Glycan 1282 (FBLN1 1282), LRP1 conjugated to Glycan 933 (LRP1 933), LRP1 conjugated to Glycan 1851 (LRP1 1851), LRP1 conjugated to Glycan 1876 (LRP1 1876), LUM conjugated to Glycan 1851 (LUM 1851), LUM conjugated to Glycan 1854 (LUM_1854), LUM conjugated to Glycan 1855 (LUM_1855), MMR conjugated to Glycan 1689 (MMR_1689), MMR conjugated to Glycan 1736 (MMR_1736), MMR conjugated to Glycan 1810 (MMR_1810), MMR conjugated to Glycan 1811 (MMR_1811), MMR conjugated to Glycan 1851 (MMR_1851), MMR conjugated to Glycan 1854 (MMR_1854), MMR conjugated to Glycan 1908 (MMR_1908), PLXDC2 conjugated to Glycan 1547 (PLXDC2_1547), PLXDC2 conjugated to Glycan 1851 (PLXDC2 1851), PLXDC2 conjugated to Glycan 1860 (PLXDC2 1860), PLXDC2 conjugated to Glycan 1890 (PLXDC2 1890), PLXDC2 conjugated to Glycan 1973 (PLXDC2 1973), PLXDC2 conjugated to Glycan 2135 (PLXDC2 2135), IC1 conjugated to Glycan 1851 (IC1 1851), IC1 conjugated to Glycan 1855 (IC1 1855), IC1 conjugated to Glycan 1860 (IC 1 1860), SHBG conjugated to Glycan 1736 (SHBG 1736), SHBG conjugated to Glycan 1810 (SHBG 1810), SHBG conjugated to Glycan 1811 (SHBG 1811), SHBG conjugated to Glycan 1851 (SHBG 1851), SHBG conjugated to Glycan 1854 (SHBG 1854), SVEP1 conjugated to Glycan 1851 (SVEP1 1851), and SVEP1 conjugated to Glycan 2142 (SVEP1_2142).

[0153] In some embodiments, step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HCC. In some embodiments, the at least one glycoform of the at least one glycoprotein is selected from the group consisting of ADMTS13_2016, ATRN 851, ATRN 854, CD109 1851, CD109 1854, CSF1R 1689, CSF1R 1851, FBLN1_1282, LRP1_1851, LUM 1851, LUMJ854, LUMJ855, MMR 1689, MMRJ810, MMR+1811, MMR 1851, MMR 1854, PLXDC2 1851, IC1_1851, IC1 1855, SHBG 1810, SHBG 1811, SHBG 1851, SHBG 1854, SVEP1 2142, ANGIO 2540, CLUS 1607, TRAN_1607, HAPT_1689, HAPT 1851, HEMO_2540, IGG 1419, A2MJ867, APOD_2175, CLUS 1689, HAPT_2321, HEMO_2175, IGGJ257, IGG 1867, TRAN 2175, VDBP_2175, AAT_2321, AGP_2321, FET_2175, FET_2540, IGG_1502, AGP_2540, AGPJ257, AGP 1219, AGP_1445, ANGIO_1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS 1257,Attomev Docket: 206085-0170-00WOCLUSJ851, CLUS 2013, CLUS 2540, HEMO J 664, HEMO J 851, HPRGJ 689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRAN 1419, and TRAN 1810.

[0154] In some embodiments, step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is decreased in the serum of the subject compared to the serum of an individual without HCC. In some embodiments, the at least one glycoform of the at least one glycoprotein is selected from the group consisting of ADAMTS13_2119, ATRN_1876, CD109_933, CD163_1282, CD163 1591, CD163 1612, CD163 1849, CD163 1868, CD163J955, CD163J995, CD163_2071, CD163_2102, CD163_2142, CD163_2468, CD42D_1994, CSF1R_19O6, CSF1R_1956, CSF1R 2071, CSF1R_21O2, CSF1R_212O, CSF1R_2134, CSF1R_214O, CSF1R 2281, LRP1 933, LRP1 1876, MMR 1736, MMR 1908, PLXDC2 1547, PLXDC2 1860, PLXDC2 1890, PLXDC2 1973, PLXDC2 2135, IC1 1860, SHBG 1736, and SVEP1 2142.

[0155] In some embodiments, the method comprises the steps of: a) obtaining a serum sample from the subject; b) determining the glycoprotein profile of the serum sample; c) producing a patient health profile comprising the glycoprotein profile; and d) detecting the presence of HCC in the subject when the health profile of the subject is greater than that of a subject without HCC.

[0156] In some embodiments, step b) further determining the serum alpha-fetoprotein (AFP) level of the subject. In some embodiments, the patient health profile further comprises at least one of the serum AFP level, the age of the subject, the glycoprotein profile of the subject, and the sex of the subject. In some embodiments, the patient health profile comprises the serum AFP level, the age of the subject, the glycoprotein profile of the subject, and the sex of the subject.

[0157] In some embodiments, the at least one glycoprotein comprises at least one selected from the group consisting of AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; ADAMTS13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

[0158] In some embodiments, the glycoprotein profile comprises at least one glycoprotein glycosylated with at least one glycan selected from the group consisting of: GlycanAttorney Docket: 206085-0170-00WO933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.

[0159] In some embodiments, the glycoprotein profile comprises the level of at least one selected from the group consisting of ANGIO_2540, CLUS_1607, TRAN_1607, HAPT_1689, HAPT 1851, HEMO 2540, IGG 1419, A2M 1867, APOD 2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN_2175, VDBP_2175, AAT_2321, AGP_2321, FET_2175, FET_2540, IGG_1502, AGP_2540, AGPJ257, AGP 1219, AGP_1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU_1257, CERU_2175, CERU 2321, CLUS_1257, CLUS 1851, CLUS_2013, CLUS_2540, HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG_2123, TRANJ419, TRAN 1810, ADAMTS13_2016, ADAMTS_2119, ATRN 1851, ATRN_1854, ATRN_1876, CD109_933, CD109 1851, CD109 1854, CD163 1611, CD163 1591, CD163 1612, CD163 1849, CD163 1868, CD163_1955, CD163_1955, CD163_2071, CD163_2102, CD163_2142, CD163_2468, CD42DJ994, CSF1R 1689, CSF1R 1851, CSF1R 1906, CSF1R 1956, CSF1R 2071, CSF1R_21O2, CSF1R_212O, CSF1R 2134, CSF1R 2140, CSF1R 2281, FBLN1 1282, LRP1 933, LRP1 1851, LRP1 1876, LUM_1851, LUMJ854, LUM 1855, MMR 1689, MMR_1736, MMR 1810, MMR 1811, MMR 1851, MMR 1854, MMR 1908, PLXDC2 1547, PLXDC2 1851, PLXDC2 1860, PLXDC2 1890, PLXDC2_1973, PLXDC2_2135, IC 1_1851, IC1_1855, IC1 1860, SHBG 1736, SHBG 1810), SHBG 1811, SHBG 1851, SHBG 1854, SVEP1 1851, and SVEP1_2142.

[0160] In some embodiments, the patient health profile of step d) comprises the age of the patient, the serum AFP level of the subject, and the level of: ANGIO_2540, CLUS_1607, and TRAN 1607.Attorney Docket: 206085-0170-00WO

[0161] In some embodiments, the patient health profile of step d) comprises the age of the patient, the serum AFP level of the subject, and the level of: ANGIO_2540, CLUS_1607, TRAN 1607, HAPT 1689, HAPT 1851, HEMO 2540, and IGG 1419.

[0162] In some embodiments, the patient health profile of step d) comprises the age of the patient, the serum AFP level of the subject, and the level of: ANGIO_2540, CLUS_1607, TRAN_1607, A2M 1867, APOD 2175, CLUS 1689, HAPT 2321, HEMO_2175, IGG_1257, IGG 1867, TRAN_2175, and VDBP_2175.

[0163] In some embodiments, the patient health profile of step d) comprises the age of the patient, the serum AFP level of the subject, and the level of: ANGIO_2540, CLUS_1607, TRAN 1607, HAPT 1689, HAPT 1851, HEMO 2540, APOD 2175, HAPT 2321, IGG 1257, AAT_2321, AGP 2321, FET_2175, FET_2540, and IGG_1502.

[0164] In some embodiments, the patient health profile of step d) comprises the age of the patient, the serum AFP level of the subject, and the level of: ANGIO_2540, CLUS_1607, TRAN_1607, HAPT 1689, HAPT 1851, HEMO_2540, A2M 1867, HAPT 2321, IGG_1257, FET_2175, FET_2540, and AGP_2540.

[0165] In some embodiments, the patient health profile of step d) comprises the level of: ANGIO 2540, CLUSJ607, TRANJ607, HEMO_2175 and IGGJ257.

[0166] In some embodiments, the patient health profile of step d) comprises the age of the patient, the serum AFP level of the subject, the sex of the subject, and the level of: ANGIO 2540, CLUS 607, TRAN 607, HEMO_2540, A2M 867, APOD_2175, HAPT 2321, HEMO 2175, IGG 1257, TRAN 2175, VDBP 2175, AGP 2321, FET 2175, IGG 1502, AGP 1257, AGP 1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS 1257, CLUS 1851,CLUS 2013, CLUS 2540, HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG_1810, IGG_2123, TRANJ419, and TRAN 1810.

[0167] In some embodiments, step c) comprises i) providing a substrate comprising a plurality of antibody spots (i.e., an affinity capture array); ii) incubating the substrate in a blocking solution; iii) incubating the substrate with the serum sample from the subject; iv) treating the substrate with an enzymatic releasing solution; and v) scanning the substrate by mass spectrometry to detect and identify the presence of glycans.Attorney Docket: 206085-0170-00WO

[0168] In some embodiments, the substrate of step i) comprises a plurality of antibody spots against at least one antigen. In some embodiments, the substrate comprises a plurality of antibody spots, wherein each antibody spot comprises antibodies that bind to a single antigen. In some embodiments, the substrate comprises a plurality of antibody spots against at least one antigen, wherein at least one antigen is selected from the group consisting of AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; ADAMTS13; ATRN; CD109; CD163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1. In some embodiments, the substrate comprises at least one antibody spot against each of AAT, AGP, A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; and HPRG.

[0169] In some embodiments, the blocking solution of step ii) is serum. In some embodiments, the serum is bovine serum albumin (BSA).

[0170] In some embodiments, step iii) comprises incubating the substrate with the serum sample from the subject for between about 1 minute and about 24 hours. In some embodiments, the substrate array is incubated with the sample for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17Attomev Docket: 206085-0170-00WO hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0171] In some embodiments, step iii) further comprises rinsing away unbound proteins and glycoproteins off the array. In some embodiments, the step of rinsing unbound proteins and glycoproteins off the array comprises at least one wash with phosphate-buffered saline (PBS). In some embodiments, the step of rinsing unbound proteins and glycoproteins off the array comprises at least one water wash. In some embodiments, the step of rinsing unbound proteins and glycoproteins off the array comprises at least one PBS wash and at least one water wash.

[0172] In some embodiments, step iv) comprises spraying the substrate with an enzymatic releasing solution comprising at least one selected from the group consisting of: trypsin, endoglycosidase H (Endo H), endoglycosidase F (EndoF), N-glycanase F (PNGase F), PNGase A, and O-glycanase.

[0173] In some embodiments, step iv) further comprises incubating the substrate after it has been sprayed with the enzymatic releasing solution. In some embodiments, the substrate is incubated for between about 1 minute and about 24 hours. In some embodiments, the substrate is incubated for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13Attomev Docket: 206085-0170-00WO hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.In some embodiments, the mass spectrometry of step v) is selected from the group consisting of: matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry. In some embodiments, step v) is preceded by a step of spraying the substrate with a MALDI matrix. In some embodiments, the MALDI matrix is selected from the group consisting of , a- cyano-4-hydroxy cinnamic acid, 2, 5 -dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid / 2- hydroxy-5-methoxybenzoic acid, 2,4,6-trihydroxyacetophenone, 6-aza-2-thiothymine, 3- hydroxypicolinic acid, 3-aminoquinoline, anthranilic acid, 5-chloro-2-mercaptobenzothiazole, 2, 5 -dihydroxy acetophenone, ferulic acid, and 2-(4-hydroxyphenylazo) benzoic acid.

[0174] Various algorithms can be used to generate models that generate a diagnosis or prediction based on the detected glycoprotein profile of the sample. In some instances, machine learning methods are applied to the generation of such models (e.g. trained classifier). In some embodiments, the model is generated by providing a machine learning algorithm with training data in which the expected output is known in advance.

[0175] In some embodiments, the systems, devices, and methods described herein generate at least one recommendation such as treatment and / or healthcare options for a subject. In some embodiments, the systems, devices, and methods herein comprise a software module providing at least one recommendation to a user. In some embodiments, the treatment and / or healthcare option are specific to the diagnosed disease or condition. For example, a recommendation can suggest a nearby hospital, doctor, or clinic with the requisite facilities or resources for treating the disease or disorder.

[0176] In some embodiments, a classifier or trained machine learning algorithm of the present disclosure comprises a feature space. In some cases, the classifier comprises two or more feature spaces. The two or more feature spaces may be distinct from one another. In someAttomev Docket: 206085-0170-00WO embodiments, a feature space comprises information such as mass spectrometry data. When training the machine learning algorithm, training data such as glycoprotein profile data is input into the algorithm which processes the input features to generate a model. In some embodiments, the machine learning algorithm is provided with training data that includes the classification (e.g. diagnostic or test result), thus enabling the algorithm to train by comparing its output with the actual output to modify and improve the model. This is often referred to as supervised learning. Alternatively, in some embodiments, the machine learning algorithm can be provided with unlabeled or unclassified data, which leaves the algorithm to identify hidden structure amongst the cases (referred to as unsupervised learning). Sometimes, unsupervised learning is useful for identifying the features that are most useful for classifying raw data into separate cohorts.

