WO2000048004A9 - High throughput mass spectrometry - Google Patents

Abstract

Apparatus and methods for high throughput mass spectrometry are provided. The methods involve sample preparation in an off-line parallel purification system. Such methods include but are not limited to the use of an appropriate buffer when generating samples or the use of a solid support for tagged components. The samples prepared in this way do not then need to be column separated. The apparatus provided includes a cell growth plate for growing cells and generating products and/or reactants, an off-line parallel purification system, a mass spectrometer, and an automatic sampler that transports samples and injects them into the mass spectrometer of the apparatus. The methods and apparatus described are used, for example, in screening enzyme reaction pathways.

Description

j HIGH THROUGHPUT MASS SPECTROMETRY

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of and priority to USSN 60/119,766, "High Throughput Mass Spectrometry," by Raillard; USSN 60/148,848 entitled "Evolution and Use of Enzymes for Combinatorial and Medicinal Chemistry," by Liu et al., filed August 12, 1999; and co-filed U.S. application, "High Throughput Mass Spectrometry," by Raillard et al., filed February 11, 2000, Attorney Docket No. 2- 029510US.

FIELD OF THE INVENTION

This invention relates to high throughput methods for mass spectrometry, for example, to monitor a plurality of samples, e.g., enzyme products generated from a library, e.g., of gene sequences.

BACKGROUND OF THE INVENTION

High throughput chemical screening, of enzyme activity for example, typically involves quantitative detection of one or more substrate and/or product. The most universal detection method to date is mass spectrometry (MS), which allows identification of a particular organic molecule based on mass to charge ratio. Traditionally, mass spectrometry is performed in tandem with liquid chromatography to purify and separate the components of interest. This purification can be considered to be on-line sequential purification. The sequential nature of the purification limits the ability of mass spectrometry to screen a large number of reaction products in a short amount of time, because the puπfication must occur in line with and previous to the mass spectrometry

DNA shuffling technology is used to create a library of related gene sequences that encode, e g . one or more enzyme that catalyzes a reaction. Such a library is constructed, e.g., by homologous exchange of DNA fragments duπng DNA shuffling In one typical set of embodiments, the library of related gene sequences is on a plasmid that has been transformed into a bacteπa. Thus a single bacteπal clone can carry a unique gene sequence representing a unique vaπant of a particular enzyme or enzyme pathway. For directed evolution, the library is screened for vaπants having a desired characteristic Evolution of enzymes and pathways involves biochemical reaction of one or more enzymes that can be detected by a chemical screening method. A chemical screening method detects the substrates and or products of the enzyme reactιon(s)

Presently, the use of mass spectrometry to analyze these enzyme reactions is extremely time consuming The time limitation is due to the need to separate and puπfy the products and reactants of the enzyme pathways before injection into a mass spectrometer. This limits the number of samples that can be analyzed to about 100 samples per day (typical purification runs (e.g., liquid chromatography) require about 10 minutes/sample. At 6 samples per hour, 144 samples can be run in a 24-hour peπod) A new high throughput system would be useful to provide a method of analyzing a library for a few mutants out of thousands that will provide the desired properties

One recently developed system is the electrospray method as descπbed in "Quantitative Electrospray Mass-Spectrometry for the Rapid Assa\ of En/yme Inhibitors," by Wu et al. in Chemistry & Biology 1997, Vol. 4 No 9, pό53~657 Electrospray lonization is a mild method of transfemng charged polar organic molecules into the gas phase for mass spectrometry analysis and is applicable for most biologically relevant organic molecules The electrospray method eliminates the need for pπor deπvatization of samples before injection into a mass-spectrometer as in GC/MS and thus shortens the analysis time for mass spectrometry However, column separation is still utilized in this technique, limiting throughput as noted above

Another recent development is descπbed in "Fast Screening for Drugs of Abuse by Solid-Phase Extraction Combined with Flow-Injection Ionspray-Tandem Mass Spectrometry," by Weinman and Svobodam, Journal of Analytical Toxicology, Vol. 22, 1998, p.319-328 The technique descπbed combined tandem mass spectrometry and electrospray methods to simultaneously detect different drugs in serum or uπne Although no column separation was used because the tandem mass spectrometry allowed detection of multiple compounds, a solid phase extraction method was necessary in the sample preparation The sample preparation steps were still too lengthy to provide high throughput screening by mass spectrometry

