WO2007082057A2 - Lectin arrays and translational applications - Google Patents

Abstract

Systems comprising lectin-based arrays and mass spectrometry means and their use in the identification and detection of aberrant glycoproteins in cancer and cancer diagnosis are provided. The lectins of the arrays comprise members collectively cognate to galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide (e.g., an array comprising PNA, ECL, and Jacalin) Glycoprotein biomarkers of cancer identified by use of the methods are also provided.

Description

Lectin Arrays And Translational Applications

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This reference claims priority benefit of U.S. Provisional Application Serial No. 60/757992 filed January 10, 2006.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [00021 This work was partially funded with NIH Research Grant DE 16275. The U.S. government may have certain rights in the invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE₃ OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK. [0003] NOT APPLICABLE

BACKGROUND OF THE INVENTION

[0004] Proteomics is a powerful approach for biomedical research because it directly studies the key functional components of biochemical systems and the cellular targets of therapeutic agents, namely proteins. Mapping proteomes, the protein complements to genomes, from tissues, cells, and body fluids is being used to validate and forward new protein targets, to explore mechanisms of action or toxicology of compounds, and to discover new disease biomarkers for clinical and diagnostic applications. Protein arrays are also rapidly becoming a powerful tool for proteomics studies (Zhu H, Bilgin M, Snyder M., Annu RevBiochem. 2003,72: 783-812).

[0005] Protein arrays are also rapidly becoming a powerful means to detect proteins, monitor their expression levels, and investigate protein interactions and functions. As a novel proteomics platform, protein arrays allow for highly parallel analysis of only a minimum of body fluid/biopsy materials and therefore have great potential in molecular analysis of human cancer as well as tumor biomarker discovery. Protein arrays can be formatted as solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, microbeads or other particles. The assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). The objective behind protein array development is to achieve high-throughput and sensitive measurements of proteins by automated means, and giving an abundance of data for a single experiment.

[0006] A bottleneck in creating antibody protein arrays, especially those for global measurements, is the production (expression and purification) of the huge diversity of antibodies which will form the array elements. A collection of high-quality expression clones is required and high-yield protein purification systems must be developed. The design of capture arrays, particularly when screening against complex samples, also needs to take into consideration the problems of cross-reactivity. Antibodies can be surprisingly cross-reactive, which can render misleading results particularly with highly multiplexed assays. Novel detection systems which further increase specificity, such as proximity ligation (Gullberg M, Gustafsdottir SM, Schallmeiner El Jarvius J, Bjarnegard M, Betsholtz C, Landegren U, Fredriksson S., Proc Natl Acad Sd USA. 2004, 101 (22): 8420-4), can help to minimize cross-reactions. MS detection is advantageous because it can resolve the bound molecules and further confirm their identities.

[0007] The majority of proteins are modified in post-translational events and one of the common modifications is glycosylation. Aberrant glycosylation has been shown as a key event in human disease progression such as cancer cell invasion and metastasis and many glycosyl epitopes actually constitute tumor-associated antigens (Hakomori S., Cancer Res. 1996, 56(23): 5309-18). In glycoproteins, carbohydrates are typically linked to serine and thereonine residues (O-link) or to asparagine residues (N-link). Protein glycosylation, and particularly N-linked glycosylation, is prevalent in proteins destined for extracellular environments (Roth J. Chem Rev. 2002,102(2): 285-303). These include proteins on the extracellular side of the plasma membrane, secreted proteins and proteins in the human body fluids such as serum, cerebrospinal fluid, saliva, lung lavage fluid and pancreatic juice. Changes in the extent of the glycosylation and the carbohydrate structure of proteins on the cell surface and in body fluids have been shown to correlate with cancer and other disease states, highlighting the clinical importance of this modification as an indicator or effector of pathological mechanisms (Durand G, Seta N., Clin Chem. 2000,46: 795-805, Spiro RG., Glycobiology. 2002, 12(4): 43R-56R). It is therefore not surprising that many clinical biomarkers and therapeutic targets are glycoproteins, including Her2/Neu in breast cancer, prostate-specific antigen in prostate cancer, CAl 25 in ovarian cancer, and carcinoembryonic antigen (CEA) in colon cancer. [0008] Aberrant glycosylation of proteins occurs in all types of human cancers and largely involves tumor invasiveness and malignancy. Therefore the provision of arrays and methods useful in detecting aberrant glycosylation of proteins in the identifying the presence of cancer and the malignant conversion potential of human pre-malignancies is vital to cancer research.

BRIEF SUMMARY OF THE INVENTION

[0009] Accordingly, in a first aspect, the invention provides methods of identifying the presence of a cancer in a subject by a) contacting a sample from the subject with an array comprising a plurality of lectins in which each member lectin of said plurality differs from other member lectins of said plurality according to their cognate saccharide, and wherein said plurality of lectins comprises members collectively cognate to galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide, and wherein said contacting is under reaction conditions capable of binding glycoproteins in said sample to a lectin cognate to a saccharide moiety of said glycoproteins; b) determining a profile of the glycoproteins bound by the lectins of the array thereby developing a lectin binding profile of the glycoproteins in the sample; and; c) comparing the profile of the sample with a profile of the glycoproteins bound by the lectins of the array for a control sample.

[0010] In a second aspect, the invention provides methods of identifying a glycoprotein whose glycosylation pattern is aberrant in cancer cells by a) contacting a sample from a subject with the cancer with an array comprising a plurality of lectins, wherein each member of the plurality is located at a predetermined locus on the array, and wherein each member lectin of said plurality differs from other member lectins of said plurality according to their cognate saccharide, and wherein said plurality of lectins comprises members collectively cognate to galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide, and wherein said contacting is under reaction conditions capable of binding glycoproteins in said sample to a lectin cognate to a saccharide group of said glycoprotein; b) determining a profile of the glycoproteins bound by the lectins of the array thereby developing a lectin binding profile of the glycoproteins in the sample; and c) comparing the profile of the sample with a profile of glycoproteins bound by the lectins of the array for a control sample; and d) determining the amino acid sequence of the aberrant proteins. [0011] In a third aspect, the invention provides methods of identifying the presence of an oral or head or neck cancer (e.g., squamous cell carcinomas of oral cavity, head or neck)) in a subject by a) contacting a sample from the subject with one, two, or three lectins selected from the group consisting of Jackalin, ECL, and PNA, or one, two, or three lectins selected non-independently to bind a saccharide moiety bound by Jackalin. ECL, and PNA; b) determining a profile of the glycoproteins bound by the lectin thereby developing a profile of the glycoproteins in the sample; and; c) comparing the profile of the sample with a profile of the glycoproteins bound by the lectin for a control sample.

[0012] For any of the above aspects, in some embodiments, the profile describes the mass/charge ratios and amounts, or relative amounts, of one or more glycoproteins bound by each locus of the array, thereby providing a lectin binding fingerprint of the glycoproteins in the sample(s). In some further embodiments, the profiles are determined using MS or MALDI-MS.

[0013] For any of the above aspects, in some embodiments, tandem mass spectrometry (MSIMS) is used to profile the glycoproteins and to sequence and identify one or more captured glycoproteins.

[0014] For any of the above aspects, in some embodiments, the control is one or more subjects who are not known or suspected of having cancer, thereby identifying the cancer. In other embodiments, the control is a previous sample from the subject at a time when the subject did not have cancer or was in remission. In some embodiments, subject is suspected of having cancer or is being screened for the possibility of having cancer. In some embodiments, the subject has previously been diagnosed and treated for the cancer and the subject is being screened for the recurrence of the cancer or the efficacy of the treatment is being evaluated by determining if the aberrant glycoprotein pattern is reverting to a control pattern. In some embodiments, one or more aberrant glycoproteins or glycoforms associated with cancer are identified in the subject. In some embodiments, of the first two aspects, one or more aberrant glycoproteins or glycoforms are detected in a fingerprint of glycoproteins wherein over 25, 50, 75, 100, 150 or more glycoproteins or glycoforms are detected according to their m/z and/or lectin binding affinity. [0015] For any of the above aspects, in some embodiments, the sample is a biological fluid taken from the subject or control. The fluid can be, for instance, saliva, cerebrospinal fluid, lymph, blood, plasma, urine, or a portion thereof. In some embodiments, the cancer is selected from the group consisting of cancers of the head and neck. For instance, the cancer can be a tumor of the oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx. In further embodiments, the cancer is one etiologically associated with tobacco or alcohol exposure.

