FIELD OF THE INVENTION

The present invention relates to methods of isolating biological material by affinity binding and analyzing bound material by mass spectrometry and X-ray crystallography.

BACKGROUND OF THE INVENTION

Advances in chemical technology have been the engine powering the biotechnology industry. Two new mass spectrometry techniques have added fresh impetus to bioresearch. These techniques are electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) . Commercial availability of these instruments is routine in the analysis of compounds, including proteins, peptides, carbohydrates, oligonucleotides, natural products, and drug metabolites.

ES-MS and MALDI-MS offer picomole to femtomole sensitivity, enabling the direct analysis of biological fluids with a minimal amount of sample preparation. These techniques can be used to measure the mass of biomolecules greater than 200,000 daltons, to provide structural information and to detect non-covalent complexes with molecular weight accuracy on the order of +/- 0.01%. The use of these mass spectrometry tools for characterization of biological materials has been carefully reviewed recently by E.J. Zaluzec et al . , Protein Expression and Purifi cation , 6: 109-123 (1995) and G. Siuzdac, Proc . Natl . Acad . Sci . U . S . A . , 91: 11290-11297 (1994) .

Several reports have shown that these mass spectrometry techniques can be useful in characterizing polypeptides that are attached to solid polymeric supports. Typically, a selectively cleavable linker molecule, usually an acid labile linker molecule that is well known in the art, such as the Rink amide linker, is initially attached to the polymeric support. Polypeptide sequences, or various organic molecules, are then synthesized directly onto the selectively cleavable linker group. B. J. Egner, et al . , J . Org . Chem . , 60: 2652-2653 (1995). The polymer-bound compounds are then placed on a MALDI-MS target, placed in the appropriate atmosphere to disassociate the compound from the linker group and consequently the polymeric bead, and subsequently analyzed by the MALDI-MS. C. L. Brummel, et al . , Science , 264: 399-400 (1994) ; B. J. Egner, et al . , J . Chem . Soc . -Chem . Commun . , 21: 2163-2164 (1995) . Alternatively, the polymer- bound compounds may be introduced into a flow injection electrospray after proper pretreatment, and analyzed via ES- MS. C. L. Brummel, et al . , Anal. Chem . , 68: 237-242 (1996) . This approach is useful for combinatorial chemistry.

In this combinatorial chemistry technique, large numbers of structurally distinct molecules are synthesized on the linker molecules or directly on the polymer support and analyzed via mass spectrometry. However, the methods known in the art are time-consuming and slow. The polypeptides, organic compounds or other structures which are attached to the beads, optionally via the linker molecule, are laboriously constructed. Nowhere does this method allow for the molecules to be analyzed to be removed directly from biological systems such as cells, biological extracts, etc., nor do the methods presently known in the art allow the quick isolation and analysis of biological materials which share a similar attribute, εuch as a binding receptor.

It is greatly desirable to provide a combinatorial system whereby biological components which share a similar structural attribute, such as a receptor, can be easily

isolated and analyzed. Such analyses include mass analysis via mass spectrometry, or structure analysis via X-ray crystallography.

SUMMARY OF THE INVENTION

The invention provides a method of analyzing a biological material by mass spectrometry, comprising the steps of (i) contacting an affinity matrix, consisting of a support having bound thereto an affinity material comprising a moiety that binds specifically to a complementary receptor, with a biological material containing the complementary receptor, whereby the receptor binds to the affinity matrix; (ii) separating unbound sample components from the receptor bound to the affinity matrix; (iii) analyzing the affinity matrix-bound receptor or receptor complex by mass spectrometry. Optionally the bound receptor or receptor may be released from the affinity matrix, recovered and analyzed by subsequent X-ray crystallographic analysis.

In one embodiment a MALDI mass spectrometer is used to ionize the bound receptor from the affinity material. In another embodiment, the bound receptor is analyzed by electrospray mass spectrometry.