Accordingly, a high throughput method of performing mass spectrometry, e g., for screening hbraπes of shuffled molecules, would be useful The present invention fulfills these and many other needs which will become apparent upon complete review of this disclosure

SUMMARY OF THE INVENTION

The invention provides a method for high throughput mass spectrometry, that is used, for example, to monitor enzyme reactions, e.g , at the rate of about 100 samples or more per hour, more preferably about 200 samples or more per hour. Using this method, many samples can be screened simultaneously so that an entire library can be screened in a week or less This provides a faster method of mass spectrometry screening than has previously existed The increase in throughput is due to a novel offline parallel puπfication system The off-line parallel puπfication eliminates the need for liquid chromatography or a separate puπfication step before injection of the sample into a mass spectrometer

In one embodiment, a method of performing high throughput mass spectrometry screening is provided In the method, one or more cells are grown Non- column-separated components of interest are puπfied from the cell colony or culture. In one aspect, the puπfication includes an off-line parallel adjustment of cell growing conditions or attachment of the non-column-separated components to a solid support. In the method, flow-mjection analysis is performed using, e g , electrospray tandem mass spectrometry, thereby obtaining mass-to-charge ratio data and providing high throughput mass spectrometry screening of the non-column-separated components of interest The growing and puπfymg steps are achieved essentially simultaneously by adjusting growing conditions or the conditions used to produce the reactants or products of interest For example, the components of interest can be produced from whole cells, from cell supernatant, from cell lysate or from puπfied enzymes with added substrates This production occurs in a volatile buffer, a buffer that reduces concentration of ionic species followed by a puπfication/clean up method such as an ion exchange resin, or the production is modified to be compatible with extraction, e.g., with an organic solvent to provide a component that can be injected directly into the mass spectrometer with no further puπfication Because these steps are in parallel, at least 100 cell colonies are screened for presence or activity of the one or more non-column-separated component in less than an hour.

Alternatively, the puπfying step is achieved by lysing cells and attaching one or more components, e.g., tagged components such as tagged enzymes, proteins, or nucleic acids, to a solid support compπsing, e.g., a tag binding moiety. The cell lysate is optionally washed from the solid support and the enzymes are contacted with one or more substrates, producing one or more products, which are optionally analyzed without further puπfication.

The one or more non-column-separated component is a protein, a protein binding molecule, a carbohydrate, a carbohydrate binding molecule, an enzyme, an enzyme substrate, a product of an enzyme catalyzed reaction, a nucleic acid, a product of a nucleic acid catalyzed reaction, a substrate with one or more hydrophobic moieties, an inorganic ion, an ohgosacchaπde, a hydrophobic molecule, a bπatme deπvative, atrazine, a polyketide, or other molecule of interest. In another embodiment, the present invention provides a method for momtoπng products or reactants, such as in enzyme reactions, by high throughput mass spectrometry by providing a cell or bacteπa that has been transformed with a plasmid containing one or more member of a library, e.g., of related gene sequences, such as related enzyme gene sequences. One or more cells or a cell colony or culture is grown from the cell; producing one or more product or reactant from the cell colony or culture in a biological matπx, thereby producing a non-column-separated sample; puπfying the non-column separated sample from the biological matπx using an off-line parallel adjustment of the biological matnx, and monitoπng the non-column separated sample by flow-injection analysis using electrospray tandem mass spectrometry, thereby monitoπng the one or more product or reactant In this way, enzyme reactions and their products can be studied at high throughput levels Alternative hbraπes are also

The products and/or reactants can be puπfied simultaneous to production, thus providing an off-line parallel puπfication system The products and/or reactants are produced, e g , using whole cells, cell supernatant, cell lysate, or from a reaction between at least one purified cell enzyme and at least one substrate The components of the sample are optionally a substrate with one or more hydrophobic moieties, an inorganic ion. a small molecule, an ohgosacchaπde, a hydrophobic molecule, a peptide, a polypeptide, a protein, a nucleic acid, a polynucleotide, a hydrophilic molecule, a tπazine deπvative. a secondary metabolite such as a polyketide, a protein, a protein binding molecule, a carbohydrate, a carbohydrate binding molecule, an enzyme, an enzyme substrate, a product of an enzyme catalyzed reaction, a nucleic acid, a product of a nucleic acid catalyzed reaction, or the like The components are optionally known or unknown components Unknown components are optionally identified and/or quantified using mass spectrometry analysis