[0016] For either of the first two aspects, in some embodiments, the array comprises a plurality of lectins having individual members which are collectively are cognate to (i.e., bind selectively to) galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide moieties. The array accordingly comprises lectins cognate to a saccharide moiety of a glycoprotein having an aberrant glycosylation pattern. Such patterns, include, but are not limited to, βl->6GlcNAc antenna in N-linked structures from increased activity; Tn and STn antigens; promiscuous 0-glycosylation and resulting peptide conformational changes; and overexpression of lacto-series type 1 and type 2 structures (often in the form of poly-LacNAc) with a variety of fiicosylation and sialylation. Additionally, poly-LacNAc with sialosyl or fucosyl substitution in glycoproteins is often ex pressed at the βl-*6GlcNAc side chain of N-linked structure or at the βl→6GlcNAc-linked side chain of the "Core 2" 0-Iinked structure. Preferably, the array comprises lectins cognate to one, two, three, four or more of these aberrant glycoforms. In some embodiments, the array has two, three, four, or from 5 to 10 lectins, 10 to 25 lectins, 20 to 30 lectins, 25 to 50 lectins, 50 to 100 lectins, or more. In yet additional embodiments, the plurality of lectins collectively can bind from 5 to 10, 10 to 30, 30 to 70 different saccharide moieties. In some embodiments, the plurality of lectins comprise one, two, or three lectins from the group consisting of Erythrina cristagalli lectin (ECL), Jacalin and PNA. In some embodiments, the array of lectins comprises at least one lectin which binds galactosamine and one lectin which binds an O-glycosidically linked oligosaccharide. In some embodiments, the lectin binding fingerprint includes an aberrant glycoprotein with a m/z ratio of about 8027 or 35358 daltons which is capable of binding to Jacalin. In other embodiments, the fingerprint includes an aberrant glycoprotein with a m/z ratio of about 4287 or 7991 Daltons which is capable of binding to PNA or ECL. In still other embodiments, the fingerprint includes all three of the above aberrant glycoproteins. In yet another embodiment, the fingerprint includes one, two, three, or four of aberrant glycoproteins having a m/z ratio of about 14096, 15445, 25767, and 28695 Daltons which are capable of binding PNA. In further embodiments of such, the subject is a human suspected of having oral cancer or having had a history oral cancer or being treated for oral cancer. In still further embodiments of any of the above, the sample is a blood sample.

[0017] For the first two aspects, in some embodiments, the plurality of lectins are located on a PDMS substrate or surface. In yet additional embodiments, each member of the plurality of lectins is located in a PDMS well.

[0018] For any of the above aspects, in some embodiments, the amount and mass of plurality of glycoproteins bound to the array is determined using mass spectroscopy or MALDI-MS.

[0019] In yet a fourth aspect, the invention provides a system for detecting aberrant glycoproteins in which the system comprises: a) an array comprising a plurality of lectins, wherein each member of the plurality is located at a predetermined locus on the array, and wherein each member lectin of said plurality differs from other member lectins of said plurality according to their cognate saccharide, and wherein said plurality of lectins comprises members collectively cognate to galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O- glycosidically linked oligosaccharide; b. means for contacting a biological sample with the array under conditions which allow the lectins of the array to bind a cognate saccharide group; and c. means for determining the profile of the glycoproteins bound to each locus of the array.

[0020] In some embodiments, the profile of the glycoproteins is the amount and mass/charge ratio mass/charge ratio of the proteins. In some embodiments, the amount and mass/charge ratio mass/charge ratio of the proteins is determined using MS or matrix-assisted laser desorption/ionization (MALDI)-MS. In some embodiments, profiling is accomplished by tandem mass spectrometry (MSIMS). In yet additional embodiments of any of the above, the array comprises a PDMS substrate. In some embodiments of any of the above, the lectins on the array format comprise Jacalin, ECL, and PNA. In some embodiments, the system comprises a PDMS array as set forth below.

[0021] In fifth aspect, the invention provides an array comprising a PDMS substrate having a silanized surface; and a plurality of lectins in which the plurality comprises jackalin, ECL, and PNA; and in which each member of the plurality is covalently bonded to the silanized surface at a specific locus. In some embodiments, the points of attachments are wells formed in the substrate. In som 1 e embodiments, the lectins are covalently cross-linked using glutaraldedyde as the crosslinker.

[0022] In still a sixth aspect, the invention provides biomarkers for cancer. In some embodiments, the biomarker is an aberrant glycoprotein with a m/z ratio of about 8027 or 35358 daltons which is capable of binding to Jacalin. In other embodiments, the biomarker is an aberrant glycoprotein with a m/z ratio of about 4287 or 7991 Daltons which is capable of binding to PNA or ECL. hi still other embodiments, the biomarker is an aberrant glycoprotein having a m/z ratio of about 14096, 15445, 25161, or 28695 Daltons which is capable of binding PNA. hi some embodiments, the invention is drawn to methods of identifying the presence of a cancer in a subject by detecting one or more of these biomarkers in a biological sample from the subject. In some embodiments, the method detects aberrant glycoproteins having a m/z ratio of about 4287, 7991 Daltons and/or 8027 and 35358 Daltons. In some embodiments, the cancer is an oral cancer.

[0023] Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1. Fabrication of PDMS wells using micromolding technique (A). A 16- well PDMS device is shown in (B). Each well is 2 mm in diameter and 200 micron in depth. The picture does not show the actual diameter of the wells.

[0025] Figure 2. Immobilization of lectins on PDMS surface. PDMS slide was initially oxidized, and then sequentially treated with 3-aminopropyltriethoxysilane (APTS) and glutaraldehyde. Lectins were immobilized through the cross-linking by glutaraldehyde.

[0026] Figure 3. MALDI-MS analysis of proteins in oral cancer and control patients' sera captured by immobilized Jacalin. (A) MALDI-TOF MS spectrum for glycoproteins captured by immobilized Jacalin, which is a tetramer protein. (B) and (C) are close-ups of (A) within different mass ranges.

[0027] Figure 4. MALDl-MS analysis of proteins in oral cancer and control patients' serum samples captured by immobilized Erythrina Cristagalli Lectin (ECL) (A) and Peanut Agglutinin (PNA) on fabricated PDMS device. ECL is a dimer while PNA is a tetramer protein.

[0028] Fig. 5. Reproducible analysis of an oral cancer patient's serum sample. The same sample was incubated with immobilized lectin Jacalin on two different PDMS wells and then measured by MALDI-TOF MS.

DETAILED DESCRIPTION OF THE INVENTION

[0029] A novel lectin array for high-throughput analysis of glycoproteins and methods of its use in detecting cancer and cancer biomarkers are provided. Applications in human oral cancer research are demonstrated.

[0030] Due to different binding characteristics of lectins to different sugar moieties, different lectins immobilized on the array can reveal distinct glycoprotein alterations. Mass spectrometry (MS) has been utilized as the readout mechanism for analysis and identification of captured glycoproteins. The developed lectin array technology is very useful for fast analysis and identification of glycoproteins, and therefore has great potential in molecular analysis of human cancers as well as biomarker discovery study. Immobilization of a panel of lectins on defined surface such as PDMS or commercially available FAST Slides to produce high-density protein arrays of the invention can readily be accomplished. Many lectins are commercially available. The developed array technology will be very useful for fast screening of glycopeptides/glycoproteins in a large number of clinical samples and discovery of biomarkers for diagnosis of human disease such as cancers. This technology is also invaluable in biopharmaceutical study because many human diseases involve aberrant protein glycosylation and disease-associated glycoproteins may be discovered as potential therapeutic targets or drugs.

[0031J MALDI-TOF MS has been utilized as the readout mechanism in the Examples herein because it allows for very flexible and high-throughput detection of captured glycoproteins. Certainly, chemiluminescence can also be developed for highly sensitive detection and profiling of glycoproteins. In addition, tandem mass spectrometry (MSIMS) can be very useful for sequencing and identification of captured glycoproteins. Sample prefractionation may be needed to enrich proteins within certain mass ranges for better detection sensitivity. Prior to lectin array analysis, high-abundance proteins in serum samples can be removed via affinity chromatography. Removal of these high-abundance proteins prior to separation and analysis may enable deeper probing of the glycoproteins and unmasking of many low-abundance glycoproteins.

[0032] Commercialization of this technology will lead to the widespread application of this novel protein array technology for molecular analysis of human diseases such as cancers. Aberrant glycosylation of proteins occurs in many human diseases including all types of human cancers. The attainment of the discriminatory glycoprotein signatures to predict the presence of human cancer and the malignant conversion potential of human pre-malignancies will be vital to human cancer research. The developed lectin array technology will be very useful for high-throughput screening of glycoproteins in minute clinical samples, and therefore has great potential in harnessing the glycoprotein alterations during the process of carcinogenesis as well as discovering glycoprotein signatures for cancer detection.