In another embodiment, the affinity material is bound to the support through a cleavable linker. Following binding of a receptor to the affinity material, the receptor- affinity material complex may be analyzed by mass spectrometry or released for recovery purposes by an agent that cleaves the linker. The affinity support may also have bound directly to it a receptor complementary to an affinity.

DETAILED DESCRIPTION

The following terms are used in this application;

Affinity material: A moiety that binds preferentially to a specific receptor. For example, proteins, peptides, polypeptides, glycoproteins, lipoproteins, phospholipids, steroids, alkyloids, or the like, e.g., hormones, lymphokines, growth factors, albumins, cytokines, enzymes, antibodies, antibody fragments, sugars, glycopeptides, or organometallic complexes, which preferentially bind receptors associated with the biological material of interest are included in the term "affinity material".

Receptor: A specific molecule or specific chemical group that is complementary to, and specifically binds to, an affinity material.

Linker molecule: A molecule which is covalently attached at one end to a solid phase support and is attached on the other end to an affinity material, which, when exposed to the appropriate stimulation, will cleave the affinity material attached thereto from the solid phase support with or without some or all of the linker molecule being attached thereto.

Affinity matrix: A solid state support having any physical shape to which an affinity material has been attached. The affinity material may be attached directly to the support, or may be attached via a linker molecule. A preferred support is a polymeric bead,

The present invention involves methods for isolating and analyzing biological materials including receptors via mass spectrometric and X-ray crystallographic techniques. The invention is based on the use of one or more affinity materials which are attached to solid state supports, the affinity material optionally being attached to the support via a selectively cleavable linker molecule, creating an affinity matrix. The affinity matrix can then be exposed to a source of biological material, some of which

contains receptors to the affinity material portion of the affinity matrix. The biological material that is unbound to the affinity bead can be washed away with any of the suitable washing procedures known in the art. The affinity material- bound receptor can then be analyzed directly in a mass spectrometer.

If the affinity material is attached to the solid state support through a cleavable linker, both the affinity material and the receptor may be cleaved from the support, isolated as a unit, and analyzed together to provide useful information regarding bonding of the affinity material to the receptor to assist in identifying and designing novel pharmaceuticals. The mass spectrum information of affinity material-bound receptor may allow the identification of different receptor subtypes provided the masses of the receptor subtypes are sufficiently different.

It is also possible to use known supports that are in the form of a soluble polymer to which the affinity material is bound, and which is later isolated by filtration or precipitation. Examples include high molecular weight polyethers and the like. Mass spectrometry and X-ray crystallographic analyses may be applied to analyze such receptor-bound affinity matrices.

The isolated affinity material and attached receptor may also be stripped from the affinity matrix and isolated in larger quantities. The isolated affinity material/receptor may be analyzed by mass spectrometry or crystallized and analyzed by X-ray crystallography. The X- ray information could further prove differences in receptor subtypes and demonstrate how the affinity material is bound to the receptor. The X-ray data could be the basis to begin a rational drug design.

Suitable solid state supports include polymeric material, silica chips, or other supports known and used in

the art. The preferred solid phase support is polymeric beads. The polymeric beads may consist of polystyrene resin (Merrifield resin) . R. B. Merrifield, J . Am . Chem . Soc , 85: 2149-2154 (1963) . However, polystyrene may be unsuitable for some uses. Polystyrene is completely hydrophobic in nature, whereas peptide chains are much more hydrophilic. This difference may induce a chain-folding effect in which the peptide attached to the polystyrene bead satisfies its own hydrogen bonding requirements rather than being solvated. Further, the hydrophobic nature of polystyrene may lead to unspecified bonding of the biological material to the bead directly, rather than specified bonding of the biological material through its receptor to the affinity material attached to the bead. Other suitable polymeric materials, such aε Sheppard's polyamide resin, may be used as these polymers are hydrophilic and can readily be solvated by dipolar aprotic solvents. E. Atherton and R. C. Sheppard, Solid phase peptide synthesis, a practical approach , I.R.L. Press at Oxford University Press (1989) . The preferred material for the solid support is Tentagel ^'1M resin (Rapp Polymere Gmbh) which consists of about 80% polyethylene glycol grafted to cross-linked polystyrene. It is generally considered that the reaction milieu within this resin is more closely related to ether and tetrahydrofuran, and consequently it has a potential for compatibility for a large range of reactions which may be used for creating the affinity beads.