The purifying system, which typically occurs in reaction conditions that mimic environmental cellular conditions, comprises alteπng or adding a buffer to the biological matrix in which the non-column-separated sample is produced, thereby producing a sample that can be injected directly into a mass spectrometer for analysis of the sample The buffer used is optionally a volatile buffer, a buffer that reduces concentration of ionic species, a buffer that allows easy parallel off-line puπfication such as an ion exchange resin, or an organic solvent extraction. Alternatively, the puπfying system comprises binding an enzyme or other component, e.g , a nucleic acid, protein, peptide, carbohydrate, or the like, to a solid support, e g., through a specific tag moiety Reactions are then performed on the solid support, which is optionally washed to remove impunties or unbound components, thereby producing samples that are sufficiently puπfied for injection into a mass spectrometer Using one of these puπfication techniques, at least about 100 library members or more are screened for presence or absence of products or reactants in less than an hour. Typically, at least about 200 or more library members are screened in about an hour Preferably, at least about 500 or more samples are screened in about an hour

In other embodiments, throughput is optionally increased, e.g , by pooling samples or components and injecting the pooled samples into the mass spectrometer for simultaneous analysis The resulting data is typically deconvoluted, e.g , using fragmentation patterns or spectra, to identify the different samples

In another embodiment, this invention provides an apparatus for high throughput mass spectrometiy screening The apparatus compπses a cell growth plate for growing cell samples and reacting enzymes, enzyme substrates, and enzyme products; an off-line parallel purification system coupled to or within the cell growth plate, for puπfying the samples; an automatic sampler coupled to the off-line parallel puπfication system; and a mass spectrometer, such as an electrospray triple quadrupole tandem mass spectrometer, coupled to the automatic sampler The automatic sampler is a sample handler that transports samples from the off-line parallel puπfication system to the mass spectrometer for injection and analysis It can transport, e.g., at least 100 samples or more in about an hour.

Using the apparatus and integrated systems of the invention, the rate of screening is determined by the maximum rate at which the automatic sampler transports samples between the off-line puπfication system and the mass spectrometer This is due to the ability of the apparatus to puπfy the samples for injection in an off-line parallel system, that is optionally a volatile buffer, a buffer that reduces concentration of ionic species, an ion exchange resin, an organic solvent, or a solid support, e.g., to bind an enzyme or other component.

In another aspect, the apparatus of the invention compπses a computer and software operably coupled to the apparatus for recording and analyzing mass spectrometer data and for controlling the automatic sampler.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the enzymatic conversion of atrazine to liydroxyatrazme by atzA.

Figure 2, panels A and B show MS/MS plots of Atrazine Figure 3 is a graph showing relative abundance versus time Figure 4 is a schematic overview of an exemplar high throughput method of the invention.

Definitions

The term "non-column-separated component" refers to components or mateπals of interest, e g., that are injected into a mass spectrometer, without prior in-line sequential separation, e.g , on a chromatography column Without a sequential in-line separation, the components are optionally analyzed in a high throughput system. Parallel systems that allow components to be puπfied or separated as they are produced allow high throughput analysis