1. Definitions

[0033] Unless otherwise stated, the following terms used in the specification and claims have the meanings given below.

[0034] It is noted here that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0035] The term "subject" references a mammal (e.g., human or chimpanzee, cow; dog; cat; rodent, guinea pig, rat, horse, mouse, hamster, rabbit. A sample may be obtained by a biopsy. The subject can have cancer or be suspected of having cancer. In preferred embodiments, the subject is human.

[0036] A "sample", as used herein, can be any biological material from a subject in which the determination of the presence or quantity of aberrant glycoproteins is carried out. A sample includes, but is not limited to, sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A sample is typically obtained from a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; rodent, guinea pig, rat, mouse, hamster, rabbit. A sample may be obtained by a biopsy. As is well known in the art, a biological sample may be directly contacted with array or added to an appropriate fluid or buffer having any ions, pH and other factors which allow the subject lectin-glycoprotein binding reaction to occur. Tris, phosphate, and HEPES buffers, for instance, may be used to adsorb a glycoprotein to a lectin over pHs from 6 to 8.

[0037] A "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (i.e., prostate, lymph node, liver, bone marrow, blood cell), the size and type of the tumor (i.e., solid or suspended (i.e., blood or ascites)), among other factors. Representative biopsy techniques include excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. A diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy" of the tumor mass, or a "fine-needle aspiration biopsy" which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison '$ Principles of Internal Medicine, Kasper, et ah, eds., 16th ed., 2005, Chapter 70, and throughout Part V.

[0038] The term "cancer" refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc. Examples of different types of cancer include, but are not limited to, breast cancer, gastric cancer, bladder cancer, ovarian cancer, thyroid cancer, lung cancer, prostate cancer, uterine cancer, testicular cancer, neuroblastoma, squamous cell carcinoma of the head, neck, cervix and vagina, multiple myeloma, soft tissue and osteogenic sarcoma, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), pleural cancer, pancreatic cancer, cervical cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, small intestine cancer, cancer of the central nervous system, skin cancer, choriocarcinoma; osteogenic sarcoma, fibrosarcoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia, and acute myelocytic leukemia.

[0039] "Head and neck squamous cell carcinoma" refers to group of cancers of epithelial cell origin originating in the head and neck, including the oral cavity and pharynx. These tumors arise from diverse anatomical locations, including the oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx, but in some cases can have in common an etiological association with tobacco and/or alcohol exposure. The oral cavity is defined as the area extending from the vermilion border of the lips to a plane between the junction of the hard and soft palate superiorly and the circumvallate papillae of the tongue inferiorly. This region includes the buccal mucosa, upper and lower alveolar ridges, floor of the mouth, retromolar trigone, hard palate, and anterior two thirds of the tongue. The lips are the most common site of malignancy in the oral cavity and account for 12% of all head and neck cancers, excluding nonmelanoma skin cancers. Squamous cell carcinoma is the most common histologic type, with 98% involving the lower lip. Next most common sites in order of frequency are the tongue, floor of the mouth, mandibular gingiva, buccal mucosa, hard palate, and maxillary gingiva. The pharynx consists of the oropharynx, nasopharynx, and hypopharynx. The most common sites of cancer in the oropharynx are the tonsillar fossa, soft palate, and base of tongue, followed by the pharyngeal wall. The hypopharynx is divided into the pyriform sinus (most common site of tumor involvement), posterior pharyngeal wall, and posted coid region.

[0040] A glycoprotein is a protein or polypeptide covalently linked to one or more saccharide moieties. Oligosaccharides are covalently linked to proteins through nitrogen or oxygen, to Ser or Thr, or to Asn in O- or N-linked oligosaccharides, respectively. In O-glycosylated proteins, for instance, the oligosaccharides range in size from 1 to >20 sugars. These oligosaccharides are usually riot uniformly distributed along the peptide chain; but are typically clustered in heavily glycosylated domains. N- Acetylgalactosamine (GaINAc) is most commonly linked to Ser or Thr. Mannose residues are never detected in normal mature O-glycans. Wild-type glycoproteins can have a number of glycosylated variants, or glycoforms, in which the same peptide sequence is associated with more than one oligosaccharide at the same glycosylation site. A single oligosaccharide may have different structures depending on the folding of the peptide portion and its recognition as an acceptor by a glycosyltransferase.

[0041] "Aberrant glycoprotein" refers to a glycoprotein whose pattern of glycosylation differs from that of normal or control population with respect to the frequency distribution of glycoforms of the glycoprotein with respect to amounts or kind. In some embodiments, the an increased amount of one, two, three, four, five, six to 10, or more glycoforms is sufficient to denote aberrant an glycoprotein. In some embodiments, the glycoform detected in a sample from a subject in comparison to a control sample from an individual known not to have cancer, for example, is 2-fold, 3-fold, 4-fold, five-fold, eight-fold, or ten-fold of the control. The aberrant glycosylation pattern may be with respect to one glycoprotein or a plurality of glycoproteins.

[0042] As used herein, the term "saccharide" may be used interchangeably with the term "carbohydrate" and refers to single simple sugar moieties or monosaccharides as well as combinations of two or more single sugar moieties or monosaccharides covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The term "saccharide" also includes N-acetylated and N-deacylated derivatives of such monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Saccharides for use in the invention may be linear or branched. Examples of saccharides include, but are not limited to, glucose (GIc) and galactose (Gal). Other examples of monosaccharides include, but are not limited to, N- acetylneuraminic acid (also called sialic acid, NANA₅ or NAN (Sia)), N-acetylglucosamine (GIcNAc), and N-acetylgalactosamine (GaINAc). The cyclic hemiacetal and hemiketal forms of the monosaccharides are contemplated within the defined term. "The saccharide moiety" of a specified glycoprotein refers to a portion or the entire saccharide moiety of the glycoprotein. For instance, a saccharide moiety of GM3 is (NeuAc)Gal-Glc-. The term"sugar" is to be construed as saccharide.

[0043] Other examples of monosaccharides include, but are not limited to, N- acetylneuraminic acid (also called sialic acid, NANA, or NAN (Sia)), N-acetylglucosamine (GIcNAc), and N-acetylgalactosamine (GaINAc). In some embodiments, the array comprises a plurality of lectins which collectively can bind to each of the above saccharides.

[0044] Saccharides include, but are not limited to, N-acetylgalactosamine or GaINAc, N- acetylglucosamine or GIcNAc, N-acetylneuraminic acid or Neu5Ac or NeuAc5,9-N,O- diacetylneuraminic acid orNeu5,9Ac2; fructose,fucose (6-deoxygalactose)or Fuc, galactitol or Gal-ol, galactosamine or GaIN, galactopyranose 3-sulfate, or Galp3S, galactose or Gal, galacturonic acid, or GaIA, glucitol or Glc-ol, glucosamine or GIcN, glucose or GIc, glucuronic acid or GIcA, N-glycoloylneuraminic acidl or Neu5Gc or NeuGc, myoinositol, inositol, mannose, methyl mannose, O-methylgalactose, rhamnose, and xylose. In some embodiments, the array comprises a plurality of lectins which collectively can bind to each of the above saccharides.

[0045] As used herein, the term "disaccharide" refers to a saccharide composed of two monosaccharides linked together by a glycosidic bond. Examples of disaccharides include, but are not limited to, lactose (Lac) (glycosidic bond between Gal and GIc), sucrose (Sue) (glycosidic bond between Frc and GIc), and maltose (MaI), isomaltose and cellobiose (glycosidic bond between GIc and GIc). In some embodiments, the array comprises a plurality of lectins which collectively can bind to each of the above saccharides.

[0046] The term "oligosaccharide" includes an oligosaccharide that has a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, an oligosaccharide is depicted herein with the non-reducing end on the left and the reducing end on the right. An oligosaccharide described herein may be described with the name or abbreviation for the non-reducing saccharide (e.g., Gal), followed by the configuration of the glycosidic bond (α or β), the ring bond, the ring position of the reducing saccharide involved in the bond, and then the name or abbreviation of the reducing saccharide (e.g., GIcNAc). The linkage between two sugars may be expressed, for example, as 2,3,2 -->3, 2-3.

{0047] The term "sialic acid" or "sialic acid moiety" refers to N-acetyl-neuraminic acid (2- keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-l-onic acid (often abbreviated as Neu5 Ac, NeuAc, or NANA).

[0048] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0049] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ά carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. W

[0050] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0051] The biomarkers of this invention are characterized by mass-to-charge ratio lectin binding capacities and were identified using mass spectrometry without knowing their amino acid sequence. If desired, biomarkers whose amino acid sequence identity is not determined can be identified by, for example, determining the amino acid sequence of the aberrant glycoproteins.