[0177] In some embodiments, at least one set of training data are used to train a machine learning algorithm. Although exemplary embodiments of the present disclosure include machine learning algorithms that use convolutional neural networks, various types of algorithms are contemplated. In some embodiments, the algorithm utilizes a predictive model such as a neural network, a decision tree, a support vector machine, or other applicable model. In some embodiments, the machine learning algorithm is selected from the group consisting of a supervised, semi -supervised and unsupervised learning, such as, for example, a support vector machine (SVM), a Naive Bayes classification, a random forest, an artificial neural network, a decision tree, a K-means, learning vector quantization (LVQ), self- organizing map (SOM), graphical model, regression algorithm (e.g. linear, logistic, multivariate, association rule learning, deep learning, dimensionality reduction and ensemble selection algorithms. In some embodiments, the machine learning algorithm is selected from the group consisting of: a support vector machine (SVM), a Naive Bayes classification, a random forest, and an artificial neural network. Machine learning techniques include bagging procedures, boosting procedures, random forest algorithms, and combinations thereof.

[0178] Illustrative algorithms for analyzing the data include but are not limited to methods that handle large numbers of variables directly such as statistical methods and methods based on machine learning techniques. Statistical methods include penalized logistic regression, prediction analysis of microarrays (PAM), methods based on shrunken centroids, support vector machine analysis, and regularized linear discriminant analysis.Attomev Docket: 206085-0170-00WO

[0179] In certain embodiments, the present invention provides a method of a diagnosing disease or condition in a subject comprising the steps of detecting the glycans present in a biological sample from a subject and using a machine learning-based model, as described above, to predict if the subject has the disease or condition. In certain embodiments, the machinelearning based model is trained using data that may include, glycan profdes of known disease states, glycan profdes of known non-disease states, and demographic data such as age, sex, nationality, race, and ethnicity.Methods of Treatment

[0180] In some aspects, the present invention provides methods of treating hepatocellular carcinoma (HCC). In some embodiments, the method comprises the steps of a) diagnosing a subject with HCC according to a method of the present invention; and b) administering to the subject at least one anticancer treatment. In some embodiments, the at least one anticancer treatment includes at least one selected from the group consisting of chemotherapy, radiation therapy, tumor ablation, surgical excision, transplant, immunotherapy, hormonal therapy, or a combination thereof.

[0181] In some embodiments, the method further comprises a step c) forming a new patient health profde during and / or after treatment. In some embodiments, step c) further comprises comparing the patient health profde to the original health profde produced during the diagnosis of step a). In some embodiments, the method comprises comparing the patient health profde to a normal control health profde.

[0182] In some embodiments, the method comprises a step d) when the patient health profde of step c) is not significantly different from the health profde produced during the diagnosis of step a). In some embodiments, the method comprises step d) when the patient health profde, in comparison to a normal control health profde, would still indicate a diagnosis of hepatocellular carcinoma (HCC).

[0183] In some embodiments, step d) comprises transitioning the patient from the initial at least one anticancer treatment of step b) to at least one different anticancer treatment. In some embodiments, the at least one anticancer treatment of step b) are continued with the addition of the at least one different anti cancer treatment. In some embodiments, at least one anticancer treatment of step b) are discontinued and only the at least one different anti cancer treatment areAttomev Docket: 206085-0170-00WO administered. In some embodiments, all anticancer treatments of step b) are discontinued and only the at least one different anticancer treatment are administered.Kits

[0184] In some aspects, the present invention provides a kit for performing a method of the present invention. In some embodiments, the kit comprises a) an affinity capture array of the present invention; b) an enzymatic releasing solution; and c) a MALDI matrix material. In some embodiments, the enzymatic releasing solutions comprises at least one selected from the group consisting of: trypsin, endoglycosidase H (Endo H), endoglycosidase F (Endo F), N-glycanase F (PNGase F), PNGase A, and O-glycanase. In some embodiments, the MALDI matrix material is at least one selected from the group consisting of a-cyano-4-hydroxy cinnamic acid, 2,5- dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid / 2-hydroxy-5-methoxybenzoic acid, 2,4,6- trihydroxyacetophenone, 6-aza-2-thiothymine, 3 -hydroxypicolinic acid, 3 -aminoquinoline, anthranilic acid, 5-chloro-2-mercaptobenzothiazole, 2, 5 -dihydroxy acetophen one, ferulic acid, and 2-(4-hydroxyphenylazo) benzoic acid.

[0185] In some embodiments, the kit further comprises instructional materials.EMBODIMENTS

[0186] Embodiment l is a method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining a glycoprotein profde of the serum sample; c) producing a patient health profde comprising the glycoprotein profde; and d) detecting the presence of HCC in the subject when the combined health profde of the subject is greater than that of an average subject.

[0187] Embodiment 2 is the method of embodiment 1, wherein step b) further comprises determining the serum alpha-fetoprotein (AFP) level of the subject.

[0188] Embodiment 3 is the method of embodiment 1 or 2, wherein the patient health profde comprises the serum AFP level of the subject, the glycoprotein profde of the subject, the age of the subject, and the sex of the subject.Attorney Docket: 206085-0170-00WO

[0189] Embodiment 4 is the method of any one of embodiments 1-3, wherein the glycoprotein profde comprises at least one glycoprotein selected from the group consisting of alpha- 1 antitrypsin (AAT); alpha- 1 -acid glycoprotein (AGP); alpha-2-macroglobulin (A2M); angiotensin (ANGIO); apolipoprotein D (APOD); apolipoprotein H (APOH); ceruloplasmin (CERU); fetuin (FET); clusterin (CLUS); haptoglobin (HAPT); hemopexin (HEMO); immunoglobulin G (IGG); transferrin (TRAN); vitamin D binding protein (VDBP); histidineproline rich glycoprotein (HPRG); a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13); attractin (ATRN); cluster of differentiation 109 (CD 109); cluster of differentiation 163 (CD 163); cluster of differentiation 42D (CD42D); colony stimulating factor 1 receptor (CSF1R); fibulin 1 (FBLN1); low density lipoprotein receptor- related protein 1 (LRP1); lumican (LUM); mannose receptor C-type 1 (MMR); plexin domain containing 2 (PLXDC2); serpin G1 (IC1), sex hormone-binding globulin (SHBG); and Susi, Von Willebrand Factor Type A (SVEP1).

[0190] Embodiment 5 is the method of any one of embodiments 1-4, wherein the glycoprotein profde comprises at least one glycan selected from the group consisting of: Glycan 933 (Hex3HexNAc2; [M+Na] m / z = 933.490), Glycan 1257 (Hex5HexNAc2; [M+Na] m / z = 1257.423), Glycan 1282 (Hex3dHexlHexNAc3; [M+Na] m z = 1282.454), Glycan 1419 (Hex6HexNAc2; [M+Na] m / z = 1419.476), Glycan 1445 (Hex4dHexlHexNAc3; [M+Na] m / z = 1444.507), Glycan 1502 (Hex4HexNAc4; [M+Na] m 'z = 1501.529), Glycan 1547 (Hex4dHexlHexNAc3; [M+SO4+2Na] m / z = 1546.539), Glycan 1591 (Hex4dHex2HexNAc3; [M+Na] m / z = 1590.558), Glycan 1607 (Hex5dHexlHexNAc3; [M+Na] m'z = 1606.560), Glycan 1611 (Hex4dHexNAc3NeuAcl; [M+2Na] m / z = 1611.604), Glycan 1664 (Hex5HexNAc4; [M+Na] m / z = 1663.581), Glycan 1689 (Hex3dHexlHexNAc5; [M+Na] m / z = 1688.613), Glycan 1736 (Hex4dHexlHexNAc3NeuAcl; [M+Na] m / z = 1735.516), Glycan 1744 (Hex8HexNAc2; [M+Na] m / z = 1743.538), Glycan 1810 (Hex5dHexlHexNAc4; [M+Na] m / z = 1809.639), Glycan 1811 (Hex5dHexlHexNAc4; [M+H+Na] m / z = 1810.639), Glycan 1849 (Hex6dHexlHexNAc3S04; [M+Nal] m / z = 1848.722), Glycan 1851 (Hex4dHexlHexNAc5; [M+Na] m z = 1850.666), Glycan 1854 (Hex5dHexNAc4NeuAcl SO4; [M+Na2] m / z = 1853.674), Glycan 1855 (Hex5dHex2NAc3SO4; [M+Na2] m / z = 1854.675), Glycan 1860 ([M+] m / z = 1859.615), Glycan 1867 (Hex5HexNAc5; [M+Na] mz = 1866.661), Glycan 1868 (Hex4HexNAc3NeuAcSO4; [M+Na3] m / z = 1867.75), Glycan 1876 (Hex5HexNAc5; [M+Na]Attorney Docket: 206085-0170-00WO m / z = 1875.611), Glycan 1890 (Hex5dHexlHexNAc4]SO4]; [M+Na] m / z = 1889.93), Glycan 1906 (Hex9HexNAc2; [M+Na] m / z = 1905.624), Glycan 1908 (Hex4HexNAc6; [M+Na] m / z = 1907.636), Glycan 1955 (Hex5HexNAc4]NeuAcl]; [M+Na] m / z = 1954.676), Glycan 1956 (Hex5dHex2HexNAc4; [M+Na] m / z = 1955.693), Glycan 1973 (Hex6dHexlHexNAc3SO4; [M+Na3] m / z = 1972.693), Glycan 1994 (Hex6HexNAc3NeuAclSC>4; [M+Na] m / z = 1993.655), Glycan 1995 (Hex6dHex2HexNAc3; [M+Na] m / z = 1994.662), Glycan 2013 (Hex5dHexlHexNAc5; [M+Na] m / z = 2012.719), Glycan 2016 (Hex6HexNAc3NeuAclSO4; [M+Na2] m / z = 2015.726), Glycan 2071 (Hex5HexNAc5SO42; [M+Na3] m / z = 2070.634), Glycan 2102 (Hex5dHex3HexNAc4; [M+Na] m / z = 2101.749), Glycan 2119 (Hex6dHex2HexNAc3NeuAclSO42; [M+Na3] m / z = 2118.609), Glycan 2120 (Hex4HexNAc5SO4; [M+Na3] m / z = 2119.605), Glycan 2123 (Hex5dHexlHexNAc4NeuAcl; [M+2Na] m / z = 2122.7173), Glycan 2134 (Hex4dHexlHexNAc6SO4; [M+Na] m / z = 2133.726), Glycan 2135 (Hex7HexNAc3(SO4)2]; [M+Na3] m / z = 2134.74), Glycan 2140 (Hex6dHexlHexNAc2NeuAcSO4]; [M+Na] m / z = 2139.709), Glycan 2142 (Hex4dHexlHexNAc5NeuAc; [M+Na] m / z = 2141.721), Glycan 2175 (Hex6dHexlHexNAc5; [M+Na] m / z = 2174.772), Glycan 2281 (Hex7HexNAc3SO42; [M+Na3] m / z = 2280.693), Glycan 2321 (Hex6dHex2HexNAc5; [M+Na] m / z = 2320.829), Glycan 2468 (Hex6dHex3HexNAc5; [M+H+Na] m / z =2467.892), and Glycan 2540 (Hex7dHexlHexNAc6+Nal; [M+H] m / z = 2539.904).