The polymeric material used may have a variety of physical shapes. Although the preferred shape is the spherical bead, with a size of approximately 50 microns in diameter, other shapes commonly known in the art may be used. These include a range of polyacrylic-grafted polyethylene extrusions called "pins" which may be paired with shapes of maximum surface area designed to optimize the capacity of the material to capture the desired biological material.

Optionally, a selectively cleavable linker molecule may be used to join the affinity material to the polymeric support material. This cleavable linker molecule is attached to the solid phase support. Such cleavable linker molecules include photolabile linkers, such as α-methylphenacyl esters, p-methoxyphenacyl esterε, and o-nitrobenzyl eεters, acid labile linkers, such as the Rink linker, or base labile linkers. The use of such cleavable linkers to connect biological material such as peptides to polymer beads to produce samples suitable for mass spectrometry analysis is known in the art. C. L. Brummel, et al . , Science, 264: 399- 402 (1994); B. J. Egner, et al . , J . Chem . Soc , Chem . Commun . , 21: 2163-2164 (1995); N. J. Haskins, et al . , Rapid Comm . Mass Spectrom . , 9: 1437-1440 (1995) ; S. Wang, J . Org . Chem . , 41(20): 3258-3261 (1976); D. H. Rich, et al . , J . Am . Chem . Soc , 97: 1575 (1975); and B. J. Egner, et al . , J . Org . Chem . , 60: 2652-2653 (1995) . The preferred linking molecule is a photolabile α-methylphenacyl-ester linker.

The affinity material may consist of protein, peptides, polypeptides, glycoproteins, lipoproteins, phospholipids, steroids, alkaloids, or the like, e.g., hormones, lymphokines, growth factors, albumins, cytokines, enzymes, antibodies, antibody fragments, sugars, glycopeptides, or organometallic complexes which preferentially bind receptors associated with the biological material of interest. Another type of affinity material useful for separation of enzymes is an enzyme substrate analogue, inhibitor, suicide substrate or the like, which binds to an enzyme but does not undergo enzyme-catalyzed transformation with subsequent rapid release of the enzyme. The affinity material will be chosen based on the receptors associated with the biological material to be isolated and analyzed. The material to be identified may consist exclusively of a receptor for the affinity material, or the biological material which comprises to the receptor.

The affinity material may be directly attached to

the solid phase support, by a selectively cleavable linker molecule, or by other means known in the art. N.K. Terret et al . , Tetrahedron 51 (30): 8135-8173 (1995) ; C. L. Brummel, et al . , Anal . Chem . 68: 237-242 (1996).

To prevent nonspecific binding of receptors to affinity matrices, nonspecific binding sites on the matrices may be preliminarily blocked by treatment with an agent such as bovine serum albumin (BSA) .

After the affinity matrices have been assembled, the beads may be placed in a column or another suitable container. The matrices are subsequently exposed to one or more biological materials containing a receptor specific for the affinity material attached to the solid support beads. After the biological material has been in contact with the affinity beads for a suitable period of time to allow specific binding of the receptor(ε) of the biological sample to the affinity matrices, the excess biological material is removed by any of the techniques commonly known in the art, e.g., saline washes or the like.