[0052] Lectins are naturally occurring carbohydrate binding proteins or glycoproteins which are selective for their cognate sugar moieties. In some embodiments, the lectin may comprise a fragment of the naturally occuring protein which binds to the cognate sugar or saccharide moiety. Lectins may bind a carbohydrate moiety as such free in solution or a carbohydrate moiety which is a part of protein body. Lectins are found throughout nature. Exemplary lectins include, but are riot limited to, Concanavalin A, Phytohaemagglutinin, Allium ascalonicum agglutinin, Aloe arborescens agglutinin, Artocarpus altilis agglutinin, Anguilla anguilla agglutinin, Aleuria aurantia agglutinin, Androctonus australis agglutinin, Agaricus bisporus agglutinin, Amphicarpaea bracteata agglutinin, Allium cepa agglutinin, Alocasia indica lectin, Amaranthus caudatus agglutinin, Agrocybe cylinbracea galectin, Amaranthus cruentus lectin, Arisaema curvatum lectin, Afimbrial adhesin, Agrocybe ylindracea, Aplysia gonad lectin, Artocarpus hirsuta agglutinin, Artocarpus integrifolia agglutinin, adhesion inhibitory receptor molecule, Japaneese eel skin mucus lectin, Artocarpus lakoocha agglutinin, Allomyrina dichtoma agglutinin, Anguilla anguilla agglutinin, Allium moly agglutinin, Arum maculatum agglutinin, spermadhesin, Aaptos papillata agglutinin, Abrus precatorius agglutinin, Aegopodium podagraria agglutinin/lectin, Allium porrum agglutinin, Aquathanatephorus pendulus lectin, Agropyrum repens agglutinin, Agropyrum repens embryo lectin, Amelia rolfsii lectin, Agropyrum repens leaf lectin, Allium sativum agglutinin/lectin, Amaranthus spinosus agglutinin, Allium ursinum agglutinin, Banana lectin, Botrytis cinerea lectin, Bryonia dioica agglutinin, Butea frondosa lectin, Biomphalaria glabrata agglutinin, bud lectin, Birgus latro agglutinin, Bowringia milbraedii agglutinin, Bauhinia purpurea agglutinin, hemolymph lectin from acorn barnacle, Bandeiraea simplicifolia agglutinin/lectin/isolectin, Brachypodium sylvaticum lectin, beletus venenatus lectin, Gallus gallus, chicken lectin, Cymbidium agglutinin, Caragana arborescens agglutinin; Cicer arietinum agglutinin, Colchicum autumnale agglutinin/lectin, Calystegia sepium agglutinin, Cyphomandra betacea lectin, Ceratobasidium cornigerum lectin; Colocasia esculenta lectin; Cucumaria echinata lectin; Cucumaria echinata haemolytic lectin; Canavalia gladiata lectin; Canna generalis lectin ; Cepaeae hortensis agglutinin, Cymbidium hybrid lectin Cladrastis lutea lectinjClivia miniata agglutinin Chelidonium majus agglutinin; Clivia miniata lectin; Cucurbita maxima agglutinin; Cytisus multiflorus agglutinin, calnexin, Con I and Con II, short form for congerin I and congerin II galectins in Conger myriaster, Convolvulus arvensis agglutinin, Concanavalin Br, Canavalia grandiflora agglutinin, Cicer arietinum agglutinin, Cucurbita pepo agglutinin, Carcinoscorpin Carcinoscoφius rotunda cauda, Croton tiglium lectin, Dioscorea batatas lectins, glutinin, Dioclea grandiflora lectin, Dioclea guianensis lectin, Datura innoxia agglutinin, Dolichos lablab agglutinin, Datura stramonium agglutinin, Elderberry lectin, Erythrina corallodendron agglutinin, Erythrina cristagalli agglutinin, Euonymus europaeus agglutinin, Epipactis helleborine agglutinin, Eranthis hyemalis lectin, Euphorbia heterophylla agglutinin, Earthworm lectin, Fucose- specific lectin, galectin, beta-galactoside specific lectins in vertebrates, invertebrates, sponge and fungus; Glucan-binding lectin, Geodia cydonium agglutinin, Geodia cydonium galactins, Galanthus nivalis agglutinin; Peanut nodule lectin, Gonatanthus pumilus agglutinin, Griffonia simplicifolia agglutinin, Gerardia savaglia lectin, Helix aspersa agglutinin, Homarus americanas agglutinin, Hura crepitans agglutinin, Helianthus tuberosus agglutinin, Hippeastrum hybrid agglutinin; Helix pomatia agglutinin; Helianthus tuberosus lectin, Hordeum vulgare lectin; Iberis amara agglutinin, Iris hybrid lectin, Jacalin, Jackfruit lectin (Artocarpus heterophyllus), Jacalin related lectin, L-I, L-II - Leaf lectins from Winged bean (Psophocarpus tetragonolobus); Laburnum alpinum agglutinin - Leptospermum archinoides agglutinin, Leucojum aestivum agglutinin Luffa acutangula agglutinin, Lima bean agglutinin; Lens culinaris agglutinin; Litchi chinensis lectin; Lycopersicon esculentum agglutinin, Loranthus europaeus lectin;; Limax flavus agglutinin, Lathyrus odoratus lectin; lathyrus ochrus isolectins, Lablab purpureus agglutinin, Lathyrus pratensis agglutinin; Limulin, Lathyrus sativum agglutinin; Lotus tetragonolobus agglutinin; Lathyrus tuberosus tuber lectin; Mung bean agglutinin; mannose-binding lectin; Maltose/mannose/maltose- binding protein; Momordica charantia agglutinin, Machaerocereus eruca lectin, Mytitus edulis lectin; Mulberry leaf lectin, wild cucumber, Peanut nodule and cotyledon lectin, Oryza sativa agglutinin, Phytolacca americana isolectins, Percea americana agglutinin, Phragmites australis lectin, Phaseolus coccineus agglutinin, PHA-E - Erythroagglutinating isolectin of PHA, PHA-L - Leucoagglutinating isolectin of PHA, PL - Pseudomonas lectin;PL-A, PL-B, PL-C, PL-D, PA, PAA; Phaseolus limensis agglutinin; PNA - Arachis hypogaea agglutinin; Ptilota plumosa agglutinin; Peanut root lectin; Pisum sativum nodule lectin 1 , Poke weed mitogen; receptor for advanced glycation endproducts; rhamnose-binding lectin, Ricinus communis agglutinin, RCA60, RCL III, RCL IV - ricin, ricin D, ricin E; Robinia pseudoaccacia seed agglutinin; Sialic acid specific lectin; Silurus asotusegg lectin; Soybean agglutinin; Sambucus canadensis lectin; Secale cereale lectin; Sambucus ebulus lectin; Solanum tuberosum agglutinin; Salvia sclarea agglutinin; Thorn apple agglutinin (Datura stramonium, Tetracarpidium conophorum lectin; Talisia esculenta pitomba lectin; Tulipa lectins; Urtica dioica agglutinin; Ulex europaeus agglutinin; Winged bean agglutinin; Wisteria floribunda agglutinin; Wheat germ agglutinin; Zea mays lectin. In some embodiments, the lectins of the array are plant lectins or insect lectins. In some embodiments, the array is populated with 10, 20, 30, 40, 50 or more lectins selected from any of the above lectin.

[0053] In some embodiments, the lectins used in forming the array are limited to one, two, or three or more of the following families of lectins in any combination: legume lectins, cereal lectins, P-, S- and C-type lectins and pentraxins. The latter four are exclusively extracted from animals. The lectins of C-type which binds calcium, and pentraxins which are lectin like serum proteins are found to play role in the defense system of animals. Lectins are mostly multivalent or, in other words, and posses at least two sugar-binding sites that enable them to agglutinate cells bearing glycosylated proteins . Actual crystal structures of different lectins indicate that most of the lectins are either dimers or tetramers. Lectins are abundant in plants than in other sources probably because of the more amount of carbohydrate present in plants which has to exist in some bound-form and they have to be transported to other parts of the cell. A vast number of lectins have been characterized by the conserved domain of binding site and their specificity of recognizing the correct sugar moiety. Accordingly, suitable galactose specific, mannose specific, maltose specific lectins have been characterized and so the code of glyco-forms have been widely deciphered.