[0191] Embodiment 6 is the method of any one of embodiments 1-5, wherein the glycoprotein profde comprises the level of at least one glycoform selected from the group consisting of angiotensin (ANGIO) conjugated to Glycan 2540 (ANGIO 2540), clusterin (CLUS) conjugated to Glycan 1607 (CLUS_1607), transferrin (TRAN) conjugated to Glycan 1607 (TRAN 1607), haptoglobin (HAPT) conjugated to Glycan 1689 (HAPT 1689), HAPT conjugated to Glycan 1851 (HAPT 1851), hemopexin (HEMO) conjugated to Glycan 2540 (HEMO 2540), and immunoglobulin G (IGG) conjugated to Glycan 1419 (IGG 1419), alpha-2- macroglobulin (A2M) conjugated to Glycan 1867 (A2M 1867), apolipoprotein D (APOD) conjugated to Glycan 2175 (APOD_2175), CLUS conjugated to Glycan 1689 (CLUS 1689), HAPT conjugated to Glycan 2321 (HAPT 2321), HEMO conjugated to Glycan 2175 (HEMO_2175), IGG conjugated to Glycan 1257 (IGG_1257), IGG conjugated to Glycan 1867 (IGG_1867), TRAN conjugated to Glycan 2175 (TRAN_2175), and vitamin D binding proteinAttorney Docket: 206085-0170-00WO(VDBP) conjugated to Glycan 2175 (VDBP 2175), alpha-1 antitrypsin (AAT) conjugated to Glycan 2321 (AAT_2321), alpha-1 acid glycoprotein (AGP) conjugated to Glycan 2321 (AGP 2321), fetuin (FET) conjugated to Glycan 2175 (FET 2175), FET conjugated to Glycan 2540 (FET_2540), IGG conjugated to Glycan 1502 (IGG_1502), AGP conjugated to Glycan 2540 (AGP_2540), AGP conjugated to Glycan 1257 (AGP_1257), AGP conjugated to Glycan 1219 (AGP_1219), AGP conjugated to Glycan 1445 (AGP_1445), ANGIO conjugated to Glycan 1851 (ANGIO 1851), apolipoprotein H (APOH) conjugated to Glycogen 1851 (APOH 1851), APOH conjugated to Glycogen 1867 (APOH 1867), APOH conjugated to Glycogen 2157 (APOH 2157), ceruloplasmin (CERU) conjugated to Glycogen 1257 (CERU_1257), CERU conjugated to Glycogen 2175 (CERU_2175), CERU conjugated to Glycogen 2321 (CERU_2321), CLUS conjugated to Glycan 1257 (CLUS_1257), CLUS conjugated to Glycan 1851 (CLUS 1851), CLUS conjugated to Glycan 2013 (CLUS_2013), CLUS conjugated to Glycan 2540 (CLUS 2540), HEMO conjugated to Glycan 1664 (HEMO 1664), HEMO conjugated to Glycan 1851 (HEMO 1851), histidine-proline rich glycoprotein (HPRG) conjugated to Glycan 1689 (HPRG 1689), HPRG conjugated to Glycan 2321 (HPRG 2321), IGG conjugated to Glycan 1607 (IGG 1607), IGG conjugated to Glycan 1689 (IGG 1689), IGG conjugated to Glycan 1744 (IGG 744), IGG conjugated to Glycan 1810 (IGG 810), IGG conjugated to Glycan 2123 (IGG_2123), TRAN conjugated to Glycan 1419 (TRAN_1419), TRAN conjugated to glycan 1810 (TRAN_1810), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) conjugated to Glycan 2016 (ADAMTS13_2016), ADAMTS13 conjugated to Glycan 2119 (ADAMTS_2119), attractin (ATRN) conjugated to Glycan 1851 (ATRN 1851), ATRN conjugated to Glycan 1865 (ATRN_1854), ATRN conjugated to Glycan 1876 (ATRN_1876), cluster of differentiation 109 (CD109) conjugated to Glycan 933 (CD109 933), CD109 conjugated to Glycan 1851(CD109 1851), CD109 conjugated to Glycan 1854 (CD109 1854), cluster of differentiation 163 (CD163) conjugated to Glycan 1282 (CD163 1611), CD163 conjugated to Glycan 1591(CD163 1591), CLDN163 conjugated to Glycan 1612 (CD163 1612), CLDN163 conjugated to Glycan 1849 (CD163 1849), CLDN163 conjugated to Glycan 1868 (CD163 1868), CLDN163 conjugated to Glycan 1955 (CD163_1955), CLDN163 conjugated to Glycan 1995 (CD163_1955), CLDN163 conjugated to Glycan 2071 (CD163_2071), CLDN163 to conjugated Glycan 2102 (CD163_2102), CLDN163 conjugated to Glycan 2142 (CD163_2142), CLDN163Attomev Docket: 206085-0170-00WO conjugated to Glycan 2468 (CD163_2468), cluster of differentiation 42D (CD42D) conjugated to Glycan 1994 (CD42D 1994), colony stimulating factor 1 receptor (CSF1R) conjugated to Glycan 1689 (CSF1R 1689), CSF1R conjugated to Glycan 1851 (CSF1R 1851), CSF1R conjugated to Glycan 1906 (CSF1R 1906), CSF1R conjugated to Glycan 1956 (CSF1R 1956), CSF1R conjugated to Glycan 2071 (CSF1R_2O71), CSF1R conjugated to Glycan 2102 (CSF1R_21O2), CSF1R conjugated to Glycan 2120 (CSF1R_212O), CSF1R conjugated to Glycan 2134 (CSF1R_2134), CSF1R conjugated to Glycan 2140 (CSF1R_214O), CSF1R conjugated to Glycan 2281 (CSF1R_2281), fibulin 1 (FBLN1) conjugated to Glycan 1282 (FBLN1 1282), low density lipoprotein receptor-related protein 1 (LRP1) conjugated to Glycan 933 (LRP1 933), LRP1 conjugated to Glycan 1851 (LRP1 1851), LRP1 conjugated to Glycan 1876 (LRP1_1876), lumican (LUM) conjugated to Glycan 1851 (LUM_1851), LUM conjugated to Glycan 1854 (LUM_1854), LUM conjugated to Glycan 1855 (LUM_1855), mannose receptor C-type 1 (MMR) conjugated to Glycan 1689 (MMR_1689), MMR conjugated to Glycan 1736 (MMR_1736), MMR conjugated to Glycan 1810 (MMR_1810), MMR conjugated to Glycan 1811 (MMR_1811), MMR conjugated to Glycan 1851 (MMR_1851), MMR conjugated to Glycan 1854 (MMR_1854), MMR conjugated to Glycan 1908 (MMR_1908), plexin domain containing 2 (PLXDC2) conjugated to Glycan 1547 (PLXDC2 1547), PLXDC2 conjugated to Glycan 1851 (PLXDC2 1851), PLXDC2 conjugated to Glycan 1860 (PLXDC2 1860), PLXDC2 conjugated to Glycan 1890 (PLXDC2 1890), PLXDC2 conjugated to Glycan 1973 (PLXDC2 1973), PLXDC2 conjugated to Glycan 2135 (PLXDC2_2135), Serpin G1 (IC1) conjugated to Glycan 1851 (IC1 1851), IC1 conjugated to Glycan 1855 (IC1 1855), IC1 conjugated to Glycan 1860 (IC 1 1860), sex hormone-binding globulin (SHBG) conjugated to Glycan 1736 (SHBG 1736), SHBG conjugated to Glycan 1810 (SHBG 1810), SHBG conjugated to Glycan 1811 (SHBG 1811), SHBG conjugated to Glycan 1851 (SHBG 1851), SHBG conjugated to Glycan 1854 (SHBG 1854), Sushi, von Willebrand Factor Type A (SVEP1) conjugated to Glycan 1851 (SVEP1_1851), and SVEP1 conjugated to Glycan 2142 (SVEP1_2142).

[0192] Embodiment 7 is the method of any one of embodiments 1-6, wherein the glycoprotein profde comprises: i) the level of: ANGIO_2540, CLUS_1607, and TRAN_1607, or ii) the level of: ANGIO_2540, CLUS_1607, and TRAN_1607, andAttorney Docket: 206085-0170-00WOA) the level of: HAPT 1689, HAPT 1851, HEMO 2540, and IGGJ419;B) the level of: A2M_1867, APOD_2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, and VDBP 2175;C) the level of: HAPT 1689, HAPT 1851, HEMO 2540, APOD 2175, HAPT 2321, IGG 1257, AAT_2321, AGP_2321, FET_2175, FET_2540, and IGG 1502;D) the level of: HAPT 1689, HAPT 1851, HEMO 2540, A2M 1867, HAPT 2321, IGG 1257, FET_2175, FET_2540, and AGP_2540;E) the level of: HEMO_2175 and IGGJ257; orF) the level of: HEMO_2540, A2M_1867, APOD_2175, HAPT_2321, HEMO 2175, IGG 1257, TRAN 2175, VDBP 2175, AGP 2321, FET 2175, IGG 1502, AGP 1257, AGP 1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS_1257, CLUS 1851, CLUS_2013, CLUS_2540, HEMO_1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRAN 1419, and TRAN 1810.

[0193] Embodiment 8 is the method of any one of embodiments 1-7, wherein step c) comprises: i) providing a substrate comprising a plurality of antibody spots; ii) incubating the substrate in a blocking solution; iii) incubating the substrate with the serum sample from the subject; iv) treating the substrate with an enzymatic releasing solution; and v) scanning the substrate by mass spectrometry to detect and identify the presence of glycans.

[0194] Embodiment 9 is the method of embodiment 8, wherein the substrate comprising a plurality of antibody spots comprises a plurality of spots against at least one antigen, wherein the antibodies of each spot bind a single antigen.

[0195] Embodiment 10 is the method of embodiment 8 or 9, wherein the at least one antigen comprises at least one selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13;Attomev Docket: 206085-0170-00WOATRN; CD109; CD163; CD42D; CSF1R; FBLN1 ; LRP1 ; LUM; MMR; PLXDC2; IC1 ; SHBG; and SVEP1.[001961 Embodiment 11 is the method of any one of embodiments 8-10, wherein the substrate comprises at least one antibody spot for each of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG.

[0197] Embodiment 12 is the method of any one of embodiments 8-11, wherein the substrate is selected from the group consisting of glass, polydimethylsiloxane (PDMS), epoxysilane-coated glass, and epoxysilane-coated PDMS.

[0198] Embodiment 13 is the method of any one of embodiments 8-12, wherein the blocking solution is bovine serum albumin (BSA).

[0199] Embodiment 14 is the method of any one of embodiments 8-13, wherein the enzymatic releasing solution comprises PNGase F.

[0200] Embodiment 15 is the method of any one of embodiments 8-14, wherein the mass spectrometry is selected from the group consisting of: matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry, matrix- assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry.

[0201] Embodiment 16 is the method of any one of embodiments 8-15, wherein the step of scanning the substrate is preceded by a step of spraying the substrate with a MALDI matrix material.

[0202] Embodiment 17 is the method of any one of embodiments 8-16, wherein the MALDI matrix solution is selected from the group consisting of: 2,5-dihydroxybenzoic acid, a- cyano-4-hydroxycinnamic acid, sinapinic acid, 1,5-diaminonaphthalene, and 9-aminoacridine.

[0203] Embodiment 18 is a device for rapid detection of hepatocellular carcinoma (HCC) comprising a solid substrate spotted with a plurality of antibodies, wherein each antibody spot comprises a plurality of antibodies that bind a single antigen,Attorney Docket: 206085-0170-00WO wherein the substrate comprises at least one antibody spot for at least one antigen selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

[0204] Embodiment 19 is device of embodiment 18, wherein the substrate comprises at least one antibody spot for each of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG.

[0205] Embodiment 20 is a kit for detecting hepatocellular carcinoma comprising: the device of embodiment 18 or 19; at least one blocking solution; at least one enzymatic releasing solution; and at least one MALDI matrix material.

[0206] Embodiment 21 is the kit of embodiment 20, wherein the blocking solution is bovine serum albumin.

[0207] Embodiment 22 is the kit of embodiment 20 or 21, wherein the enzymatic releasing solution comprises PNGase F.

[0208] Embodiment 23 is the kit of any one of embodiments 20-22, wherein the MALDI matrix solution is a-cyano-4-hydroxycinnamic acid.

[0209] Embodiment 24 is a method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; and d) determining that the subject has HCC when the level of the at least one glycan is altered compared to that in the serum of an individual without HCC.

[0210] Embodiment 25 is the method of embodiment 24, wherein the at least one glycoprotein comprises at least one selected from the group consisting of: AAT, AGP, A2M, ANGIO, APOD, APOH, CERU, FET, CLUS, HAPT, HEMO, IGG, TRAN, VDBP, HPRG,Attomev Docket: 206085-0170-00WOADAMTS13, ATRN, CD109, CD163, CD42D, CSF1R, FBLN1 , LRP1 , LUM, MMR, PLXDC2, IC1, SHBG, and SVEP1.[002111 Embodiment 26 is the method of embodiment 24 or 25, wherein the at least one glycan is selected from the group consisting of: Glycan 933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.

[0212] Embodiment 27 is the method of any one of embodiments 24-26, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycan is increased in the serum of the subject compared to the serum of an individual without HCC, and wherein the one glycan is selected from the group consisting of: Glycan 1257, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1607, Glycan 1664, Glycan 1689, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1867, Glycan 2013, Glycan 2123, Glycan 2175, Glycan 2321, and Glycan 2540.

[0213] Embodiment 28 is the method of any one of embodiments 24-27, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycan is decreased in the serum of the subject compared to the serum of an individual without HCC, and wherein the one glycan is selected from the group consisting of: Glycan 933, Glycan 1736, Glycan 1860, Glycan 1876, Glycan 2071, Glycan 2102, and Glycan 2142.

[0214] Embodiment 29 is a method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; andAttomev Docket: 206085-0170-00WO d) determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein in the serum of the subject is altered compared to the serum of an individual without HCC.

[0215] Embodiment 30 is the method of embodiment 29, wherein the at least one glycoprotein comprises at least one selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

[0216] Embodiment 31 is the method of embodiment 29 or 30, wherein the at least one glycan is selected from the group consisting of: Glycan 933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.

[0217] Embodiment 32 is the method of any one of embodiments 29-31, wherein the at least one glycoform of the at least one protein comprises at least one selected from the group consisting of: ANGIO 2540, CLUS 1607, TRAN 1607, HAPT 1689, HAPT 1851, HEMO_2540, IGG 1419, A2M_1867, APOD_2175, CLUS_1689, HAPT_2321, HEMO_2175, IGG 1257, IGG 1867, TRAN 2175, VDBP 2175, AAT 2321, AGP 2321, FET 2175, FET_2540, IGG 1502, AGP_2540, AGP 1257, AGP 1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS_1257, CLUS 1851, CLUS_2013, CLUS_2540, HEMO_1664, HEMO_1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRAN_1419, TRAN_1810, ADAMTS13_2016, ADAMTS_2119, ATRN_1851, ATRN_1854, ATRNJ876, CD109_933, CD109J851, CD109J854, CD163 1611, CD163 1591, CD163 1612, CD163 1849, CD163 1868, CD163 1955, CD163J955, CD163_2071, CD163_2102, CD163_2142, CD163_2468, CD42D_1994, CSF1R_1689, CSF1R 1851, CSF1R 1906, CSF1R 1956, CSF1R_2O71, CSF1R_21O2, CSF1R_212O, CSF1R_2134,Attomev Docket: 206085-0170-00WOCSF 1 R_2140, C SF 1 R 2281 , FBLN1 _1282, LRP 1 933, LRP 1 1851, LRP 1 J 876, LUM J 851, LUMJ854, LUMJ855, MMR_1689, MMR 1736, MMR_1810, MMR 1811, MMR_1851, MMR 1854, MMR 1908, PLXDC2 1547, PLXDC2 1851, PLXDC2 1860, PLXDC2 1890, PLXDC2 1973, PLXDC2 2135, IC 1 1851, IC 1 1855, IC1 1860, SHBG 1736, SHBG 1810), SHBG 1811, SHBG 1851, SHBG 1854, SVEP1 1851, and SVEP1 2142.