The characteristics of the bound receptors may be determined by mass spectrometry. Alternatively, the bound material may be isolated by nondestructive mass spectrometry methods, and the isolated material crystallized for analysis by X-ray crystallography. Suitable mass spectrometry techniques include electrospray mass spectrometry, matrix- assisted laser desorption/ionization mass spectrometry, time- of-flight secondary mass spectrometry. The MALDI mass spectrometer may be used in conjunction with a time-of-flight mass analyzer. Time-of-flight analysis is well-suited to the pulsed nature of laser desorption in MALDI. Additionally, time-of-flight analysis has virtually no upper mass range, and is therefore very compatible with the analysis of large biopolymers which may be detected by the present invention. The preferred mass spectrometric techniques are electrospray mass spectrometry and matrix-assisted laser

desorption/ionization mass spectrometry.

To analyze the material by MALDI-MS, one or more affinity matrices having a receptor bound thereto are placed on a suitable target substrate, such as stainless steel. The target biological molecule may be initially dissociated from the affinity bead prior to mass spectrometric analysis, or may be analyzed without pretreatment of the affinity bead to induce cleavage of the target molecule. If dissociation of the target molecule prior to analysis is desired, one approach would be to attach the affinity material to an acid- cleavable linker and then use acid to achieve dissociation. Other linkers which are cleavable with specific groups, e.g. , hydrazine or hydroxylamine, may also be used. Another approach is to dissociate the receptor from the affinity material, e.g., by lowering the pH of the solution, e.g., with a solution of dilute (0.1% -1.0%) trifluoroacetic acid (TFA) . The TFA may be dissolved in an aqueous solution, or dissolved in other suitable solvents known in the art, including CH ₂ C1 ₂ .

The target molecule (i.e., affinity matrix with attached target molecule) is placed together with an appropriate reagent for MALDI-MS analysis. Any appropriate reagent known in the art may be used. Suitable reagents include sinapinic acid, 2 ,5-dihydroxybenzoic acid, and a- cyano-4-hydroxycinnamic acid. A preferred reagent is a- cyano-4-hydroxycinnamic acid. When acid reagents are used, it is desirable to remove amine protecting groups on the affinity bead, e.g., Boc groups, to permit ion pairing between the amine groups and the carboxyl groups of the matrix. This appears to improve the quality of the mass spectrometric results.

The reagent:target molecule molar ratio is preferably approximately 10,000:1, giving a final volume of 0.5-2.0 microliters. The mixture is applied to a MALDI-MS

probe tip of the spectrometer, and allowed to dry by either ambient evaporation, heating with a stream of warm air, or under vacuum. Alternatively, if the affinity bead comprises a photocleavable linker molecule, the affinity bead with the bound target biological molecule may be analyzed directly, with the laser radiation being used to simultaneously cleave and desorb the affinity material/ biological target conjugate.

Laser radiation is applied to the probe tip or to the affinity bead comprising a photocleavable linker molecule, to effect desorption of the target biological material or affinity material/biological target conjugate. The laser may be a nitrogen laser, Nd-YAG laser, or other laser known and used in the art, with a preferred frequency of 337 nm or 355 nm. The laser is pulsed a number of times, typically about 30-50 pulses, and ions comprising the target material are generated. The ions generated are captured in an electric field and passed through to the detector. Because MALDI-MS is nondestructive to the target sample, a sufficient amount of the target material may also be isolated, crystallized and analyzed by X-ray crystallography, using methods well known in the art.

For ES-MS, one or more affinity beads having the target biological material specifically bound thereto is placed in an appropriate container, and the target biological material separated from the affinity bead(s) , e.g., by using a 0.1-1.0% TFA solution as previously described. The resulting solution is dried and extracted into a suitable solvent, such as CH^CN, and an aliquot of 0.5-2.0 microliters introduced into an appropriate electrospray mass spectrometer and analyzed.

The mass spectrometry data can be used to determine the molecular weight of the isolated biological material, or the mass spectrometry sample can be crystallized and the

resulting crystal structure determined by X-ray crystallography techniques known and used in the art.