[0054] In some embodiments, the plurality of lectins comprise members who collectively bind at least mannose, fucose, and galactose or N-acetylgalactosamine. Mannose binding lectins include, but are not limited to, conconavalin A which binds to branched α -mannosidic structures; high-mannose type, hybrid type and biantennary complex type N-Glycans; lentil lectin which binds fucosylated core region of bi- and triantennary complex type N-Glycans; and snowdrop lectin which binds α 1-3 and α 1-6 linked high mannose structures. Galactose / N-acetylgalactosamine binding lectins include, but are not limited to, PNA which binds Galβl-3GalNAcαl-Ser/Thr (T-Antigen); coral tree lectin which binds Galβl-4GlcNAcβl-R; and Ricinus communis Agglutinin, RCA 120 which binds GaIβl-4GlcNAcβl-R; Jacalin which binds (Sia)Galβl-3GalNAcαl-Ser/Thr (T-Antigen); and hairy vetch lectin which binds GalNAcα-Ser/Thr (Tn- Antigen). Sialic acid / N-acetylglucosamine binding lectins include, but are not limited to, elderberry agglutinin which binds Neu5Acα2-6Gal(NAc)-R, wheat germ agglutinin which binds GlcNAcβl-4GlcNAcβl -4GIcNAc, Neu5Ac (sialic acid), and Maackia amurensis lectin which binds Neu5Ac/Gcα2-3Galβl-4GlcNAcβl-R. Fucose binding lectins include, but are not limited to, Ulex europaeus agglutinin which binds to Fucαl-2Gal-R; Aleuria aurantia lectin which binds to Fucαl-2Galβl-4(Fucαl-3/4)Galβl- 4GIcNAc.

[0055J Aberrant glycosylation observed in experimental and human cancers, include but are not limited to, βl— »6GlcNAc antenna in N-linked structures from increased activity; Tn and STn antigens; promiscuous 0-glycosylation and resulting peptide conformational changes; and overexpression of lacto-series type 1 and type 2 structures (often in the form of poly-LacNAc) with a variety of fucosylation and sialylation. Additionally, poly-LacNAc with sialosyl or fucosyl substitution in glycoproteins is often ex pressed at the βl— >6GlcNAc side chain of N-linked structure or at the βl->6GlcNAc-linked side chain of the "Core 2" 0- linked structure, (see, Hakomori S., Cancer Res. 1996, 56(23): 5309-18). In some embodiments, the array is populated with lectins which can detect each of these aberrant proteins. Suitable aberrant glycoproteins and glycans to be targeted for detection according to the methods of the invention include, but are not limited to, those disclosed in Yamashita, K., et al., J. Biol. Chem., 259: 10834-10840 (1984); Dennis, J. W. and Laferte, S. Asn-linked oligosaccharides and the metastatic phenotype. In: CL. Reading. S. Hakomori, and D.M. Marcus (eds.) Altered Glycosylation in Tumor Cells, pp. 257-267, New York: Alan R. Liss, 1988; Springer, G.F., Science (Washington, D.C.), 224:1198-1206 (1984); Hirohashi, S., et al., Proc. Natl. Acad. ScL USA, 52:7039-7043 (1985); Kjeldsen, T.B., et al., Cancer Res., 48:2214-2220 (1988); Kurosaka, A., et al., J. Biol.^' Chem., 263:8724-876 (1988); and Hakomori, S., Adv. Cancer Res., 52:257-331 (1989), Muramatsu, T., Glycobiology, 3:294- 296 (1993); Matsuura, H, et al., J. Biol. Chem. 263:3314-3322 (1988); Lloyd, K.O., Ann,. NY Acad. ScL, 690:50-58 (1993); and Fukuda, M., Cancer Res., 55.2237-2244 (1996), each of which is incorporated in its entirety to the extent not inconsistent with the present disclosure and, in particular, with respect to the aberrant glycoproteins and glycans and glycoforms disclosed therein. [0056] The term lectin does not include antibodies. "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding. A suitable antibody is one which specifically binds and recognizes a saccharide or glycan moiety present on a glycoprotein.

[0057] Immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

[0058] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)⁵ ₂, a dimer of Fab which itself is a light chain joined to VH-C_HI by a disulfide bond. The F(ab)⁵2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)⁵2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments maybe synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990))

[0059] The phrase "specifically (or selectively) binds" to a lectin or "specifically (or selectively) refers to a binding reaction that is determinative of the presence of a saccharide or glycan moiety on a protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified lectins bind to a particular glycoprotein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an lectin under such conditions requires the lectin is selected for its specificity for a particular glycan group. Lectins and their binding affinities are well known in the art and the cognate saccharide structures recognized by a lectin are easily characterized by one of ordinary skill in the art. A variety of assay formats may be used to select lectins specifically binding with a particular glycan.

[0060] "Therapeutic treatment" and "cancer therapies" refers to apoptosis-mediated and non-apoptosis mediated cancer therapies including, without limitation, chemotherapy, hormonal therapy, radiotherapy, immunotherapy, and combinations thereof.

[0061] By "therapeutically effective amount or dose" or "sufficient amount or dose" herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0062] The use of controls and standard curves for determining the concentration of an analyte are well known fundamentals in the art.

[0063] An "array" has a plurality of lectins for separately analyzing a plurality of glycoproteins. Arrays can be based on spatial separation of biomolecular recognition elements or on the differential labeling of such elements. For example, in a differential labeling array, lectins may be uniquely labeled so that each lectin is uniquely identified by the label. In a spatial array, for example, each lectin may be uniquely localized (e.g., immobilized to a uniquely labeled particles or spot such as a microtiter well), thereby forming an array.

[0064] Arrays permit many assays to be performed in parallel. For example, array-based biosensors are used for multianalyte sensing (see Michael K.L et al, (1998) Anal Chem 70: 1242-6). Current bioinstruments (reviewed in Cunningham, AJ. (1998) Introduction to Bioanalytical Sensors, John Wiley and Sons, New Youk) include integrated microsystems of higher speeds. Lectins that recognize different analytes may be immobilized on spatially W

separated zones or positioned into separate chambers. A variety of different arrays and detectors can be employed in the practice of the present invention. Arrays used in the subject invention can be microspot and microwell, microfluidic arrays.

[0065] The substrates of the various arrays can be fabricated from a variety of materials, including plastics, polymers, ceramics, metals, membranes, gels, glasses, silicon and silicon nitride, and the like. The arrays can be produced according to any convenient methodology known to the art. A variety of array and detector configurations and methods for their production are known to those skilled in the art and disclosed in United States Patent Nos. 6,043,481; 6,043,080; 6,039,925; 6,025,129; 6,025,601; 6,023,540; 6,020,110; 6,017,496; 6,004,755; 5,976,813; 5,872,623; 5,846,708; 5,837,196; 5,807,522; 5,736,330; 5,770,151; 5,711,915; 5,708,957; 5,700,637; 5,690,894; 5,667,667; 5,633,972; 5,653,939; 5,658,734; 5,624,711, 5,599,695; 5,593,839; 5,906,723; 5,585,639; 5,584,982; 5,571,639; 5,561,071; 5,554,501; 5,534,703; 5,529,756; 5,527,681; 4,472,672; 5,436,327; 5,429,807; 5,424,186; 5,412,087; 5,405,783; 5,384,261; 5,474,796; 5,274,240; and 5,242,974. The disclosures of these patents are incorporated by reference herein.

[0066] The arrays may be positioned into the bottom of microwells, microchannels or on the surfaces such as planar waveguides. The area of Micro-Total Analysis Systems (mu TAS), otherwise known as "microsystems" or "Lab-on-a-chip", is used to describe miniaturized sensing devices and systems that integrate microscopic versions of the devices necessary to process chemical or biochemical samples, thereby achieving completely automated and computer controlled analysis on a microscale. Micro/miniaturized total analysis systems developed so far may be classified into two groups. One is a MEMS (Micro Electro Mechanical System), which uses pressurized flow controlled by mechanical flow control devices (e.g., microvalves, micropumps or centrifugal pumps). The other types use electrically driven liquid handling without mechanical elements. Currently, microsystems are being produced in both academic and commercial settings. The term "microsystem" is used herein to describe both types of miniaturized systems. A variety of integrated microsystems, MEMS, and microsystem devices are well known to the art. See, for example, United States Patent Nos. 6,043,080; 6,042,710; 6,042,709; 6,036,927; 6,037,955; 6,033,544; 6,033,546; 6,016,686; 6,012,902; 6,011,252; 6,010,608; 6,010,607; 6,008,893; 6,007,775; 6,007,690; 6,004,515; 6,001,231; 6,001,229; 5,992,820; 5,989,835; 5,989,402; 5,976,336; 5,972,710; 5,972,187; 5,971,355; 5,968,745; 5,965,237; 5,965,001; 5,964,997; 5,964,995; 5,962,081; 5,958,344; 5,958,202; 5,948, 684; 5,942,443; 5,939,291; 5,933,233; 5,921,687; 5,900,130; 5,887,009; 5,876,187; 5,876,675; 5,863,502; 5,858,804; 5,846,708; 5,846,396; 5,843,767; 5,750,015; 5,770,370; 5,744,366; 5,716,852; 5,705,018, 5,653,939; 5,644,395; 5,605,662; 5,603,351; 5,585,069; 5,571,680; 5,410,030; 5,376,252; 5,338,427; 5,325,170; 5,296,114; 5,274,240; 5,250,263; 5,180,480; 5,141,621; 5,132,012; 5,126,022; 5,122,248; 5,112,460; 5,110,431; 5,096,554; 5,092973; 5,073239; 4,909,919; 4,908,1 12; 4,680,201; 4,675,300; and 4,390,403, all of which are incorporated by reference herein.