[0218] Embodiment 33 is the method of any one of embodiments 29-32, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HCC, wherein the at least one glycoform of the at least one glycoprotein is selected from the group consisting of: ADMTS13_2016, ATRN_1851, ATRN_1854, CD109 1851, CD109 1854, CSF1R 1689, CSF1R 1851, FBLN1 1282, LRP1 1851, LUM 1851, LUM_1854, LUM 1855, MMR 1689, MMRJ810, MMR+1811, MMR 1851, MMR 1854, PLXDC2 1851, IC1 1851, IC1 1855, SHBG 1810, SHBG 1811, SHBG 1851, SHBG 1854, SVEP1_2142, ANGIO_2540, CLUS 1607, TRAN_1607, HAPT_1689, HAPT 1851, HEMO 2540, IGG_1419, A2M_1867, APOD_2175, CLUS 1689, HAPT_2321, HEMO 2175, IGG 1257, IGG 1867, TRAN_2175, VDBP_2175, AAT_2321, AGP_2321, FET_2175, FET_2540, IGGJ502, AGP_2540, AGP 1257, AGP 1219, AGP_1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS_1257, CLUS_1851, CLUS_2013, CLUS_2540, HEMO_1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG_2123, TRANJ419, and TRAN_1810.

[0219] Embodiment 34 is the method of any one of embodiments 29-33, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HCC, wherein the at least one glycoform of the at least one glycoprotein is selected from the group consisting of: ADAMTS13_2119, ATRN_1876, CD109_933, CD163_1282, CD163 1591, CD163 1612, CD163 1849, CD163 1868, CD163_1955, CD163 1995, CD163_2071, CD163_2102, CD163_2142, CD163_2468, CD42DJ994, CSF1R 1906, CSF1R 1956, CSF1R_2O71, CSF1R_21O2, CSF1R_212O, CSF1R_2134, CSF1R_214O, CSF1R 2281, LRP1_933, LRP1 1876, MMR_1736, MMR 1908,Attomev Docket: 206085-0170-00WOPLXDC2J547, PLXDC2 1860, PLXDC2J 890, PLXDC2J973, PLXDC2 2135, IC1J860, SHBG 1736, and SVEP1 2142.EXPERIMENTAL EXAMPLES

[0220] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0221] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present disclosure, and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.Example 1 : Identification of Diagnostic Models for Early Detection of Hepatocellular Carcinoma

[0222] N-glycosylation of glycoproteins is known to be dysregulated in many cancer types and has been shown to be aberrant in both tissue and serum of patients with HCC (Ji et al., 2016, Anal Bioanal Chem, 408:7761-7774; Zhu et al., 2014, J Proteome Res, 13:2986-2997; Cheng et al., 2016, Oncotarget, 7:61199-61214; Zhang et al., 2015, J Proteome Res, 14:5388- 5395; Liang et al., 2018, Sci Rep, 9: 11580; Aoyagi et al., 1998, Cancer, 83:2076-2082; Buamah et al., 1986, Lancet, 1 :922-923; Aoyagi et al., 1985, Biochim Biophys Acta, 830:217-223; Naitoh et al., 1999, J Gastroenterol Hepatol, 14:436-445; Comunale et al., 2010, PLoS One, 5:el2419; Yin et al., 2015, J Proteome Res, 14:4876-4884; West et al., 2018, J Proteome Res, 17:3454- 3462) N-glycosylation on the tumor surface has been demonstrated to impact both HCC tumor proliferation and metastasis through the upregulation of growth factor receptor signaling (Wang et al., 2015, FASEB J, 29:3217-3227). Previous work has shown an increase in branched and fucosylated N-glycans in HCC tissue when compared to cirrhotic or normal liver tissue (West et al., 2018, J Proteome Res, 17:3454-3462) Additionally, abnormal N-glycosylation has been observed in HCC serum, which includes the differential N-glycosylation of several identified biomarkers of HCC, such as AFP-L3 (Buamah et al., 1986, Lancet, 1 :922-923; Aoyagi et al.,Attomev Docket: 206085-0170-00WO1985, Biochim Biophys Acta, 830:217-223) However, prior studies have not connected tissue N- glycosylation with serum N-glycosylation. Aberrant N-glycosylation is often heterogeneous between tumors, and there is limited understanding in how tumor-specific N-glycosylation translates to serum glycoproteins, even though many of those glycoproteins originate from the liver. Recent advancements have been made for the analysis of N-glycans via spatial-omics Matrix Assisted Laser Desorption-Ionization (MALDI) Imaging Mass Spectrometry (IMS) for both tissue and serum samples, including for the specific analysis of serum glycoproteins through antibody arrays (West et al., 2018, J Proteome Res, 17:3454-3462; Black et al., 2018, Anal Chem, 91 :8429-8435; Blaschke et al., 2020, J Am Soc Mass Spectrom, 31 :2511-2520; DelaCourt et al., 2021, Mol Cancer Res, 1868-1877).

[0223] A sample cohort of 53 early-stage HCC tissue samples including 23 samples with of matched tumor tissue, background liver tissue, and serum samples were utilized to address the disconnect between HCC-correlated tissue N-glycosylation and HCC-correlated serum N- glycosylation. These samples were analyzed with MALDI-IMS N-glycan imaging workflows and identified specific glycans that were altered in tissue and serum. Subsequently, glycoproteomics identified the serum glycoproteins that contained these glycan changes. These biomarkers were then analyzed using a novel high throughput slide based glycan analysis method, called the GlycoTyper (Black et al., 2019, Curr Protoc Protein Sci, 98:e99). This allowed for the simultaneous glycan analysis of the identified glycoproteins in 201 patients, 100 of whom had HCC in the background of cirrhosis and 101 with cirrhosis alone. Combinations of altered glycans on the identified glycoproteins were incorporated into a machine learning algorithm, that also included age, gender and AFP. These spatial omics based, machine learning (SGML) algorithms were able to differentiate HCC from cirrhosis with an area under the receiver operating characteristic (AUROC) in internal cross validation as high as 0.94 in all patients and in those patients with early-stage HCC, greatly improving on the non-invasive detection of HCC when compared to AFP alone.

[0224] Patient tissues and serum: Formalin-Fixed Paraffin-Embedded (FFPE, 5 pm thick) tissues were sectioned from 53 HCC tissue blocks that were obtained through surgical resection of HCC in the background of liver cirrhosis. Additionally, 23 serum samples that matched to patients of obtained tissue samples, were also obtained HCC tissues were subtyped according to the Hoshida classification system as done previously (Hoshida et al., 2009, CancerAttomev Docket: 206085-0170-00WORes, 69:7385-7392). All tissues were H&E stained following MALDI-IMS analysis and tumor regions were annotated by a liver pathologist. Patient characteristics of the 53 tumors and 23 cases with matching serum are found in Table 1.1) HCC was defined using AASLD criteria. 2) Subset of HCC patients from whom serum and tissue was available. 3) Gender with tire number in each group. 4) The number in each group as stated. 5) HB V, hepatitis b virus. HCV. hepatitis C virus, NASH, non-alcoholic steato-hepatitis, unknown, liver disease from unknow etiology. Actual numbers given. 6) Child-Pugh score for degree of liver disease, actual number given. 7) Number of lesions as detected by MRI or CT-Scan. Mean with range is given 8) Size of all tumors. Mean with range is given. 9). The Barcelona Clinic Liver Cancer (BCLC) Staging System. Actual numbers in each group give, stage 0: very early, stage A: early, stage B: immediate, stage C: advanced, stage D: end-stage. 10) AFP. alpha-fetoprotein.

[0225] A second set of serum samples was obtained (Wang et al., 2016, Biochem Biophys Res Commun, 476: 140-145; Wang et al., 2017, Cancer Epidemiol Biomarkers Prev, 26:795-803; Wang et al., 2022, Cancers (Basel), 14) and consisted of 100 samples from patients with HCC in the background of cirrhosis and 101 samples from patients with cirrhosis alone.. Demographic and clinical information was obtained, and a blood sample was collected from each subject. A 20-ml blood sample was drawn from each subject, spun, aliquoted, and serum stored at -80 °C until testing. Blood samples for HCC patients were drawn prior to initiation of HCC treatment.

[0226] For all patients, diagnosis of cirrhosis was based on liver histology or clinical, laboratory and imaging evidence of hepatic decompensation or portal hypertension. Each non-Attorney Docket: 206085-0170-00WOHCC patient had a normal ultrasound; if serum AFP were elevated, a CT or MRI showed no liver mass. For HCC patients, the diagnosis of HCC was made per AASLD guidelines (Singal et al., 2023, Hepatology, 78: 1922-1965) using histopathology or characteristic diagnostic imaging. Early-stage HCC was defined using the Milan Criteria (Mazzaferro et al., 1996, N Eng J Med, 334:693-699). As previously described, demographic and clinical information were obtained, and a blood sample was collected from each subject prior to initiation of any HCC treatment (Wang et al., 2022, Cancers (Basel), 14). A 20-ml blood sample was drawn from each subject, spun, aliquoted, and serum stored at -80 °C until testing.

[0227] FFPE tissue preparation for MALDI-IMS: HPLC grade methanol, ethanol, acetonitrile, xylene, and water were obtained from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid and a-cyano-4- hydroxycinnamic acid were obtained from Sigma-Aldrich (St. Louis, MO). Peptide-N-glycosidase F (PNGase F) Prime and Sialidase Prime were obtained from N-Zyme Scientific (Doylestown, PA).

[0228] FFPE tissues were prepared according to a previously published protocol (Powers et al., 2013, Anal Chem, 85:9799-9806). Tissue Tack microscope slides were purchased from Polysciences Inc (Warrington, PA, Catalog No. 24216). Slides were taken through dewaxing and wash steps, before being processed by antigen retrieval in a Decloaking Chamber in 10 mM citraconic anhydride buffer, pH 3. PNGase F Prime and Sialidase Prime were simultaneously applied using a M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC), and enzymes were incubated for 2 hours at 37 °C. MALDI matrix a- cyano-4-hydroxycinnamic acid (0.042 g CHCA in 6 mL 50% acetonitrile / 49.9% water / 0.1% TFA) was sprayed by the M5 TM-Sprayer.

[0229] HPLC grade methanol, ethanol, acetonitrile, xylene, and water were obtained from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid and a-cyano-4- hydroxycinnamic acid were obtained from Sigma-Aldrich (St. Louis, MO). Peptide-N-glycosidase F (PNGase F) Prime and Sialidase Prime were obtained from N-Zyme Scientific (Doylestown, PA).

[0230] N-Glycomic total serum preparation: Serum samples were prepared for total serum N-glycan MALDI-IMS analysis through a previously published protocol (Blaschke et al., 2020, J Am Soc Mass Spectrom, 31 :2511-2520). Hydrogel-coated slides (Nexterion Slide H) were obtained from Applied Microarrays (Tempe, AZ). Serum samples were spotted and immobilized on the slide surface, and then washed to remove salts and lipids from the sample.Attomev Docket: 206085-0170-00WOEach sample was spotted in triplicate. N-glycans were then enzymatically released through the same methodology of spraying / incubating PNGase F Prime / Sialidase Prime as tissue samples.

[0231] GlycoTyper: Serum samples were prepared for glycoprotein-specific N-glycan MALDI-IMS analysis through a previously published antibody array protocol (Black et al., 2018, Anal Chem, 91 : 8429-8435). Glycerol free antibodies were tested for their ability to bind to target glycoprotein, but not any other glycoproteins, using methods described elsewhere (Black et al., 2018, Anal Chem, 91 :8429-8435). Only antibodies specific to their target were used for analysis. Antibodies were spotted onto nitrocellulose-coated microscope slides (Grace-Bio Labs, Bend, OR). The following antibodies were used for the GlycoTyper experiment and purchased from Abeam (Cambridge, MA): Rabbit anti-Alpha 1 Antitrypsin (#ab240375), Rabbit anti- Ceruloplasmin (#ab249323), Rabbit anti-Clusterin (#ab229445). Mouse anti-Human Alpha 1B- Glycopotein (#MAB7757), Goat anti-Human alphal-Acid Glycoprotein (#AF3694), Mouse antiHuman Apolipoprotein H (#MAB5087), Mouse anti-Human Fetuin A / AHSG (#MAB1184), Mouse anti-Human HPRG (#MAB1869), Mouse anti-Human Vitamin D Binding Protein ( MAB3778) antibodies were obtained from R&D Systems (Minneapolis, MN). Mouse anti- Alpha-2- Macroglobulin (#2 -MM3 -Pl) antibody was obtained from NeoBiotechnologies (Union City, CA). Mouse anti-angiotensinogen II / III (#NB 100-62346) antibody was obtained from Novus Biologicals (Centennial, CO). Mouse anti-Apolipoprotein D (#10R-A137b) and Goat anti-Haptoglobin (#70R-7558) antibodies obtained from Fitzgerald (Acton, MA). Mouse antiHemopexin ( ABA-133202) antibody was obtained from Fisher Scientific (Hampton, NH). Goat anti-Human IgG (#A80-104A) and Goat anti-Human Transferrin (#A80-128A) antibodies were obtained from Bethyl Laboratories (Montgomery, TX).

[0232] The antibodies were first diluted in Phosphate Buffered Saline IX solution (PBS 10X, BP39920, and HPLC grade water W5-4 from Fisher Scientifics) at a final concentration of 40 ng / pl and using a Picus Electronic Single Channel Pipette from Sartorius (Goettingen, Germany), lul of each antibody was spotted on Nitrocellulose slides (SKU470639 from Grace Bio-Labs) on the top of which were clipped ProPlate 8 well slide modules (Path Protein Microarray Slides, 25x25xlmm, #805025 from Grace Bio-Labs, Bend, OR). Well chambers were mounted to the slide to create separated regions for serum incubation. Each region had eight unique antibodies spotted at specified positions, with 16 total unique antibodies forAttomev Docket: 206085-0170-00WO analysis. Serum samples were incubated in duplicate in each of two antibody arrays. N-glycans were enzymatically removed via the same protocol as tissues and total serum analysis.