In the present invention the term "off-line parallel adjustment of cell growing conditions" or "off-line parallel puπfication system" or "off-line parallel adjustment of biological matrix" is used to refer to a new method of sample preparation The method is used to prepare complex samples for injection into a mass spectrometer without a time-consuming sequential purification and/or separation step. In this method, the samples and their reaction conditions are adjusted or modulated, e.g , in a cell growth plate in parallel with the production of products and reactants of interest. Cell growing conditions and reaction conditions are optimized, e.g., to obtain products with sufficient puπty for, e g , mass spectrometry, by routine alteration and optimization of reaction parameters and conditions The system is not coupled to a column separation system that functions in line with and sequential to the mass spectrometer. In one embodiment, the non-column separated components are puπfied without separation. Alternatively, the offline puπfication system comprises a reactor, e.g., an enzyme reactor, e.g., a solid support for binding or attaching a library of components, e.g., tagged enzymes are optionally bound to a solid support compπsing tag-binding molecules For example, cells that have been transformed with genes encoding enzyme sequences with specific tags, e.g., biotin, are typically lysed after expression of the enzyme. The enzymes are bound to the support or enzyme reactor, e.g., a solid support compπsing streptavidin, and the cell lysate is optionally removed, e g., by removing the solid support from the cell lysate or by filteπng the cell lysate from the solid support Substrates are provided to perform enzyme reactions on the support, thereby producing products that are sufficiently pure for injection into a mass spectrometer. The solid support optionally compπses a set of magnetic, agarose, or polystyrene beads, pms, a membrane, or the like For example, beads are optionally placed in a sample well, e.g., on a cell growth plate When the cells are lysed, tagged components bind to the beads, e.g., via a tag-bindmg moiety on the beads. The beads are then optionally removed from the sample well for further reaction or identification. Alternatively, the cell lysate is removed or washed from the beads. Pins are optionally lifted in and out of a sample well to bind to and/or remove tagged components from a sample Similarly, a membrane is optionally used to bind components Other non-tagged components are optionally washed from the membrane or the membrane is removed, e g.. from the sample well to provide puπfied components. "Product or reactant" is used herein to refer to products or reactants, e.g., of enzyme catalyzed reactions. The product or reactant is optionally a protein, a peptide, a protein or peptide binding molecule, a carbohydrate, a carbohydrate binding molecule, a nucleic acid molecule, a polynucleotide, a nucleic acid or polynucleotide binding molecule, or a product of a nucleic acid catalyzed reaction. Additionally, the product and or reactant is optionally an enzyme or enzyme substrate. The product or reactant is any molecule of interest that is to be analyzed by the methods of the invention.

A "cell growth plate" is used herein to refer to a plate on which cells can be grown in an appropπate media. Exemplar plates include 1536, 384 or 96-well microtiter plates. The plates are used to grow cell colonies. For example cell colonies containing gene hbraπes are picked directly from transformation plates into 1536, 384 or 96-well microtiter plates with appropπate growth media using, for example, a Q-bot from Genetix Additionally, the off-line parallel punfication and/or adjustment of reaction conditions occurs on the cell growth plate when the products or reactants of interest are generated. All product generation and puπfication steps optionally occur in the wells of the cell growth plate. In some embodiments, the cell growth plate compπses a solid support, e.g., particles, beads, a membrane, a set of pms, or the like, for binding one or more components, e.g., enzymes, e.g., after cells are lysed. For example, each well of a microtiter plate optionally compπses one or more agarose beads, e.g., beads compπsing avidin or streptavidin to which enzymes compπsing a biotin tag will bind. Alternatively, a set of pms is optionally introduced into the wells of the cell growth plate to bind to or remove tagged enzymes from the cell lysate

A "mass spectrometer" is an analytical instrument that can be used to determine the molecular weights of vaπous substances, such as proteins and nucleic acids. It can also be used in some applications, e.g., to determine the sequence of protein molecules and the chemical composition of virtually any matenal. Typically, a mass spectrometer compπses four parts- a sample inlet, an lonization source, a mass analyzer, and a detector. A sample is optionally introduced via vaπous types of inlets, e g., solid probe, GC, or LC, in gas, liquid, or solid phase The sample is then typically ionized in the lonization source to form one or more ions The resulting ions are introduced into and manipulated by the mass analyzer Surviving ions are detected based on mass to charge ratio. In one embodiment, the mass spectrometer bombards the substance under investigation with an electron beam and quantitatively records the result as a spectrum of positive and negative ion fragments Separation of the ion fragments is on the basis of mass to charge ratio of the ions. If all the ions are singly charged, this separation is essentially based on mass. A quadrupole mass spectrometer uses four electπc poles for the mass analyzer These techniques are descπbed generally in many basic texts, e.g., Quadrupole Mass Spectrometry and its Applications, by Peter Dawson, Spπnger Nerlag, 1995; and Spectrometπc Identification of Organic Compounds, by Silverstem, Bassler and Momll, Fourth Edition, 1981. In an electrospray mass spectrometry system, lonization occurs by an electπc field that is used to generate charged droplets and subsequent analyte ions by ion evaporation for TIS analysis. See, Richard B. Cole (1997) "Electrospray lonization Mass Spectrometry" John Wiley and Sons, Inc