[0067J It is now possible to fabricate complex miniaturized systems for total biochemical analysis. This technology represents a combination of several disciplines that include microfabrication, microfluidics, microelectronmechanical systems, chemistry, biology, and engineering. Miniaturized devices can be- electrical, such as microelectrodes and signal transducers; optical such as photodiodes and optical waveguides; and mechanical, such as pumps. In the new field of microfluidics, the integration of automated microflow devices and sensors allow very precise control of ultra-small flows on microchip platforms (Gravesen et al. (1993) J. Micromech. Microeng^ 3:168-182; Shoji and Esashi (1994) J. Micromech. Microeng._4:157-I71). Many different flows can be combined in all sorts of ways and mixed on the same chip. Existing technology also allows the integration of intersecting channels, reaction chambers, mixers, filters, heaters, and detectors to perform on-chip reactions in sub- nanoliter volumes in a highly controlled and automated manner with integrated data collection and analysis (Colyer et al. (1997) Electrophoresis 18:1733-1741 ; Effenhauser et al (1997) Electrophoresis 12:2203-2213).

[0068] Generally, array technology is a method of choice for high throughput analysis. Microarrays, wherein thousands of different bioaffinity molecules are immobilized on a surface in a defined and spatially resolvable fashion (usually as spots 10-100 μm in diameter) and used to capture ligands, have been developed for both nucleic acids and proteins (Fodor etal (1991) Science 251:767-773; Lueking e/ a/. (1999) Anal. Biochem. 270:103-111). This microarray technology can be applied in the present invention to provide amino acid analysis microchips.

[0069] Fabrication methods for protein arrays include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography. A number of commercial arrayers including manual equipments are available for spotting proteins on defined surfaces. Bacterial colonies can be robotically grided onto PVDF membranes for induction of protein expression in situ. Fluorescence and chemiluminescence detection are widely used in protein array. Protein analytes binding to antibody arrays may be detected via a secondary antibody in a sandwich assay format. Label-free detection methods, such as atomic force microscopy, surface plasma resonance and scanning Kelvin nanoprobe, have been developed (Seong SY, Choi CY, Proteomics 2003, 3 (11): 2176-89; Jones VW, Kenseth JR, Porter MD, Mosher CL, Henderson E., Anal. Chem. 1998,70,1233-1241; Thompson M, Cheran LE, Zhang M, Chacko M, Huo H, Sadeghi S., Biosens Bioelectron. 2005, 20(8): 1471-81). These methods avoid alteration of protein analytes.

[0070] One of the most common formats in protein array is the capture array, in which the capture reagents (antibodies, protein scaffolds, peptides, etc) are used to detect target molecules in mixtures such as serum or tissue lysate. These form the basis of diagnostic chips and arrays for expression profiling. In diagnostics, capture arrays can be used to carry out multiple immunoassays in parallel, testing for several analytes in individual serumlbiopsy sample or many serumlbiopsy samples simultaneously. In proteomics, capture arrays are used to quantify and compare the levels of proteins in cells and tissues under different conditions (health, disease, differentiation, drug treatment, etc). Protein array technology is also very promising in studying protein-protein interactions and protein post-translational modifications (using specific antibodies), in a high-throughput fashion.

[0071] Rapid, automated and simultaneous testing of multiple samples are commonly performed in microwell formats. The microtiter plate has become a popular format for biological assays because it is easy to use, is readily integrated into an automated process and provides multiple simultaneous testing on a simple disposable device. The traditional 96- well format is being replaced with microwells with larger numbers of smaller wells. These provide plates with 192-20,000 wells with volumes that range from 125 microliters to 50 nanoliters (Reviewed in Kricka (1998) Clinical Chemistry 44:2008-2014). A range of new micropipetting systems based on ink-jet principles have been developed for delivery of nanoliter volumes of samples and reagents to microwells (for example, see, Rose and Lemmo (1997) Lab Automat News : 2:12-9; Fischer-Fruholz (1998) American Lab; Feb 46-51). The new high-density, low volume microwell format has been adapted for a diverse range of analytical methods.

[0072] Advantageous properties of substrates for the microarrays of the subject invention are those for substrates of traditional microarrays: ease of manufacture and processing, compatibility with detection systems, good material strength, and low nonspecific biomolecule adsorption. The substrate material should allow efficient immobilization of biomolecules either directly or through an intermediate surface coating. Glass, silicon, and plastic substrates are commonly used for microarray production and are examples of suitable substrates for use in some preferred embodiments of the subject invention. Glass has a number of favorable qualities. These include transparency, and the compatibility with radioactive and fluorescent samples. However, a variety of other materials are suitable substrates. Polypropylene also has favorable physical and chemical properties. For example, Boehringer Mannheim uses small disposable polystyrene carriers onto which microdots are deposited using inkjet technology (Ekins (1998) Clin. Chem. 44:2015-2030). As mentioned above, biomolecule immobilization on chips may be accomplished by various means including, but not limited to, adsorption, entrapment, and covalent attachment. Covalent attachment is the preferred method for "permanent" immobilization. Functionalized organosilanes have been used extensively as an intermediate layer for biomolecule immobilization on glass and silicon substrates. Silanes are commercially available that contain an ever-increasing number of reactive functional groups suitable for biomolecule conjugation either directly or via a cross-linker.

[0073] The microarrays of the current invention can be made using existing technologies for array construction. The microarrays of the current invention may be produced, for example, by deposition of tiny amounts of lectin bearing solutions in a predetermined pattern on a surface using arraying robots (As reviewed, for example, in Schena (ed) (2000) " Microarray Biochip Technology" Eaton Publishing, Natick, MA; Schena (ed) (2000) "DNA Microarrays A Practical Approach", Oxford University Press). The volume delivered is typically from 100 microliters to nanoliters.

[0074] The technologies for spotting arrayed materials onto a substrate fall into two categories: noncontact and contact dispensing. Noncontact dispensing involves the ejection of drops from a dispenser onto the surface. Contact printing involves direct contact between the printing mechanism and the solid support. For example, to construct a lectin microarrays of the current invention, a high-precision contact-printing robot may be employed to deliver volumes of lectin solutions to surfaces yielding spots of about 150 to 200 micrometers in diameter.

[0075] A variety of chemically derivatized substrates can be printed and imaged by commercially available arrayers and scanners. For example, slides that have been treated with an. aldehyde-containing silane reagent are commonly available (e.g., from TeleChem International, Cupertino, CA). The aldehydes react with primary amines on proteins or amine modified nucleic acids to form a SchifPs base linkage. Substrates for microarray construction may be coated by a protein layer and the proteins to be spotted may be attached to this protein layer using chemical crosslinking. For example, MacBeath et al. (2000), supra, teach a method for spotting proteins on microarrays. The proteins are printed in phosphate-buffered saline with 40% glycerol included to prevent evaporation of the nanodroplets. They attached a layer of bovine serum albumin (BSA) to the surface of a glass substrate. Glass treated with an aldehyde-containing silane reagent readily react with amines on a protein's surface to form a covalent attachment forming a molecular layer of BSA. The BSA on the surface is then activated using a chemical cross-linking reagent (e.g., N₅N'- disuccinimidyl carbonate). The activated residues on the BSA then react with residues on the printed protein to form covalent linkages. Printed proteins are displayed on top of the BSA monolayer rendering them accessible to macromolecules in solution.

[0076] Another example of a known method for microarray construction involves the in situ synthesis of unique oligonucleotides on a solid support. Proteins or other biomolecules may be attached to oligonucleotides having complimentary sequences to those positioned on the array in known locations. These oligonucleotide bearing biomolecules are then bound to the arrays in known locations by complimentary base pairing (for a review of this method, see, Niemeyer et al.{\ 998) Analytical Biochem. 268, 54-63.)