[0233] MALDI-IMS N-glycan imaging: Slides were imaged on a MALDI-TOF (timsTOF flex, Bruker Daltonics) mass spectrometer in positive ion mode (m / z 600-5000 for tissue and m / z 700-4000 for serum assays). For both tissue and serum, images were collected at a 150 pm raster with 200 laser shots per pixel and at 100 pm raster for serum assay. Data was visualized and analyzed using SCiLS™ Lab 2022 for tissue and SCiLS™ Lab 2024a for serum (Bruker). Peaks were assigned to N-glycan structures utilizing a previously developed database with consideration for biosynthetic pathways of N-glycans (West et al., 2018, J Proteome Res, 17:3454-3462; Drake et al., 2018, Curr Protoc Protein Sci, 94:e68). Not all N-glycans observed in tissue were also seen in serum samples. Proposed N-glycan structures and corresponding m / z values can be found in Figure 5.

[0234] GlycoProteomics for identification of fucosylated peptides: Serum samples were prepared using the EasyPep 96 MS Sample Prep Kit (Thermo Fisher Scientific, Waltham, MA) with minor modifications to the manufacturer’s instructions. Briefly, 20 pL of serum were denatured with 80 pL of lysis solution. Cysteine residues were reduced with iodoacetamide and alkylated at 50 °C for 30 minutes. Proteins were incubated with Endoglycosidase F3 (Endo F3; N-Zyme Scientific) for 1 hour at 37 °C. Subsequently, samples were incubated with PNGase F (N-Zyme Scientific) for 2 hours at 37 °C (Cao et al., 2022, Nat Commun, 13:3910). One pg of trypsin-Lys C endoprotease digestion mixture was added to each sample and proteins digested overnight at 37 °C with shaking at 300 rpm. The reaction was quenched with stop solution and the samples were transferred to C18 spin columns to remove salts and detergents. Eluted peptides were dried by vacuum centrifugation and resuspended in lx PBS. HexNAc Fuc modified peptides were enriched with a recombinant Aleuria aurantia lectin (AAL N224Q) coupled to nickel beads (Romano et al., 2011, Biochemical and Biophysical Research Communications, 414:84-89). Following enrichment, samples were acidified, and desalted by solid-phase extraction using ZipTip with 0.6 pL C18 resin (Millipore, Burlington, MA).Enriched peptides were dried under vacuum and resolubilized in 5% acetonitrile, 0.2% formic acid in LC-MS / MS grade water immediately prior to LC-MS / MS analysis.

[0235] LC-MSMS acquisition: Peptides were separated and analyzed on with an Easy nLC 1200 in-line with the Orbitrap Exploris 480 mass spectrometer (Thermo Scientific,Attorney Docket: 206085-0170-00WOWaltham, MA) with instrument control software v. 4.2.28.14. Two pg of peptides were pressure loaded onto a C18 reversed phase column (Acclaim PepMap RSLC, 75 pm x 25 cm (2 pm, 100 A) Thermo Fisher cat. # 164941) and separated using a gradient of 0-35% B in 90 min at a flow rate of 300 nL / min. Solvent A was 5% acetonitrile, 0.2% formic acid and solvent B was 80% acetonitrile, 0.2% formic acid. Mass spectra were acquired in data-dependent mode with a high resolution (60,000) FTMS survey scan, mass range of m / z 375-1575, followed by tandem mass spectra (MS / MS) of the most intense precursors with a cycle time of 3 seconds. The automatic gain control was set to 300% for the survey MS scan and 100% for the MS / MS scan. HCD fragmentation was performed with a precursor isolation window of 1.4 m / z, a maximum injection time of 40 ms, and HCD collision energy of 33%. The MS / MS scan was acquired at 15,000 resolution. Monoisotopic-precursor selection was set to “peptide”. Precursors within 10 ppm mass tolerance were dynamically excluded from resequencing for 20 sec. Advanced peak determination was enabled. Precursor ions with charge states that were undetermined, 1, or >6 were excluded.

[0236] Database searching and quantitation: Raw files were searched using MaxQuant v2.4.2.0 (Max Planck Institute) against a human UniProt protein database (20,422 sequences, updated March 2023) and a reversed, decoy database. Fixed modification of cysteine with carboxyamidomethylation and variable oxidation of methionine, phosphorylation of serine or threonine, and protein N-terminal acetylation were included. Custom modifications for O- HexNAcylated serine or threonine (OGlcNAc_NL_D) and N-linked HexNAc fucose modification of asparagine (HexNAcFuc) were created in Andromeda. The O-linked HexNAc included neutral loss of C8H13NO5 (203.0794 Th) and diagnostic HexNAc ions. The N-linked HexNAcFuc modification had a composition of C14H23NO9 (349.1373 Th), a neutral loss composition of C6H10O4 (146.0579 Th), and diagnostic HexNAc ions. HexNAc ion masses are listed in Table 2. Two missed cleavages were permitted. The final precursor and fragment mass tolerances were less than 4.5 ppm. For identification a 1% false discovery rate (FDR) threshold was required at the spectrum, peptide, and protein levels. Matching between runs was enabled. Data were processed in Perseus vl.6.15.0 (Max Planck Institute). Peptide intensity values from the modification specific peptide table were log2 transformed and median normalized. After normalization a value of 15 was added to each intensity to avoid negative intensity values. The list of peptides was filtered to remove reversed database hits, peptides without HexNAc Fucose,Attorney Docket: 206085-0170-00WO and peptides with more than 3 modifications. The fold change in mean log2 peptide intensities from HCC and cirrhotic serum samples compared and visualized by plotting against the Student’s t test -loglO p value. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2022, Nucleic Acids Res, 50:D543-D552) with the dataset identifier PXD047546.Table 2; Diagnostic ions

[0237] Statistical data analysis: In both tissue and serum, all N-glycans of the peak list were normalized to relative abundance to yield a relative quantification, this normalization enhanced robustness. Data distributions of each N-glycan were visualized, outliers were identified, they were re-valuated if required.

[0238] Univariate statistical methods were applied to each N-glycan data, such as the distribution of each N-Glycan, change of mean and median between HCC and cirrhosis, and corresponding AUC were evaluated. The association between the “interesting” glycans were analyzed by clustering and correlation analysis.

[0239] The data distribution of glycan from matched tissue and serum were tested by Shapiro-Wilk test. If the results indicated they were not significantly different from a normal distribution, Pearson correlation coefficients were derived and the p-value obtained.

[0240] Predictive modeling with machine learning algorithm: To avoid a bias caused by the scale of the different N-glycans in the building of machine learning models, z-transformation was applied to each variable. The data used for the development of the SGML was considered high dimensional data with 16 proteins, each with 83 N-glycans, for a total of 1,328 variables. Feature selection was applied to combat issues often related to high dimension of Omics data. Filter techniques, Information Gain, Importance, Joint Mutual Information Maximization, andAttomev Docket: 206085-0170-00WOAUC of each N-Glycan were evaluated; redundant and non-informative variables were removed from analysis.

[0241] Subsequently, wrapper methods were applied to search subsets of glycans to optimize performance of discrimination and prediction (internal Cross-Validation) criterion by AUC. Sensitivity and specificity, and partial ROC-90 (Wang et al., 2022, Cancers (Basel), 14) were the criteria for further search.

[0242] For initial analysis, an exhaustive search for feature selection was avoided, and random search, sequential search, genetic search, and recursive elimination features with machine learning algorithms were mainly applied. These had good performance in previous glycan analysis (Mehta et al., 2018, PLoS One, 13:e0203149; Wang et al., 2016, Cancer Prev Res (Phila), 9: 172-179), such as, support vector machines, Gradient Boosting Machine, Logistic regression, Random Forest, Bayesian additive regression Tree, Gradient Boosting with Component-wise Linear model, Gradient Boosting for Additive Model, Multivariate Adaptive Regression Splines, Extreme Gradient Boosting, Logistic Model Tree, etc.

[0243] The optimal subsets of glycans derived by feature selection and corresponding machine learning algorithms were evaluated by Apparent Validation(AP) for its discriminative ability. The predictive ability of each algorithm was evaluated by Leave-One-Out-Cross- Validation(LOOCV), 3-fold Cross-Validation, 5-fold Cross-Validation. To ensure robustness of the selected algorithm, random shuffling of the samples was performed 200 times for the 3 -fold and 5-fold CV (repeated analysis) to ensure the robustness of final algorithms.

[0244] Support Vector machine with linear kernel model performed better or equally compared with all other candidate models, with good robustness in discriminative and predictive performances. All statistical analysis was conducted using either R (Version 4.3.0) and / or GraphPad Prism (10.1.0).

[0245] 53 HCC tissue sections were analyzed by spatial MALDI glycan imaging (Table1). Nearly all patients (98.1%) had early-stage HCC, with a median of 1.00 lesions (25%:1.0,75%: 1.0; SD: 0.28) and median tumor size of 2.8 cm (25%:2.1, 75%:4.2; SD:2.5cm). Most patients (89%) had Child Pugh A cirrhosis, and the most common etiologies were hepatitis C infection, alcohol induced liver disease and NASH. As expected, HCC tissue was associated with significant glycan heterogeneity. Tumors had a mean of 15.7 (SD: 9.7) N-glycan alterations with a 1.5-fold or greater change per tumor. All tumors had at least one alteration in N-linkedAttomev Docket: 206085-0170-00WO glycosylation, with the mean of the most altered N-glycan being 11 .8-fold increased (range 1 .6 to 220.9).

[0246] Two of the most common N-glycan alterations in HCC tissue were increases in fucosylation and increases in branching. In Figure 1, imaging mass spectrometry data is shown with a color intensity scale for all images (red is most abundant and blue is least abundant). Figure 1 A-Figure IE highlights five representative HCC tissues that were associated with alterations in fucosylation, along with the other N-glycan changes found concomitantly with these specific tumors. For each panel, the H&E-stained tissue is shown with four representative images of altered glycans in the tumor tissue as compared to the surrounding non-transformed tissue. In all cases, the tumor tissue is highlighted on the H&E-stained image. As Figures 1 A- Figure IE show, pathology annotated HCC tissue was associated with increased amount of fucosylated N-glycan at m / z 2174.774 (Hex6HexNAc5Fucl), m / z 2320.829 (Hex6HexNAc5Fuc2), m / z 2539.907 (Hex7HexNAc6Fucl), m / z 2685.964 (Hex7HexNAc6Fuc2) and m / z 2905.040 (Hex8HexNAc9Fucl). Based upon the compositions, these are presumed to be branched (triantennary and higher) and fucosylated N-glycan, but other fucosylated N-glycan changes were also observed (Figure7). Often, this tissue also had alterations in high mannose N-glycan (m / z 1257.424; Hex5HexNAc2 and m / z 1419.479; Hex6HexNAc2), and these are shown in Figure 1A- Figure 1DD. Of the 53 HCC tissues, increased levels of branched N-glycan (tri-antennary or higher) containing fucose were found in 52% of the tissue samples examined. The complete list of glycans identified in all issue sections is also provided in Figure 5.

[0247] Tissues that lacked major changes in fucosylated N-glycans were associated primarily with increased levels of a N-glycan at m / z 2393.848 (Hex7HexNAc6), presumed to be a tetra-antennary N-glycan devoid of fucosylation. Figures 1F-J highlight five representative HCC tissues that fell into this category. While all these have increased levels of the 2393.848 N- glycan within the HCC tissue, very few other N-glycan alterations were observed. Increased levels of the 2393.848 N- glycan were found in 53% of the tissue samples examined. The most abundant secondary glycans were high mannose glycans (m / z 1257.424, 1419.479, 1743.587; Hex7HexNAc2) but there was significant heterogeneity in the other glycans altered in this tissue.

[0248] Figure IK is a heat map of the glycans that were observed in all tissues examined. Every patient had at least one N-glycan that was increased by 1.5-fold in the HCC tissue asAttomev Docket: 206085-0170-00WO compared to the adjacent normal tissue. When all tissues were examined, the most abundant type of N-glycan alteration was fucosylation (on any core structure), which was observed in the tumor regions of 60% of tissue samples example. N-glycan alterations were weakly associated with tumor subtype. Increased fucosylated N-glycan were found in 71% of the Hoshida SI type tumors, 64% of the S2 type tumors but only 31% of the S3 type tumors. In contrast S3 type tumors had a greater level of a tetra-antennary branched N-glycan devoid of fucosylation (50%) as compared to the SI (21%) or S2 (18%) tumors. Figure IL highlights that alterations in fucosylation on specific structures were associated with broad overall alterations in fucosylation.