"High throughput mass spectrometry" is used herein to refer to a mass spectrometry system that is capable of screening samples at a rate of from about 100 or 200 samples per day to about 15,000 samples per day In the present invention, systems are provided that screen about 200 samples in less than an hour, e.g., 200 samples are injected into a mass spectrometer and analyzed in less than an hour. In addition, high throughput mass spectrometry refers to the pooling of samples, e.g., into a single injection. For example, multiple samples are pooled into a single injection. This increases the rate of screening of the mass spectrometer because multiple samples are simultaneously injected. About 2 to about 1000 samples are optionally pooled. Typically about 5 to about 500 samples are pooled or about 5 to about 100 samples. In other embodiments, about 5 to about 20 samples are pooled For example, 100 samples are optionally pooled into a single injection and 200 injections are optionally made in about an hour, thereby screening a total of 20,000 samples by MS in about an hour. In other words, samples, e.g., clones or library members, are screened at a rate of about 480,000 samples per day. This is well over the typical MS screening rate of about 100 to about 200 samples per day. A "high throughput system" typically has throughput rates as descπbed above. Systems of interest in the present case, include, but are not limited to, mass spectrometry systems, magnetic resonance systems, IR and UN spectroscopy systems, and the like A "cell colony" is used herein to refer to the in vitro propagation of cells isolated from living tissues A cell colony, as used herein, is typically a growth of cells on a solid medium or in a liquid culture, typically one that is visible to the eye without magnification The one or more cells or clones (cells having the same genetic makeup) from a cell colony may be analyzed as whole cells or in the form of a complete cell lysate or a cell supernatant A puπfied cell lysate is the product of cell lysis or the complete or partial disintegration or breaking up of the cell wall The cells may be lysed before use in the present invention and the resulting cell lysate used to geneiate the products or reactants of interest Alternatively, the cell supernatant is used to generate components of interest For interest secreted proteins are optionally obtained or puπfied from cell supernatant and used in the methods of the invention

As used herein, "puπfied cell enzymes with added substrates" refers to enzymes that have been previously purified from cells or other sources Substrates are then added to the puπfied enzymes to produce reaction products of interest This is in contrast to the generation of reaction products from whole cells or cell lysates When the puπfied enzymes are attached to a solid support, e g , an enzyme reactor, the reaction products are optionally puπfied by washing the solid support or by removal of the enzymes from the reaction mixture, e g , by removal of the solid support For example when enzymes are puπfied from a cell lysate using p s compπsing a tag-bindmg moiety, the pins are optionally placed into a reaction mixture for the enzyme reaction and then removed at the conclusion of the reaction, leaving behind a puπfied product

"Nucleic acid" refers to deoxyπbonucleotides or nbonucleotides and polymers thereof in either single- or double-stranded form The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurπng, and non-naturally occurnng, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides Examples of such analogs include, without limitation, phosphorothioates, phosphoramidites, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl nbonucleotides, peptide -nucleic acids (PNAs) 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 analog or mimetic of a corresponding naturally occurnng ammo acid, as well as to naturally occurnng amino acid polymers

The term "amino acid" refers to naturally occurπng and synthetic ammo acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids Naturally occurnng amino acids are those encoded by the genetic code, as well as those ammo acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoseπne Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurnng amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoseπne, 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 occurnng 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 function in a manner similar to a naturally occurnng amino acid.

As used herein, "product of an enzyme catalyzed reaction" refers to any product generated by a reaction that has been catalyzed by an enzyme. Enzymes react with substrate molecules to produce reaction products that are of interest in the present invention. For example, to evolve a new functional enzyme, the identity and detection of products of the new enzyme's reaction with substrates will provide important information regarding the functionality of the new enzyme. The products are optionally known compounds or unknown compounds.