[0077] In some embodiments of the current invention, it will be necessary to immobilize proteins and nucleic acids. Conventional methods for protein and nucleic acid immobilization may be used in these embodiments. Proteins and nucleic acids have been immobilized in a vast number of ways over the last 30 years and many references can be found describing various immobilization techniques. Proteins and nucleic acids have been immobilized on biosensors, microarrays, microspheres, nanoparticles, and a multitude of other supports. Adsorption, entrapment, encapsulation, cross-linking and covalent attachment are among the techniques employed for immobilization of biomolecules. Proteins and nucleic acids may be encapsulated by enveloping the molecules in various forms of semipermeable membranes, entrapped in gel lattices, adsorbed onto or covalently attached to surfaces. For example, proteins and nucleic acids may be entrapped in gels along with fluorescent or other indicators (Flora and Brernnan (1999) Analyst 124:1455-1462). These biomolecules may be encapsulated into sol-gel derived materials prepared either as monoliths or beads. A support-free type of immobilization is crosslinking. This method involves joining of proteins to each other to form three-dimensional complex structures. Chemical methods for crosslinking normally involve covalent bond formation between the proteins by means of a bi-or multi-functional reagent, such as glutaraldehyde. Strategies for reversible immobilization of proteins include reversible chemical interactions (Tyagi, et α/.(1994) Biotechnol. Appl. Biochem. 20:93-99) in particular metal chelation (Gritsch et α/.(1995) Biosens. Bioelectron. 10: 805-812) or disulfide cleavage ( Batistaviera et α/.(1991) Appl. Biochem. Biotech. 31: 175-195), protein-ligand interactions (Phelps et al. (1995) Biotechnol. Bioeng. 46, 514-524) and nucleic acid hybridization (Niemeyer et al. (1994) Nucleic Acids Res. 22: 5530-5539).

[0078] Methods for site-selective immobilization of biomolecules on surfaces have been developed. This will facilitate the fabrication of spatially defined lectin arrays. For example, immobilization of immunoglobulins was achieved by photolithography techniques (Rozsnyai, et al. (\992) Angew Chem. Int. Ed. Engl 31, 759). Nucleic acid-directed immobilization of proteins provides a single site-selective process for the immobilization of proteins and other biomolecules under mild chemical conditions (Niemeyer et α/.(1998) Anal. Biochem. 268, 54-63). Oligonucleotide arrays are widely used for DNA analysis (e.g., Kozal et al. (1996) Nat. Med. 2: 753-759) and such arrays are used as standard array templates for the constructing of arrays of any biomolecule that can be attached to a single stranded nucleic acid. The single stranded nucleic acid is then hybridized to its complimentary strand immobilized in a known location on a surface. This method of arraying proteins and nucleic acids may be employed in some embodiments of the subject invention.

[0079] Other methods for immobilizing functionally active proteins on microarrays are known. For example, Arenkov et al. ( 2000) Anal. Biochem. 278: 123 teach methods of arraying functionally active proteins using microfabricated polyacryl amide gel pads. And MacBeath et al (2000) Science 289: 1760-1763 teach methods for spotting proteins onto chemically derivatized glass slides at high spatial densities. A high-precision robot was used to spot proteins onto chemically derivatized slides at high spatial densities. The proteins are attached covalently to the slide surface, yet retain their ability to interact specifically with other proteins or small molecules. [0080] Protein or nucleic arrays of the subject invention may be created using any of the known microarray methods as reviewed in Schena et al.(ed) DNA Microarrays A Practical Approach, Oxford University Press;

[0081] Methods used for immobilizing proteins or nucleic acids are described in the following references, and others (Mosbach (1976) Meth. Enzymol. 44:2015-2030; Hermanson, G. T. (1996) Bioconj'ugate Techniques, Academic Press, NY; Bickerstaff, G. (ed) (1997) Immobilization of Enzymes and Cells , Humana Press, NJ; Cass and Ligler (eds.) (1998) Immobilized Biomolecules in Analysis, Oxford University Press; Watson et al. (1998) Curr. Opin. Biotech. 609:614; Ekins (1998) Clin.Chem. 44:2105-2030; Roda ef a/. (2000) Biotechniques 28: 492-496; Wong (1993) Chemistry of Protein Conjugation and Cross- linking CRC Boca Raton, FL;. Taylor, (1991) Protein Immobilization: fundamentals and applications Marcel Dekker, Inc New York; Hutchens (ed) (1989,) Protein recognition of immobilized ligands, VoI 83 Alan R Liss, Inc; Sleytr U.B. (ed) (1993) Immobilized macromolecules, application potentials VoI 51. Springer series in applied biology, Springer- Verlag, London; Wilchek and Bayer (eds) (1990) Avidin-Biotin Technology. Academic Press, San Diego; Ghosh et al. (1987) Nucleic Acids Res. 15: 5353-5372; Burgener et al. (2000) Bioconjug. Chem. 11 : 749-754; Steel et al. (2000) Biophvs J 79:975-981 ; Afanassiev et α/.(2000) Nucleic Acids Res. 28: E66; Roda et al. (2000) Biotechniques 28: 492-496; Shena (ed.) (2000) DNA Microarrays, a practical approach (Oxford University Press); Schena (ed.) (2000) Microarray Biochip Technology. (Eaton Publishing Natick, MA); MacBeath et al. (2000) Science 289:1760-1763; Schena et al (1998) Trends in Biotechnol.16: 301-306; and Ramsey (1998) Nat. Biotechnol. 16: 40-44; all of which are incorporated by reference herein.

[0082] Many coupling agents are known in the art and can be used to immobilize biomolecules in the current invention. Over 300 cross-linkers are currently available. These reagents are commercially available (e.g., from Pierce Chemical Company (Rockford, II). A cross-linker is a molecule which has two reactive groups with which to covalently attach a protein, nucleic acids or other molecules. In between the reactive groups is typically a spacer group. Steric interference with the activity of the biomolecule by the surface may be ameliorated by altering the spacer composition or length. There are two groups of cross- linkers, homobifunctional and heterobifunctioal. Tn the case of heterobifunctional crosslinkers, the reactive groups have dissimilar functionalities of different specificies. On the other hand, homobifunctional cross linkers' reactive groups are the same. A through review of cross-linking can be found in Wong, 1993, Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton. Bi functional cross-linking reagents may be classified on the basis of the following (Pierce Chemical Co. 1994): functional groups and chemical specificity, length of cross-bridge, whether the cross-linking functional groups are similar (homobifunctional) or different (heterobifunctional), whether the functional groups react chemically or photochemically, whether the reagent is cleavable, and whether the reagent can be radiolabeled or tagged with another label.

[0083] When macromolecular ligands are used, the biomolecules should be immobilized in such a way as to reduce steric hindrances generated by the support. A variety of methods for achieving this are known in the art. For example, the active site or other binding region of the biomolecule can be orientated away from the surface (Reviewed in Bickerstaff, (ed.) (1997; Immobilization of Enzymes and Cells, pp. 261-275).

[0084] When it is necessary to reduce steric problems of an immobilized lectin, suitable spacer arm may optionally be used to immobilize the biomolecule to a surface. Suitable spacer arms may include, but are not limited to, carbon spacers, poly ethylene glycol polymers, peptides, dextrans, proteins, and nucleic acids. For example, Maskos et α/.(1992) teach methods of immobilizing oligonucleotides to chips.

[0085] The fingerprints of this invention can be obtained by mass spectrometry (MS). Suitable MS methods include, but are not limit to, time-of-flight, quadrupole filter, ion trap, ion cyclotron resonance, magnetic sector, and electrostatic sector analyzer. In some embodiments, MALDI-MS methods are used as exemplified herein.

EXAMPLES

[0086] The lectin protein array can be immobilized as a panel of lectins on defined surface using the lectins as the bait for glycoproteins. Subsequently MS (MALDI-MS and MSIMS) is used to detect captured glycoproteins. We have fabricated a proto-type lectin array and used it to analyze the glycoproteins in serum samples from oral cancer and control subjects.

Example 1. Fabrication of PDMS device and immobilization of lectins on PDMS surface

[0087] An array of poly (dimethylsiloxane) (PDMS) wells having immobilized lectin on the surface of PDMS wells through covalent bonding was fabricated. The use of PDMS elastomer for miniaturized bioassays has numerous advantages over silicon and glass. PDMS as a material is inexpensive, flexible, compatible with biological studies (e.g., nontoxic to cells) and optically transparent down to 230 ran (and therefore compatible with many optical methods for detection). It is also very simple to fabricate a PDMS device and bond it to other surfaces. As shown in Fig. IA, a micromolding technique was used to fabricate the PDMS device. A polystyrene mold was produced in the machine shop of UCLA School of Engineering. Two components of PDMS rubber (RTV615A & B, General Electric) were mixed at a 10:1 volume ratio (RTV615A:RTV615B) in an evacuated flask. The two components were stirred with a magnetic stir bar at room temperature. To eliminate the voids in the cured PDMS elastomer, the mixed PDMS rubber components were degassed by exposing to a vacuum for about 1 hour. The degassed liquid was then poured over the polystyrene mold and cured in an oven at 65 ⁰C for 4 hours. A 16-well PDMS device is shown in Fig. IB. Each well is 2 mm in diameter and 500 μm in depth, which amounts to ~1.5 μl in volume. The thickness of the device is ~1 mm.