[0249] Identification of cancer specific N-linked glycans in matching tissue and serum. Paired serum samples were available in 23 patients to allow correlation between the glycans found in tissue and those found in serum. Characteristics of these 23 patients were similar to the entire HCC cohort (Table 1). Specifically, over 90% of patients had Child Pugh A cirrhosis and early-stage HCC. Serum samples from 23 patients that had matching tissues were analyzed for total N-glycosylation content using a recently developed MALDI-IMS serum N-glycan profiling method (Figure 2A) (Blaschke et al., 2020, J Am Soc Mass Spectrom, 31 :2511-2520). The goal was to determine which glycans in serum correlated with the level of N-glycan in the matched tissue. Figures 2B-D shows an example of the N-glycan heterogeneity observed in 14 representative HCC patients. Figure 2B-D highlights the abundance of N-glycan at m / z 2320.829, 2393.848 m / z, and 2539.907 in these patients. As these Figures show, patients had variations in the glycans expressed (patients were analyzed in triplicate). For example, Patient 1, has a low level of the N-glycan at 2320.84, very high expression of the N-glycan at 2393.848 and no expression of a N-glycan at 2539.907. In contrast, patient 12, has high expression of the N- glycan at 2539.907 but lower levels of the glycans at 2320.829 and 2393.848. Pearson correlations were determined for all N-glycans in the m / z peak list, which allowed for the correlation between total serum N-glycosylation and tumor-specific tissue N-glycosylation. Figure 2E shows the relationship between the glycans whose level in HCC tissue and in serum was correlated. Results showed that 13 / 73 (23%) N-glycans were positively correlated between tumor and serum. The majority of these (11 / 13) were fucosylated N-glycan (indicated with the asterisks). All of these were positively correlated, which suggests that HCC tumor and serum N- glycosylation patterns are related and importantly, trends on tumor tissue are represented in serum.Attomev Docket: 206085-0170-00WO

[0250] Glycoproteomics to identify altered glycoproteins. The major N-glycan changes observed in both tissue and matching serum included fucosylation and, given the nature of the N- glycan (on agalactosylated structures or multiply fucosylated) it was presumed that many, if not most, were core fucosylated. To identify those serum proteins that contained these N-glycan changes, a glycoproteomic method was utilized (Figure 3A) that utilized sequential enzymatic digestion of glycoproteins with Endoglycosidase F3 (Endo F3) followed by PNGase F (Car et al., 2022, Nat Commun, 13:3910). A volcano plot shows all the glycopeptides that were altered in HCC and as Figure 3B shows, the number of fucosylated glycopeptides increased in the HCC samples. Indeed, 541 glycopeptides (peptide with an attached N-acetyl-glucosamine and fucose residue) were identified and 360 of these were increased in the HCC samples (67%). Figure 3C- E highlights some of the glycopeptides from the proteins identified including glycopeptides from clusterin (Figure 3C); alpha-2 macroglobulin (Figure 3D) and hemopexin (Figure 3E). Of these, the fucosylated glycopeptides from hemopexin were found to be decreased in the HCC samples, as compared to the cirrhotic samples. Changes in the relative abundance of the glycopeptides could also result from alterations in protein abundance or other modifications of the peptides such as methionine oxidation which was elevated in the cirrhotic samples. It is noted that many of these glycoproteins have been previously identified as part of the fucosylated proteome (Comunale et al., 2010, PLoS One, 5:el2419; Block et al., 2005, Proc Natl Acad Sci USA, 102:799-784; Comunale et al., 2006, J Proteome Res, 5:308-315; Comunale et al., 2009, J Proteome Res, 8:595-602; . Wang et al., 2009, Cancer epidemiology, biomarkers & prevention, 18: 1914-1921; Comunale et al., 2011, Cancer Epidemiol Biomarkers Prev, 20:1222-1229;Norton et al., 2016, Proteomics, 16:3126-3136).

[0251] To orthogonally analyze the identified glycoproteins, the same patient samples used for glycoproteomics were analyzed by a recently developed MALDI-IMS antibody array workflow which allows for the specific capture, and subsequent N-glycan analysis, of glycoproteins of interest (Black et al., 2019, Curr Protoc Protein Sci, 98:e99); thus allowing for validation of altered glycosylation. Figure 3F outlines the workflow of this approach and Figures 3G-3I highlights the same three glycoproteins shown in Figures 3C-3E. For two of these, the trend is the same, increased levels of total fucosylation are observed in most notably observed on a N-glycan at m / z 2539.907 (Hex7HexNAc6Fucl; Figure 3G) or 2174.772 (Hex6HexNAc5Fucl; Figure 3H), which were glycans also observed as being increased in tissueAttomev Docket: 206085-0170-00WO(Figure 1). Surprisingly, hemopexin, which was decreased in the glycoproteomics, also had increased levels of fucosylation, which was most clearly observed with the N-glycan at 2539.907 (Figure 31). This implies that the fucose present on this hemopexin N-glycan was not on the core N-acetylglucosamine but attached to one of the antennae N-acetylglucosamines or the total level of hemopexin was reduced, which would impact the glycoproteomics but not the GlycoTyper.

[0252] Development of spatial omic based machine learning (SOML) algorithms for the early detection ofHCC. To examine the biomarker potential of these abundant serum glycoforms, the same method in Figure 3F was utilized for the simultaneous examination of glycoproteins in a case-control patient set including 100 cases with HCC and cirrhosis and 101 controls with cirrhosis (Table 3). Most patients were male and had underlying hepatitis C infection. Patients were diverse regarding race / ethnicity with one-third White, 30% Black, and 30% Hispanic.Table 3; Patients Used for Biomarker Analysis1) Scrum from patients with HCC or cirrhosis used for algorithm discovery. 2) Scrum from patients with HCC or cirrhosis used for algorithm validation. 3) Gender with the % in each group. 4) Mean age with patient range. 5) Ethnicity with % in each group. 6) The Barcelona Clinic Liver Cancer (BCLC) Staging System. % in each group given. 5) % of disease etiology. HBV. hepatitis b virus, HCV, hepatitis C virus, alcohol, chronic alcohol use, NAFLD, non-alcoholic fatty liver disease, unknown, liver disease from unknow etiology. X) AST, aspartate aminotransferase, 9) ALT, alanine aminotransferase; 10) ALK, Alkaline phosphatase, 11) Albumin level.12) AFP, alpha-fetoprotein. 13) AUROC of AFP in this patient set with 95% confidence interval. 14) AFP-L3, the core fucosy lated version of AFP given as a % of total AFP. 14) AUROC of AFP-L3 in this patient set with 95%Attorney Docket: 206085-0170-00WO confidence interval.

[0253] Sixteen glycoproteins were selected for analysis based on results of the glycoproteomic experiments in Figure 3 and availability of appropriate antibodies. Tn total there were 83 N-linked glycans that were associated with the 16 glycoproteins analyzed. The major N- glycans found to be altered on the examined glycoproteins included glycans such as 2320.829, 2539.908, and 1257.424, which were also found directly on HCC tissue and in the matching serum (Figure 4B-Figure 4K). AFP-L3 had the highest AUROC among all single glycoforms at 0.74 in this sample set to distinguish HCC from cirrhosis. However, when the unique glycoforms were combined, biomarker algorithms could be obtained. Table 4 presents seven potential algorithms that were created using a support vector machine with a linear kernel. These were referred to as Spatial-Omics associated Machine learning (SOML) HCC algorithms. Glycoform combinations had AUROC values ranging from 0.90 to 097 in the derivation analysis and 0.89 to 0.94 in internal cross validation analyses (Table 4). Combinations included 7 to 39 glycoforms from up to 15 different glycoproteins (Figure 6). In accordance with previous methods (Wang et al., 2017, Cancer Epidemiol Biomarkers Prev, 26:795-803; Wang et al., 2022, Cancers (Basel), 14; Wang et al., 2016, Cancer Prev Res, 9: 172-179; Wang et al., 2012, Proceedings. TEE International Conference of Bioinformatics and Biomedicine, 2012; Wang et al., 2013, BMC medical genomics, 6 Suppl 3:S9; Wang et al., 2018, J Immunol Methods, 462:59-64), age, gender and AFP were also included in the algorithm to improve diagnostic performance (Singal et al., 2015, Hepatology, 75:541-549; Singal et al., 2022, Glin Gastroenterol Hepatol , 20:953- 955 e2). As Table 4 and Figure 4L show, the best algorithm had an AUROC in internal leave one out cross validation (LOOCV) of 0.94 in all patients. The best SOML algorithm increased the sensitivity of HCC detection at 90% specificity to 88%. As a comparison, AFP had an AUROC of 0.78 in the sample set, with a sensitivity of 47% at 90% specificity.Table 4: SOML AlgorithmsAttomev Docket: 206085-0170-00WO1) Seven diagnostic algorithms were created using a support vector machine with a linear kernel. 2) The number of glycoforms (proteins with unique glycans in each model). 3) Clinical factors such as age, AFP or gender used in model. 4) AP. apparent validation. 5) Area under the curve for each model for apparent v alidation. 6) SE, standard error. 7) 95%CI-LL. lower limit of 95% confidence interval. 8) 95%CI-UL, upper limit of 95% confidence interval.9) LOOCV, leave one out cross validation. 10) 3-fold CV, three-fold cross validation. 11) 5- fold CV, five-fold cross validation.

[0254] Of the 100 patients with HCC, 41% had early-stage cancer (Table 3), and the best SGML algorithms had a similar performance in this sub-set of patients, as compared to all patients. As Figure 4L shows, the AUROC of the model G algorithm was 0.95, with a sensitivity of 88% at 90% specificity. In contrast, AFP performance was more limited in this sub-set with an AUROC of 0.74, with a sensitivity of 39% at 90% specificity. The best SOML algorithms improved upon this with an AUROC of 0.95, with a sensitivity of 88% at 90% specificity.Additional glycoforms identified as significant between HCC and cirrhosis, and their differentiation capabilities, are listed in Table 5.Table 5: Potential Protein Glycoform BiomarkersAtorney Docket: 206085-0170-00WOAttorney Docket: 206085-0170-00WO'Protein Gene name and m / z value. ADAMTS13. a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; ATRN, attractin; CD109. cluster of differentiation 109; CD 163, cluster of differentiation 163; CD42D, cluster of differentiation 42D; CSF1R, colony stimulating factor 1 receptor; FBLN1, fibulin 1; FBLN2. fibulin 2; LRP1, low density lipoprotein receptor-related protein 1; LUM, lumican; MMR, maimose receptor C-type 1 (also MRC1); PLXDC2, plexin domain containing 2; IC1. serpin Gl; SHBG, sex hormone-binding globulin; SVEP1, Sushi, von Willebrand Factor Type A.2Mean value of glycofomi in 10 hepatocellular carcinoma (HCC) samples.3Mean value of glycoform in 10 cirrhotic samples.4Area under the receiver operator curve (AUC) in differentiating HCC from cirrhotic.5p-value for AUC as compared to no discrimination (AUC = 0.5).6p-value for mean values of HCC and cirrhotic.

[0255] In recent years, aberrant N-glycosylation has been a major focus of cancer biomarker development, which includes altered N-glycosylation of known biomarkers such as AFP, PSA and CA-19-948. It has been clearly demonstrated that N-glycosylation of cancer is widely dysregulated from non-cancer, although much of this work has been performed on serum, with no link to what is occurring in the tissue. Recently, tissue-based N-glycan spatial-omics has been used to examine the glycans found in tissue but again, this work was independent of matched serum. To extend this work to clinically relevant biomarker development, it must be demonstrated that cancer-caused dysregulation of N-glycosylation found within the tumor, is apparent in matched serum as well as tumor tissue. To accomplish this, novel MALDI-IMS techniques have been developed, aiming to analyze N-glycosylation and correlate with spatial glycomic analyses of tumor tissue. In this work, serum MALDI-IMS techniques were utilized to analyze patient-matching serum and tissue samples to connect tumor-related tissue N- glycosylation to tumor-related serum N-glycosylation and create a novel liquid biopsy for N- linked glycans.

[0256] Regarding serum N-glycosylation, there was a remarkable amount of correlation between total serum N-glycosylation and tumor-specific N-glycosylation, with 23% of individual N-glycans significantly positively correlated between the two. Considering that many of the glycoproteins within serum did not originate from the tumor, or even from the liver, that level of correlation suggests that there is significant HCC tumor biomarker potential contained within theAttorney Docket: 206085-0170-00WO serum N-glycome. Tt is noted that this is the first study examining both HCC tissue and serum for a protein or glycan biomarker.

[0257] However, not surprisingly, glycosylation associated with HCC displayed a great degree of heterogeneity, with no single N-glycan, or even N-glycan class, being found in more than 60% of patient samples. Many groups have examined the glycosylation of a single protein, often with a single glycan change, as biomarkers of HCC, but these have all had limited performance. Indeed, AFP-L3 is a version of AFP with a bi-antennary core fucosylated glycan65. Thus, it is not surprising that N-glycan biomarker development has been difficult, with many groups looking for changes in single glycans that could identify all cancers. That is, while alterations in glycosylation have been associated with many types of cancers (Comunale et al., 2010, PLoS One, 5:el2419; Wang et al., 2017, Cancer Epidemiol Biomarkers Prev, 26:795-803; Comunale et al., 2006, J Proteome Res, 5:308-315; Wang et al., 2009, Cancer epidemiology, biomarkers & prevention, 18:1914-1921; Callewaert et al., 2003, Glycobiology, 13:367-475; Callewaert et al., 2004, Nat Med, 10:429-434; Liu et al., 2008, Chinese journal of hepatology, 16:74-75; Blomme et al., 2009, J Hepatol, 50:592-603; Vanderschaeghe et al., 2008, Molecular & cellular proteomics, 8:986-94; Debruyne et al., 2010, Clin Chem, 56:823-831;Vanderschaeghe et al., 2010, Anal Chem, 82: 7408-7415; Blomme et al., 2011, Gastrointestinal and liver physiology, 300:G833-842; Blomme et al., 2012, Dig Liver Dis, 44:315-322;Vanderschaeghe et al., 2013, Methods Mol Biol, 919:249-257; Verheist et al., 2017, Clin Cancer Res, 23:2750-2758; Block et al ., 2005, Proceedings of the National Academy of Sciences of the United States of America, 102:779-784; Marrero et al., 2005, J Hepatol, 43: 1007-1012; Mehta et al., 2008, J Virol, 82: 1259-1270; Norton et al., 2008, J Cell Biochem, 104: 136-149; Comunale et al., 2009, Journal of Proteome Research, 8:595-602; Comunale et al., 2011, Cancer epidemiology, biomarkers & prevention, 20: 1222-1229; Mehta et al., 2012, Cancer epidemiology, biomarkers & prevention, 21 :925-933; Comunale et al., 2013, Proteomics Clin Appl, 7:690-700; Lamontagne et al., 2013, PLoS ONE, 8:e64992; Betesh et al., 2017, Proteomics Clin Appl, 11; Shah et al., 2015, Mol Cell Proteomics, 14:2753-2763; Hu et al., 2020, Cell Rep, 3: 108276), the development of N-glycan specific biomarkers has been limited. The reason for this was that N-glycan analysis often required the purification of a protein of interest before analysis, which was not feasible for the analysis of the hundreds or thousands of samples needed for clinical evaluation or, importantly, clinical use. Additionally, this oftenAttorney Docket: 206085-0170-00WO limited the analysis to a single protein at a time. Studies that utilize just total serum are easier but lose all links to protein identification and in turn have diminishing clinical utility. The method utilized here allows for the simultaneous analysis of all glycans on multiple targets. Indeed, while N-glycan analysis was performed on 16 glycoproteins, that number could easily be increased. Current antibody array platforms often incorporate hundreds or thousands of antibodies, and such a platform could be used in the future to allow for targeting of more serum glycoproteins. This could include HCC glycoprotein biomarkers like AFP and GP73, whose glycoforms have shown promise as improved cancer biomarkers (Block et al., 2005, Proceedings of the National Academy of Sciences of the United States of America, 102:779-784; Jiang et al., 2016, Glycoconj J, 32:657-664; Li et al., 2001, Clin Chim Acta, 313: 15-9).