As used herein, "product of a nucleic acid catalyzed reaction" refers to any product generated by a reaction that has been catalyzed by a nucleic acid functioning as an enzyme, e.g., the cleavage product of a hammerhead or hairpin nbozyme.

The term "protein binding molecule" is used herein to refer to any molecule which binds or interacts with a protein It includes, but is not limited to, other proteins, carbohydrates, lipids, nucleic acids and the like.

The term "carbohydrate" includes any of a large class of carbon-hydrogen- oxygen compounds. It includes but is not limited to sugars and their polymers, e.g., starch, glycogen, glucose, and cellulose, and polyhydroxyaldehydes, polyhydroxyketones, or their deπvatives Most but not all carbohydrates are represented chemically by the formula, C_x(H₂O)_n, where "n" is three or higher

"Carbohydrate binding molecule" is used herein to refer to any molecule or compound that binds or interacts with a carbohydrate, either specifically or nonspecifically. It includes but is not limited to other carbohydrates, proteins, lipids, nucleic acids and the like The term "enzyme," as used herein, generally refers to a protein which acts as a catalyst to reduce the activation energy of a chemical reaction in other compounds or "substrates "

The term "substrate with one or more hydrophobic moieties" is used herein to refer to a substrate that comprises a molecule that has at least one, and possibly more, hydrophobic group or portion

An "inorganic ion" is an ion which does not compπse an organic component

"Ohgosacchande" refers to a relatively short molecular chain made up of about 10 to about 100 simple sugars or monosacchaπde units.

The term "hydrophobic molecule" refers to any molecule or portion of a molecule which has an affinity for oil at an oil-water interface A "hydrophilic molecule" refers to molecule or any portion of a molecule that has an affinity for water at an oil- water interface The term "library" is used herein to refer to gene hbraπes, e.g., produced by mutagenesis, recombination, directed evolution, shuffling, or other diversity generating techniques; enzyme hbranes, combinatoπal or chemical hbranes; naturally occurπng hbranes; e.g., of microorganisms, hbranes of non-biological compounds, and the like "Library of related gene sequences" is used herein to refer to a group of similar gene sequences, for example gene sequences encoding enzymes or enzyme subunits that have been evolved or shuffled to create new and/or related genes that encode enzymes with the ability to act on a new substrate, or for enhanced catalytic properties with an old substrate, either alone or in combination with other genes. In some embodiments, a library compnses a group of genes that have been fused to a sequence encoding a specific tag, e.g., a biotin tag. For example, the expression products of such a library are then optionally bound to a solid support compnsing a tag-bindmg moiety, e.g., avidin or streptavidin, that binds the specific tag

As used herein, "biological matrix" refers to the fluid, substance, or reaction mixture or growth medium in which a cell is grown The products and reactants of interest in the invention are optionally generated and/or punfied in the biological matπx. The biological matπx is typically similar to the native environmental conditions of the enzyme or substance of interest. In some embodiments, the enzymes, e.g., tagged enzymes, are removed from the biological matπx by binding to a solid support, e.g , polystyrene or magnetic beads in a cell growth plate or pins dipped into the wells in which the cells were grown and lysed

"Transformed" as used herein, refers to a cell that has been transfected or transduced with a nucleic acid A cell has been "transformed" by an exogenous nucleic acid when such exogenous nucleic acid has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. The exogenous DNA may be maintained on an episomal element, such as a plasmid Transformation refers to any way of getting a nucleic acid across a cell membrane, including electroporation, ballistics, injection, using hpid-nucleic acid complexes, etc

By "host cell" is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coh, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells

A "plasmid" is a DNA molecule with an oπgin of replication. The plasmid is capable of being replicated in one or more host cell types. Because a plasmid is usually small and relatively simple, they are commonly used in recombinant DNA expeπments as acceptors of foreign DNA. The term "simultaneously" refers to two events that occur at essentially the same time For example, the generation of the products or reactants of interest in the present invention occurs simultaneously with the puπfication in the off-line parallel puπfication system. The two events are both done at the same time in the same location, e.g., the cell growth plate, to save time in the analysis, thus allowing a high throughput mass spectrometry screening to occur.