[0088] The overall procedures for covalent immobilization of lectins on PDMS surface is illustrated in Fig. 2. PDMS slides were thoroughly rinsed with ethanol, dried with air gun, and then placed in an oxygen asher (Tegal) for 5-min oxidization. Immediately after removal from the oxygen asher, PDMS slides were soaked in 10% 3-aminopropyltriethoxysilane (APTS, Sigma) (pH 7.0) for silanization at 80 ⁰C for 3 hours. Then the slide was rinsed with distilled water for at least 30 seconds. To allow for cross-linking, the PDMS slide was soaked in 10% glutaraldehyde (Sigma) at room temperature for 1 hour. Finally, PBS was used to thoroughly wash the slides for three times and then the slides were dried with air gun to remove excess buffer on the surface.

[0089] Each well of the PDMS slide was incubated with 0.5 μl 75 μg/ml lectin in PBS (pH 7.4) at 37°C for overnight. A glass cover slide was used to cover the PDMS device and avoid evaporation during the incubation. The slide was then washed with PBS containing 0.1 % (v/v) Tween 20 (pH7.4) for 3 times. The prepared array was kept in PBS with sodium azide to prevent bacteria growth.

Example 2. Lectin array analysis of serum glycoproteins from oral cancer patient [0090] Three lectins, including Erythrina Cristagalli Lectin (ECL), Jacalin (JAC) and Peanut Agglutinin (PNA) (Vector Laboratories, UK), were immobilized on the surface of fabricated PDMS wells. Prior to serum analysis, the PDMS slides were washed with PBS to remove sodium azide. Both oral cancer and control patients' serum samples were diluted 5 times with PBS buffer (pH 7.4). 0.5 μl of each sample was added to the PDMS well and incubation was carried out at 37 ⁰C for 2 hours. After the incubation, the slide was first washed with 0.1% Tween-20 in PBS (pH 7.4) for three times and then with water for three times to remove PBS and Tween-20. The PDMS slide was then air-dried and 0.5 μl of 2, 5- Dihydroxybenzoic acid (DHB) matrix (20 mg/ml in 0.2% TFA and 50% ACN) was directly layered on each PDMS wells. After the crystallization, the PDMS slide was attached to the MALDl plate using double-sided adhesive tape and subsequent MALDI-MS (Applied Biosystems DE-STR) measurement was performed at linear mode.

[0091] The obtained results clearly indicate that lectin array can harness distinct serum glycoprotein changes associated with oral cancer, although these findings need to be further validated. Fig. 3 A depicts the MALDI-MS spectra for glycopeptides/glycoproteins captured by Jacalin. The upper trace was from a control subject while the bottom one was from an oral cancer patient. The close-ups within different mass ranges are shown in Fig. 3 B & C. The overall patterns are similar, but two proteins (m/z, 8027 & 35358 Da) were only found in cancer samples. Several other proteins were obviously at higher levels in the cancer subject than in the control subject.

[0092] Due to distinct binding characteristics, different lectins showed different glycoprotein patterns for the same sample. Fig. 4 depicts the MALDI-MS spectra for glycoproteins captured by lectins ECL and PNA from the oral cancer and control serum samples. More glycoproteins were detected in the cancer sample. Interestingly, both ECL and PNA captured two proteins, m/z, 4287 and 7991 Da, in cancer but not in control sample. Moreover, PNA captured a panel of proteins, m/z, 14096, 15445, 25767 and 28695 Da, only from oral cancer sample. To identify the proteins with respect to amino acid sequence, they can be digested in the PDMS wells and subsequently MALDI-QqTOF or LC-QqTOF analysis of resulting peptide may identify the proteins

[0093] MALDI-MS detection was used in this study because it provides accurate mass measurement and high resolving power for the captured proteins. Fig.5 shows the MALDI- MS spectra for the same serum sample incubated with immobilized Jacalin on two different PDMS wells. Almost identical spectra were observed, indicating that the measurements were reproducible.

[0094] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the extent not inconsistent with the present disclosure. No reference to a publication herein should be construed as an admission that such is prior art.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying cancer in the subject, said method comprising: a. contacting a sample from the subject with an array comprising a plurality of lectins, wherein each member of the plurality is located at a predetermined locus on the array, and wherein each member lectin of said plurality differs from other member lectins of said plurality according to their cognate saccharide, and wherein said plurality of lectins comprises members collectively cognate to galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide, and wherein said contacting is under reaction conditions capable of binding glycoproteins in said sample to a lectin cognate to a saccharide group of said glycoprotein; b. determining the profile of the glycoproteins bound by each locus of the array,; and c. comparing the profile with a control profile, thereby identifying a subject with cancer.

2. The method of claim 1, wherein the subject is suspected of having the cancer.

3. The method of claim 1 , wherein the subject has previously been diagnosed and treated for the cancer.

4. The method of claim 1, wherein the amount and mass of glycoproteins bound to the lectins of the array is determined using MALDI-MS.

5. The method of claim 1 , wherein the sample is a biological fluid taken from the subject.

6. The method of claim 1, wherein the fluid is saliva, cerebrospinal fluid, lymph, blood, plasma, urine, or a portion thereof.

7. The method of claim 1 , wherein the cancer is selected from the group consisting of cancers of the head and neck.

8. The method of claim 1, wherein the cancer is a tumor of the oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx.

9. The method of claim 1 wherein the cancer is one etiologically associated with tobacco or alcohol exposure.

10. The method of claim 1 , wherein an aberrant glycosylation associated with cancer is identified in the subject.

11. The method of claim 1, wherein the plurality of lectins are located on a PDMS surface.

12. The method of claim 1, wherein each member of the plurality of lectins is located in a PDMS well.

13. The method of claim 1, wherein the lectin cognate to galactosamine is Erythrina cristagalli lectin (ECL).

14. The method of claim 1, wherein the lectin cognate to O-glycosidically linked oligosaccharide is jacalin or PNA.

15. The method of claim 1, wherein the cancer is oral cancer.

16. The method of claim 1, wherein the profile is determined by MS.

17. The method of claim 1, wherein the profile is determined by MS and provides the mass/charge ratios and amounts of one or more glycoproteins bound by each locus of the array.

18. A method of identifying a glycoprotein whose glycosylation pattern is aberrant in cancer cells, said method comprising: a. contacting a sample from a subject with the cancer with an array comprising a plurality of lectins, wherein each member of the plurality is located at a predetermined locus on the array, and wherein each member lectin of said plurality differs from other member lectins of said plurality according to their cognate saccharide, and wherein said plurality of lectins comprises members collectively cognate to galactosamine, N-acetyl-galactosamine, N-acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide, and wherein said contacting is under reaction conditions capable of binding glycoproteins in said sample to a lectin cognate to a saccharide group of said glycoprotein; b. determining the profile of the glycoproteins bound by each locus of the array; and c. comparing the profile with a profile for a control sample(s) from subject(s) who are not known or suspected of having the cancer, thereby identifying aberrant glycoproteins associated with the cancer state; and d. determining the amino acid sequence of the aberrant proteins.

19. The method of claim 18, wherein the profile is determined by MS or MALDI-MS and provides the mass/charge ratios and amounts of one or more glycoproteins bound by each locus of the array.

20. A system for detecting aberrant glycoproteins, said system comprising: a. an array comprising a plurality of lectins, wherein each member of the plurality is located at a predetermined locus on the array, and wherein each member lectin of said plurality differs from other member lectins of said plurality according to their cognate saccharide, and wherein said plurality of lectins comprises members collectively cognate to galactosamine, N-acetyl-galactosamine, N- acetyl neuraminic acid, mannose, and O-glycosidically linked oligosaccharide; b. means for contacting a biological sample with the array under conditions which allow the lectins of the array to bind a cognate saccharide group; and c. means for determining the amount and mass/charge ratio of the glycoproteins bound to each locus of the array.

21. The method of claim 20, wherein the means for determining comprises MALDI-MS.

22. The method of claim 20, wherein the array comprises a PDMS surface.

23. An array comprising: a) a PDMS substrate having a silanized surface; b) a plurality of lectins, wherein the plurality comprises jackalin, ECL, and PNA; and wherein each member of the plurality is covalently bonded to the silanized surface at a specific locus.