[0258] It is noted that antibody microarrays are often used for detecting protein expression from various biofluids and have been used in combination with lectins for N-glycan information (Chen et al., 2007, Nat Methods, 4:437-444). However, lectins only recognize a subset of N-glycan features, many of which are unknown, and their binding is mediated by several factors that can alter their binding affinity (such as protein structure). The GlycoTyper method addresses this limitation and allows for compositional information to be determined for every protein captured.

[0259] Additional methods allowing for the analysis of sialic acid in multiple MALDL IMS workflows are being developed. One such approach that has been used with immune cell antibody array slides is an amidation reaction to stabilize sialic acids, as well as distinguish their isomeric linkages (Dressman et al., 2023, Anal Chem, 95:10289-10297). Further, a longitudinal study will be performed to determine how this algorithm will be used in the management of patients at risk of developing HCC.

Claims

Attomev Docket: 206085-0170-00WOCLAIMSWhat is claimed is:

1. A method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining a glycoprotein profde of the serum sample; c) producing a patient health profile comprising the glycoprotein profile; and d) detecting the presence of HCC in the subject when the combined health profile of the subject is greater than that of an average subject.

2. The method of claim 1, wherein step b) further comprises determining the serum alpha-fetoprotein (AFP) level of the subject.

3. The method of claim 2, wherein the patient health profile comprises the serum AFP level of the subject, the glycoprotein profile of the subject, the age of the subject, and the sex of the subject.

4. The method of claim 1, wherein the glycoprotein profile comprises at least one glycoprotein selected from the group consisting of alpha-1 antitrypsin (AAT); alpha-l-acid glycoprotein (AGP); alpha-2-macroglobulin (A2M); angiotensin (ANGIO); apolipoprotein D (APOD); apolipoprotein H (APOH); ceruloplasmin (CERU); fetuin (FET); clusterin (CLUS); haptoglobin (HAPT); hemopexin (HEMO); immunoglobulin G (IGG); transferrin (TRAN); vitamin D binding protein (VDBP); histidine-proline rich glycoprotein (HPRG); a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13); attractin (ATRN); cluster of differentiation 109 (CD 109); cluster of differentiation 163 (CD 163); cluster of differentiation 42D (CD42D); colony stimulating factor 1 receptor (CSF1R); fibulin 1 (FBLN1); low density lipoprotein receptor-related protein 1 (LRP1); lumican (LUM); mannose receptor C-type 1 (MMR); pl exin domain containing 2 (PLXDC2); serpin G1 (IC1), sex hormone-binding globulin (SHBG); and Susi, Von Willebrand Factor Type A (SVEP1).Attorney Docket: 206085-0170-00WO5. The method of claim 1 , wherein the glycoprotein profile comprises at least one glycan selected from the group consisting of: Glycan 933 (Hex3HexNAc2; [M+Na] m'z = 933.490), Glycan 1257 (Hex5HexNAc2; [M+Na] m / z = 1257.423), Glycan 1282 (Hex3dHexlHexNAc3; [M+Na] m / z = 1282.454), Glycan 1419 (Hex6HexNAc2; [M+Na] m z = 1419.476), Glycan 1445 (Hex4dHexlHexNAc3; [M+Na] m / z = 1444.507), Glycan 1502 (Hex4HexNAc4; [M+Na] m / z = 1501.529), Glycan 1547 (Hex4dHexlHexNAc3; [M+SO4+2Na] m / z = 1546.539), Glycan 1591 (Hex4dHex2HexNAc3; [M+Na] m / z = 1590.558), Glycan 1607 (Hex5dHexlHexNAc3; [M+Na] m / z = 1606.560), Glycan 1611 (Hex4dHexNAc3NeuAcl; [M+2Na] m / z = 1611.604), Glycan 1664 (Hex5HexNAc4; [M+Na] m / z = 1663.581), Glycan 1689 (Hex3dHexlHexNAc5; [M+Na] m / z = 1688.613), Glycan 1736 (Hex4dHexlHexNAc3NeuAcl; [M+Na] m / z = 1735.516), Glycan 1744 (Hex8HexNAc2; [M+Na] m / z = 1743.538), Glycan 1810 (Hex5dHexlHexNAc4; [M+Na] m / z = 1809.639), Glycan 1811 (Hex5dHexlHexNAc4; [M+H+Na] m / z = 1810.639), Glycan 1849 (Hex6dHexlHexNAc3S04; [M+Nal] m / z = 1848.722), Glycan 1851 (Hex4dHexlHexNAc5; [M+Na] m'z = 1850.666), Glycan 1854 (Hex5dHexNAc4NeuAclSO4; [M+Na2] m / z = 1853.674), Glycan 1855 (Hex5dHex2NAc3SO4; [M+Na2] m / z = 1854.675), Glycan 1860 ([M+] m / z = 1859.615), Glycan 1867 (Hex5HexNAc5; [M+Na] m / z = 1866.661), Glycan 1868 (Hex4HexNAc3NeuAcSO4; [M+Na3] m / z = 1867.75), Glycan 1876 (Hex5HexNAc5; [M+Na] m / z = 1875.611), Glycan 1890 (Hex5dHexlHexNAc4]SO4]; [M+Na] m / z = 1889.93), Glycan 1906 (Hex9HexNAc2; [M+Na] m / z = 1905.624), Glycan 1908 (Hex4HexNAc6; [M+Na] m / z = 1907.636), Glycan 1955 (Hex5HexNAc4]NeuAcl]; [M+Na] m / z = 1954.676), Glycan 1956 (Hex5dHex2HexNAc4; [M+Na] m / z = 1955.693), Glycan 1973 (Hex6dHexlHexNAc3SO4; [M+Na3] m / z = 1972.693), Glycan 1994 (Hex6HexNAc3NeuAclSO4; [M+Na] m / z = 1993.655), Glycan 1995 (Hex6dHex2HexNAc3 ; [M+Na] m / z = 1994.662), Glycan 2013 (Hex5dHexlHexNAc5; [M+Na] m'z = 2012.719), Glycan 2016 (Hex6HexNAc3NeuAc 1 SO4; [M+Na2] m / z = 2015.726), Glycan 2071 (Hex5HexNAc5SO42; [M+Na3] m / z = 2070.634), Glycan 2102 (Hex5dHex3HexNAc4; [M+Na] m / z = 2101.749), Glycan 2119 (Hex6dHex2HexNAc3NeuAclSO42; [M+Na3] m / z = 2118.609), Glycan 2120 (Hex4HexNAc5SO4; [M+Na3] m / z = 2119.605), Glycan 2123 (Hex5dHexlHexNAc4NeuAcl; [M+2Na] m / z = 2122.7173), Glycan 2134 (Hex4dHexlHexNAc6SO4; [M+Na] m / z = 2133.726), Glycan 2135 (Hex7HexNAc3(SO4)2]; [M+Na3] m / z = 2134.74), Glycan 2140 (Hex6dHexlHexNAc2NeuAcSO4]; [M+Na] m / z =Attorney Docket: 206085-0170-00WO2139.709), Glycan 2142 (Hex4dHexlHexNAc5NeuAc; [M+Na] m / z = 2141 .721), Glycan 2175 (Hex6dHexlHexNAc5; [M+Na] m / z = 2174.772), Glycan 2281 (Hex7HexNAc3SC>42; [M+Na3] m / z = 2280.693), Glycan 2321 (Hex6dHex2HexNAc5; [M+Na] m / z = 2320.829), Glycan 2468 (Hex6dHex3HexNAc5; [M+H+Na] m / z =2467.892), and Glycan 2540 (Hex7dHexlHexNAc6+Nal; [M+H] m / z = 2539.904).

6. The method of claim 1, wherein the glycoprotein profile comprises the level of at least one glycoform selected from the group consisting of angiotensin (ANGIO) conjugated to Glycan 2540 (ANGIO_2540), clusterin (CLUS) conjugated to Glycan 1607 (CLUS_1607), transferrin (TRAN) conjugated to Glycan 1607 (TRAN 1607), haptoglobin (HAPT) conjugated to Glycan 1689 (HAPT 1689), HAPT conjugated to Glycan 1851 (HAPT 1851), hemopexin (HEMO) conjugated to Glycan 2540 (HEMO 2540), and immunoglobulin G (IGG) conjugated to Glycan 1419 (IGG_1419), alpha-2-macroglobulin (A2M) conjugated to Glycan 1867 (A2M_1867), apolipoprotein D (APOD) conjugated to Glycan 2175 (APOD 2175), CLUS conjugated to Glycan 1689 (CLUS 1689), HAPT conjugated to Glycan 2321 (HAPT 2321), HEMO conjugated to Glycan 2175 (HEMO 2175), IGG conjugated to Glycan 1257 (IGG 1257), IGG conjugated to Glycan 1867 (IGG_1867), TRAN conjugated to Glycan 2175 (TRAN_2175), and vitamin D binding protein (VDBP) conjugated to Glycan 2175 (VDBP 2175), alpha-1 antitrypsin (AAT) conjugated to Glycan 2321 (AAT_2321), alpha-1 acid glycoprotein (AGP) conjugated to Glycan 2321 (AGP_2321), fetuin (FET) conjugated to Glycan 2175 (FET_2175), FET conjugated to Glycan 2540 (FET_2540), IGG conjugated to Glycan 1502 (IGG_1502), AGP conjugated to Glycan 2540 (AGP 2540), AGP conjugated to Glycan 1257 (AGP 1257), AGP conjugated to Glycan 1219 (AGP_1219), AGP conjugated to Glycan 1445 (AGP_1445), ANGIO conjugated to Glycan 1851 (ANGIO 1851), apolipoprotein H (APOH) conjugated to Glycogen 1851 (APOH 1851), APOH conjugated to Glycogen 1867 (APOH 1867), APOH conjugated to Glycogen 2157 (APOH 2157), ceruloplasmin (CERU) conjugated to Glycogen 1257 (CERUJ257), CERU conjugated to Glycogen 2175 (CERU_2175), CERU conjugated to Glycogen 2321 (CERU_2321), CLUS conjugated to Glycan 1257 (CLUSJ257), CLUS conjugated to Glycan 1851 (CLUS_1851), CLUS conjugated to Glycan 2013 (CLUS_2013), CLUS conjugated to Glycan 2540 (CLUS_2540), HEMO conjugated to Glycan 1664 (HEMO 1664), HEMO conjugated to Glycan 1851 (HEMO 1851), histidine-proline richAttorney Docket: 206085-0170-00WO glycoprotein (HPRG) conjugated to Glycan 1689 (HPRG 1689), HPRG conjugated to Glycan 2321 (HPRG 2321), IGG conjugated to Glycan 1607 (IGG 1607), IGG conjugated to Glycan 1689 (IGG 1689), IGG conjugated to Glycan 1744 (IGG 1744), IGG conjugated to Glycan 1810 (IGG 1810), IGG conjugated to Glycan 2123 (IGG 2123), TRAN conjugated to Glycan 1419 (TRAN_1419), TRAN conjugated to glycan 1810 (TRAN_1810), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) conjugated to Glycan 2016 (ADAMTS13_2016), ADAMTS13 conjugated to Glycan 2119 (ADAMTS_2119), attractin (ATRN) conjugated to Glycan 1851 (ATRN_1851), ATRN conjugated to Glycan 1865 (ATRN_1854), ATRN conjugated to Glycan 1876 (ATRN_1876), cluster of differentiation 109 (CD 109) conjugated to Glycan 933 (CD109 933), CD 109 conjugated to Glycan 1851 (CD109 1851), CD109 conjugated to Glycan 1854 (CD109 1854), cluster of differentiation 163 (CD163) conjugated to Glycan 1282 (CD163 1611), CD163 conjugated to Glycan 1591 (CD163 1591), CLDN163 conjugated to Glycan 1612 (CD163 1612), CLDN163 conjugated to Glycan 1849 (CD163 1849), CLDN163 conjugated to Glycan 1868 (CD163 1868), CLDN163 conjugated to Glycan 1955 (CD163_1955), CLDN163 conjugated to Glycan 1995 (CD163_1955), CLDN163 conjugated to Glycan 2071 (CD163_2071), CLDN163 to conjugated Glycan 2102 (CD163_2102), CLDN163 conjugated to Glycan 2142 (CD163_2142), CLDN163 conjugated to Glycan 2468 (CD163_2468), cluster of differentiation 42D (CD42D) conjugated to Glycan 1994 (CD42D 1994), colony stimulating factor 1 receptor (CSF1R) conjugated to Glycan 1689 (CSF1R 1689), CSF1R conjugated to Glycan 1851 (CSF1R 1851), CSF1R conjugated to Glycan 1906 (CSF1R 1906), CSF1R conjugated to Glycan 1956 (CSF1R 1956), CSF1R conjugated to Glycan 2071 (CSF1R 2071), CSF1R conjugated to Glycan 2102 (CSF1R_21O2), CSF1R conjugated to Glycan 2120 (CSF1R_212O), CSF1R conjugated to Glycan 2134 (CSF1R_2134), CSF1R conjugated to Glycan 2140 (CSF1R_214O), CSF1R conjugated to Glycan 2281 (CSF1R_2281), fibulin 1 (FBLN1) conjugated to Glycan 1282 (FBLN1 1282), low density lipoprotein receptor-related protein 1 (LRP1) conjugated to Glycan 933 (LRP1 933), LRP1 conjugated to Glycan 1851 (LRP1 1851), LRP1 conjugated to Glycan 1876 (LRP1_1876), lumican (LUM) conjugated to Glycan 1851 (LUM_1851), LUM conjugated to Glycan 1854 (LUM_1854), LUM conjugated to Glycan 1855 (LUM_1855), mannose receptor C-type 1 (MMR) conjugated to Glycan 1689 (MMR_1689), MMR conjugated to Glycan 1736 (MMR_1736), MMR conjugated to Glycan 1810 (MMR_1810), MMR conjugated to GlycanAttomev Docket: 206085-0170-00WO181 1 (MMR 1811), MMR conjugated to Glycan 1851 (MMRJ 851 ), MMR conjugated to Glycan 1854 (MMR_1854), MMR conjugated to Glycan 1908 (MMR_1908), plexin domain containing 2 (PLXDC2) conjugated to Glycan 1547 (PLXDC2 1547), PLXDC2 conjugated to Glycan 1851 (PLXDC2 1851), PLXDC2 conjugated to Glycan 1860 (PLXDC2 1860), PLXDC2 conjugated to Glycan 1890 (PLXDC2 1890), PLXDC2 conjugated to Glycan 1973 (PLXDC2_1973), PLXDC2 conjugated to Glycan 2135 (PLXDC2_2135), Serpin G1 (IC1) conjugated to Glycan 1851 (IC 1 1851), IC1 conjugated to Glycan 1855 (IC 1 1855), IC1 conjugated to Glycan 1860 (IC 1 1860), sex hormone-binding globulin (SHBG) conjugated to Glycan 1736 (SHBG 1736), SHBG conjugated to Glycan 1810 (SHBG 1810), SHBG conjugated to Glycan 1811 (SHBG 1811), SHBG conjugated to Glycan 1851 (SHBG 1851), SHBG conjugated to Glycan 1854 (SHBG 1854), Sushi, von Willebrand Factor Type A (SVEP1) conjugated to Glycan 1851 (SVEP1 1851), and SVEP1 conjugated to Glycan 2142 (SVEP1_2142).