An "automatic sampler" is a robotic handler that transports samples from one location to another An automatic sampler is used for example, to transport samples from a cell growth plate and inject them into a mass spectrometer for analysis. Examples of automatic samplers include the Gilson 8-probe microtiter autosampler and the microtiter autosampler from CTC analytics. Automatic samplers optionally include robotic handlers that are used to pick colonies, such as a Q-bot, and/or add or remove reagents to or from the cell growth plate DETAILED DISCUSSION

Mass spectrometry has been used to detect metabolites in biological fluids and to monitor enzyme reactions. See, e.g., "Quantitative Electrospray Mass Spectrometry for the Rapid Assay of Enzyme Inhibitors. Wu et al., Chemistry and Biology, 9/19/97, 4, p653 In one embodiment, the present invention uses the inherent capacity of electrospray MS to monitor enzyme reactions and their reaction products by adapting a high throughput flow injection analysis. Using the method of the present invention, a sample is injected directly into a mass spectrometer without any column separation and analyzed instantly. The speed of the analysis is limited only by the motonc movements of the autosampler used to inject the samples Thus, an entire 96- well microtiter plate of samples is typically run in less than an houi Autosampler companies, such as Gilson and CTC Analytics are currently working to increase the throughput to one plate in 10 minutes, which would then allow for about 570 injections per hour or about 13,000 injections into a mass spectrometer in a day. If samples are pooled, e.g., about 2 to about 1000 samples are combined and injected simultaneously, then the screening rate increases to about 1000 samples per hour to about 575,000 samples per hour or about 25,000 samples per day to about 13 million samples or more per day. One aspect of the present mass spectrometry method is that the samples are punfied off-line so that an in-line sequential chromatography step is not necessary. A liquid chromatography (LC) step, to separate the components, is usualh coupled to the mass spectrometer (MS) in a sequential fashion so that the limiting factor in mass spectrometry throughput is the speed at which the LC can process t-omponenb, With an off-line punfication system, such as the one herein, the speed of mas^ spectrometry analysis is not limited by a sequential puπfication step The mass spectrometry throughput in this invention is typically rate dependent on how fast the automatic sampler can transport and inject the samples into the mass spectrometer.

To analyze enzyme reactions using high-throughput mass spectrometry, first a single colony of cells must be picked and grown. Second, enzyme products are generated using whole cells, complete or partial cell lysates, or punfied enzymes to which substrates have been added Third, the products generated from the biological matnx are puπfied in an off-line parallel purification system Fourth, flow injection analysis is performed using tandem mass spectrometry Applications for high-throughput MS include but are not limited to screening plasma, uπne or cerebral spinal fluid, or the like for, i.e , identification of metabolites that correlate with cancer susceptibility or presence, event specific testing of exposure to toxins, momtonng effects of drug tπals, monitoπng effects of prescπbed drug use, creation of a metabolite encyclopedia that contains metabolite profiles for every type of cell in the human body Additionally, testing and analysis can be performed on non- human animals, plants, and food and dnnk items, such as gra or wine. In another aspect, high throughput (HTP) MS is used in plant genetics for identification of the gene pathways responsible for synthesis of commercially valuable plant products, such as drugs, and oils, and for identification of the effects of gene transformation on metabolite phenotype, or for screening plants for the presence of desired natural products. High- throughput MS is also useful for similar analyses in bacteπal and viral systems In a particularly useful aspect of present invention, high throughput mass spectrometry (HTP- MS) is used to screen hbranes of cells, e.g., for an expression product of a shuffled nucleic acid or for screening a library for enzyme activity e.g., a library produced from directed evolution or shuffling