EXAMPLE 1. ATTACHMENT OF AN α-METHYLPHENACYL-ESTER PHOTOLABILE LINKER

MOLECULE TO POLYSTYRENE BEADS

Polystyrene beads bearing 2-bromopropionyl groups are available from Advanced Chemtech, Louisville, KY. Boc- Gly-OH (0.52 g, 4.2 mmol) was dissolved in a mixture of 4.5 mL EtOH and 1 mL of H ₂ 0. Cesium carbonate (612.9 mg. 1.88 mmol) in 2 mL water was added dropwise to the Boc-Gly-OH solution to pH 7.1. The mixture was then evaporated to dryness (35°C) and the residual solid was taken up in fresh 25 mL DMF and evaporated three times.The cesium salt was then dissolved in 15 mL DMF and mixed (vortex) with 2.0021 g of 2- bromopropionylpolystyrene resin (1.02 mmol/g) for 48 h. The resin was then washed with 4 x 50 mL water, 4 x 30 mL DMF, 4 x 30 mL MeOH, 4 x 30 mL methylene chloride, 4 x 30 mL DMF, and 4 x 30 mL methylene chloride to give Boc-Gly-α- methylphenacyl-ester-resin.

EXAMPLE 2 ATTACHMENT OF BIOTIN TO α-METHYLPHENACYL-ESTER LINKER

The TFA salt was removed from the resin by washing the resin with 2 x 8 mL 10% diisopropylethylamine in methylene chloride. The resin was then washed with 3 x 8 mL NMP. The deprotected resin (421.3 mg) was then mixed with Boc- Lys(Fmoc) -OH (1.1309 g, 2.41 mmol) , hydroxybenzotriazole (334.1 mg, 2.47 mmol), diisopropylcarbodiimide (0.38 mL, 2.43 mmol), and 6.5 mL NMP for 1.7 h. The resin waε then washed with 6 mL NMP, 6 mL isopropanol, and 4 x 6 mL NMP.

The protecting Fmoc group was removed from the lysine e-amine by mixing with 25% piperadine in DMF for 5 min., followed by a second treatment with piperidine for 10 min.

The resin was then washed with 6 mL NMP, 6 mL isopropanol, then 4 x 6 mL NMP.

The resultant e-NH ₂ -α-Boc-Lys-Gly-OCH(CH,) -CO-C _ή H ₄ -bead was reacted with biotin (603.8 mg, 2.47 mmol) , HBTU (1.1091,

2.92 mmol), 2 mL diisopropylethylamine, 6 mL DMF, and 4 mL

NMP for 2 h. The resin was then washed with 2 x 8 mL methylene chloride.

The resin was washed with 2 x 8 mL methylene chloride. The Boc protecting group was removed by mixing the resin with 5 mL of methylene chloride and 5 mL TFA for 15 min. The TFA salt was removed from the resin by 2 8 mL of 10% diisopropylethylamine in methylene chloride. The resin was then washed with 3 6 mL NMP.

EXAMPLE 3 PRODUCTION OF STREPTAVADIN-BIOTIN CONJUGATE

Streptavadin (11.7 mg) was dissolved in 1 ml of 0.05 molar phosphate buffer (pH 7.5) . Beads prepared as in Example 2 were incubated with the streptavidin solution for 2 hours at room temperature. The beads were then washed 7 times with 3 ml of distilled water to remove unbound streptavadin.

EXAMPLE 4

MALDI MASS SPECTROMETRIC ANALYSIS OF STREPTAVADIN-BIOTIN CONJUGATE

A 2 microliter ethanol/bead sample containing approximately 50 affinity beads having the streptavidin- biotin conjugate were deposited on a MALDI stainless steel sample plate, prior to the addition of α-cyano-4- hydroxycinnamic acid. Samples were dried at room temperature and then analyzed on a PerSeptive Voyager Elite MALDI spectrometer equipped with a nitrogen laser radiating at 337 nm.