7. The method of claim 3, wherein the glycoprotein profile comprises: i) the level of: ANGIO_2540, CLUS_1607, and TRAN_1607, or ii) the level of: ANGIO_2540, CLUS_1607, and TRAN 607, andA) the level of: HAPT 1689, HAPT 1851, HEMO 2540, and IGG 1419;B) the level of: A2M_1867, APOD_2175, CLUS_1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, and VDBP 2175;C) the level of: HAPT 1689, HAPT 1851, HEMO 2540, APOD 2175, HAPT 2321, IGG 1257, AAT 2321, AGP 2321, FET 2175, FET 2540, and IGG 1502;D) the level of: HAPT 1689, HAPT 1851, HEMO 2540, A2M 1867, HAPT 2321, IGG 1257, FET 2175, FET_2540, and AGP_2540;E) the level of: HEMO 2175 and IGG 1257; orF) the level of: HEMO_2540, A2M_1867, APOD_2175, HAPT_2321, HEMO 2175, IGG 1257, TRAN 2175, VDBP 2175, AGP_2321, FET 2175, IGG 1502, AGP 1257, AGP 1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU 2321, CLUS 257, CLUS_1851, CLUS_2013, CLUS_2540, HEMO 1664,Attomev Docket: 206085-0170-00WOHEMO J 851, HPRGJ689, HPRG 2321, IGGJ607, IGGJ689, IGGJ744, IGG 1810, IGG 2123, TRAN 1419, and TRAN 1810.

8. The method of claim 1, wherein step c) comprises: i) providing a substrate comprising a plurality of antibody spots; ii) incubating the substrate in a blocking solution; iii) incubating the substrate with the serum sample from the subject; iv) treating the substrate with an enzymatic releasing solution; and v) scanning the substrate by mass spectrometry to detect and identify the presence of glycans.

9. The method of claim 8, wherein the substrate comprising a plurality of antibody spots comprises a plurality of spots against at least one antigen, wherein the antibodies of each spot bind a single antigen.

10. The method of claim 9, wherein the at least one antigen comprises at least one selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

11. The method of claim 8, wherein the substrate comprises at least one antibody spot for each of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG.

12. The method of claim 8, wherein the substrate is selected from the group consisting of glass, polydimethylsiloxane (PDMS), epoxysilane-coated glass, and epoxysilane-coated PDMS.

13. The method of claim 8, wherein the blocking solution is bovine serum albumin(BSA).Attorney Docket: 206085-0170-00WO14. The method of claim 8, wherein the enzymatic releasing solution comprises PNGase F.

15. The method of claim 8, wherein the mass spectrometry is selected from the group consisting of: matrix-assisted laser desorption / ionization imaging Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption / ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry.

16. The method of claim 15, wherein the step of scanning the substrate is preceded by a step of spraying the substrate with a MALDI matrix material.

17. The method of claim 16, wherein the MALDI matrix solution is selected from the group consisting of: 2,5-dihydroxybenzoic acid, a-cyano-4-hydroxycinnamic acid, sinapinic acid, 1,5-diaminonaphthalene, and 9-aminoacridine.

18. A device for rapid detection of hepatocellular carcinoma (HCC) comprising a solid substrate spotted with a plurality of antibodies, wherein each antibody spot comprises a plurality of antibodies that bind a single antigen, wherein the substrate comprises at least one antibody spot for at least one antigen selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH;CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD109; CD163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.Attomev Docket: 206085-0170-00WO19. The device of claim 18, wherein the substrate comprises at least one antibody spot for each of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG.

20. A kit for detecting hepatocellular carcinoma comprising: the device of claim 18; at least one blocking solution; at least one enzymatic releasing solution; and at least one MALDI matrix material.

21. The kit of claim 20, wherein the blocking solution is bovine serum albumin.

22. The kit of claim 20, wherein the enzymatic releasing solution comprises PNGase F.

23. The kit of claim 20, wherein the MALDI matrix solution is a-cyano-4- hydroxycinnamic acid.

24. A method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; and d) determining that the subject has HCC when the level of the at least one glycan is altered compared to that in the serum of an individual without HCC.

25. The method of claim 24, wherein the at least one glycoprotein comprises at least one selected from the group consisting of: AAT, AGP, A2M, ANGIO, APOD, APOH, CERU, FET, CLUS, HAPT, HEMO, IGG, TRAN, VDBP, HPRG, ADAMTS13, ATRN, CD109, CD163, CD42D, CSF1R, FBLN1, LRP1, LUM, MMR, PLXDC2, IC1, SHBG, and SVEP1.Attomev Docket: 206085-0170-00WO26. The method of claim 24, wherein the at least one glycan is selected from the group consisting of: Glycan 933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.

27. The method of claim 24, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycan is increased in the serum of the subject compared to the serum of an individual without HCC, and wherein the one glycan is selected from the group consisting of: Glycan 1257, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1607, Glycan 1664, Glycan 1689, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1867, Glycan 2013, Glycan 2123, Glycan 2175, Glycan 2321, and Glycan 2540.

28. The method of claim 24, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycan is decreased in the serum of the subject compared to the serum of an individual without HCC, and wherein the one glycan is selected from the group consisting of: Glycan 933, Glycan 1736, Glycan 1860, Glycan 1876, Glycan 2071, Glycan 2102, and Glycan 2142.

29. A method of detecting hepatocellular carcinoma (HCC) in a subject comprising: a) obtaining a serum sample from the subject; b) determining the levels of at least one glycoprotein in the serum sample; c) determining the level of at least one glycan conjugated to the at least one glycoprotein; andAttomev Docket: 206085-0170-00WO d) determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein in the serum of the subject is altered compared to the serum of an individual without HCC.

30. The method of claim 29, wherein the at least one glycoprotein comprises at least one selected from the group consisting of: AAT; AGP; A2M; ANGIO; APOD; APOH; CERU; FET; CLUS; HAPT; HEMO; IGG; TRAN; VDBP; HPRG; AD AMTS 13; ATRN; CD 109; CD 163; CD42D; CSF1R; FBLN1; LRP1; LUM; MMR; PLXDC2; IC1; SHBG; and SVEP1.

31. The method of claim 29, wherein the at least one glycan is selected from the group consisting of: Glycan 933, Glycan 1257, Glycan 1282, Glycan 1419, Glycan 1445, Glycan 1502, Glycan 1547, Glycan 1591, Glycan 1607, Glycan 1611, Glycan 1664, Glycan 1689, Glycan 1736, Glycan 1744, Glycan 1810, Glycan 1811, Glycan 1849, Glycan 1851, Glycan 1854, Glycan 1855, Glycan 1860 , Glycan 1867, Glycan 1868, Glycan 1876, Glycan 1890, Glycan 1906, Glycan 1908, Glycan 1955, Glycan 1956, Glycan 1973, Glycan 1994, Glycan 1995, Glycan 2013, Glycan 2016, Glycan 2071, Glycan 2102, Glycan 2119, Glycan 2120, Glycan 2123, Glycan 2134, Glycan 2135, Glycan 2140, Glycan 2142, Glycan 2175, Glycan 2281, Glycan 2321, Glycan 2468, and Glycan 2540.

32. The method of claim 29, wherein the at least one glycoform of the at least one protein comprises at least one selected from the group consisting of: ANGIO_2540, CLUS_1607, TRAN 1607, HAPT 1689, HAPT 1851, HEMO 2540, IGG 1419, A2M 1867, APOD 2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, VDBP 2175, AAT_2321, AGP 2321, FET_2175, FET_2540, IGG_1502, AGP_2540, AGP 1257, AGP 1219, AGP 1445, ANGIOJ851, APOH 1851, APOH 1867, APOH 2157, CERU_1257, CERU 2175, CERU 2321, CLUSJ257, CLUS_1851, CLUS_2013, CLUS_2540,HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRANJ419, TRAN 1810, ADAMTS13_2016, ADAMTS_2119, ATRN_1851, ATRN_1854, ATRNJ876, CD109_933, CD109 1851, CD109 1854, CD163 1611, CD163 1591, CD163 1612, CD163 1849, CD163 1868, CD163 1955, CD163 1955, CD163 2071, CD163_2102, CD163_2142, CD163_2468, CD42D 1994,Attorney Docket: 206085-0170-00WOCSF1RJ689, CSF1RJ851, CSF1R 1906, CSF1R 1956, CSF1R 2071, CSF1R 2102, CSF1R_212O, CSF1R_2134, CSF1R_214O, CSF1R 2281, FBLN1_1282, LRP1_933, LRP1 1851, LRP1 1876, LUM 1851, LUM 1854, LUM 1855, MMR 1689, MMR 1736, MMR 810, MMR 811, MMR 1851, MMR 1854, MMR 1908, PLXDC2 547, PLXDC2 1851, PLXDC2 1860, PLXDC2 1890, PLXDC2 1973, PLXDC2 2135, IC1 1851, IC1 1855, IC1 1860, SHBG 1736, SHBG_1810), SHBG 1811, SHBG_1851, SHBG_1854, SVEP1 1851, and SVEP1_2142.

33. The method of claim 29, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HCC, wherein the at least one glycoform of the at least one glycoprotein is selected from the group consisting of: ADMTS13_2016, ATRNJ851, ATRN_1854, CD109 1851, CD109 1854, CSF1R 1689, CSF1R 1851, FBLN1_1282, LRP1 1851, LUM_1851, LUM 1854, LUMJ855, MMR_1689, MMR 810, MMR+1811, MMR 851, MMR 1854, PLXDC2 1851, ICl l 851, IC1 1855, SHBG 1810, SHBG 1811, SHBG 1851, SHBG 1854, SVEP1 2142, ANGIO 2540,CLUS 1607, TRANJ607, HAPT 1689, HAPT 1851, HEMO_2540, IGG_1419, A2MJ867, APOD 2175, CLUS 1689, HAPT 2321, HEMO 2175, IGG 1257, IGG 1867, TRAN 2175, VDBP 2175, AAT_2321, AGP_2321, FET_2175, FET_2540, IGG 1502, AGP_2540, AGP 1257, AGP 1219, AGP 1445, ANGIO 1851, APOH 1851, APOH 1867, APOH 2157, CERU 1257, CERU 2175, CERU_2321, CLUS_1257, CLUS_1851, CLUS_2013, CLUS_2540, HEMO 1664, HEMO 1851, HPRG 1689, HPRG 2321, IGG 1607, IGG 1689, IGG 1744, IGG 1810, IGG 2123, TRANJ419, and TRANJ810.

34. The method of claim 29, wherein step d) comprises determining that the subject has HCC when the level of the at least one glycoform of the at least one glycoprotein is increased in the serum of the subject compared to the serum of an individual without HCC, wherein the at least one glycoform of the at least one glycoprotein is selected from the group consisting of: ADAMTS13_2119, ATRNJ876, CD109_933, CD163_1282, CD163 1591, CD163_1612, CD163_1849, CD163 1868, CD163 1955, CD163 1995, CD163 2071, CD163_2102, CD163_2142, CD163_2468, CD42D 1994, CSF1R 1906, CSF1R 1956, CSF1R 2071,Atorney Docket: 206085-0170-00WOCSF1R_21 O2, CSF1R 2120, CSF1R_2134, CSF1R_214O, CSF1R 2281, LRP1 933, LRP1 1876, MMR_1736, MMR 1908, PLXDC2_1547, PLXDC2_1860, PLXDC2 1890, PLXDC2 1973, PLXDC2 2135, IC1 1860, SHBG 1736, and SVEP1 2142.

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