I. Integrated system elements

Making Libranes The present invention typically utilizes DNA shuffling or directed evolution technologies to make libranes which are screened by the high throughput methods of the invention, but other types of hbranes are also available and are optionally screened by the present methods A "library" of compositions or compounds in the present invention is a large collection of samples, e.g., composed of proteins, expression products, genes, nucleic acids, cells, pharmacologically active compositions, e.g., drugs, small organic molecules, peptides, and the like. Libranes include, but are not limited to, a library of biological or chemical compositions, such as a library of expression products or vanant genes or a library of mutagenized cells. Such hbranes are optionally generated by DNA shuffling, random mutagenesis, transposon mutagenesis, or combinatonal gene assembly Gene hbranes are optionally expressed to produce hbranes of expression products which are screened by MS The present methods are optionally uses to screen any desired group of compounds or molecules. Techniques for the production of libranes are well known to those of skill in the art. Making libraries typically includes the construction of recombinant nucleic acids and the expression of genes in transfected host cells Molecular cloning techniques to achieve these ends are known in the art A wide vaπety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well known to persons of skill. General texts which descπbe molecular biological techniques useful herein, including mutagenesis, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed ), Nol 1-3, Cold Spπng Harbor Laboratory, Cold Spnng Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds , Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ("Ausubel")) Examples of techniques sufficient to direct persons of skill through in vitro amplification methods (useful for making library nucleic acids), including the polymerase chain reaction (PCR) the hgase chain reaction (LCR), Q -rephcase amplification and other RNA polymerase mediated techniques (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, id., as well as in Mulhs et al, (1987) U.S Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc San Diego, CA (1990) (Innis), Arnheim & Levinson (October 1, 1990) C&EN 36-47, The Journal Of NIH Research (1991) 3, 81-94; Kwoh et al. (1989) Proc Natl. Acad. Sci USA 86, 1173, Guatelh et al (1990) Proc Natl Acad. Sci USA 87, 1874; Lomell et al (1989) J Chn Chem 35, 1826; Landegren et al , (1988) Science 241, 1077-1080; Nan Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barπnger et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564 Improved methods of cloning in vitro amplified nucleic acids are descπbed in Wallace et al , U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleic acids by PCR are summanzed in Cheng et al (1994) Nature 369' 684-685 and the references therein, in which PCR amphcons of up to 40kb are generated One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restnction digestion, PCR expansion and sequencing using reverse transcnptase and a polymerase. See, Ausubel, Sambrook and Berger, all supra

Methods of transducing cells, including plant and animal cells, with nucleic acids as in library construction are generally available, as are methods of expressing proteins encoded by such nucleic acids In addition to Berger, Ausubel and Sambrook, useful general references for culture of animal cells include Freshney (Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley- Liss, New York (1994)) and the references cited therein, Humason (Animal Tissue Techniques, fourth edition W.H. Freeman and Company (1979)) and Ricciardelh, et al., In Nitro Cell Dev Biol. 25:1016-1024 (1989). References for plant cell cloning, culture and regeneration include Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc New York, NY (Payne); and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Spnnger Lab Manual, Spπnger-Nerlag (Berlin Heidelbeig New York) (Gamborg) A vanety of Cell culture media are descnbed in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton. FL (Atlas) Additional information for plant cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma- Aldπch, Inc (St Louis, MO) (Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldnch, Inc (St Louis, MO) (Sigma-PCCS)

A vanety of diversity generating/product screening reactions are optionally used to produce libranes that are optionally screened by the methods provided herein. For example libranes of related enzyme encoding genes are optionally expressed and the products of the enzyme reactions punfied and analyzed in a high throughput format by mass spectroscopy as descπbed herein One important class of such diversity generating reactions is so called "nucleic acid shuffling" or "DNA shuffling". In these methods, any of a vanety of recombmation-based diversity generating procedures can be used to diversify starting nucleic acids, or organisms compnsing nucleic acids, or even to diversify character stnngs which are "in silico" (in computer) representations of nucleic acids Diverse nucleic acids/character stnngs/organisms which are generated are typically screened for one or more activity Nucleic acids, character stnngs, or organisms which comprise nucleic acids are then used as substrates in subsequent recombination reactions, the products of w hich are, again, screened for one or more activity. This process is repeated recursively until one or more desirable product is produced A vanety of diversity generating protocols, including nucleic acid shuffling protocols, is available and fully descπbed m the art The following publications descπbe a variety of recursive recombination procedures and/or methods which can be incorporated into such procedures, as well as other diversity generating protocols: Stemmer, et al., (1999) "Molecular breeding of viruses for targeting and other clinical properties. Tumor Targeting" 4:1-4; Nesset et al. (1999) "DNA Shuffling of subgenomic sequences of subtihsin" Nature Biotechnology 17:893-896; Chang et al. (1999) "Evolution of a cytok e using DNA family shuffling" Nature Biotechnology 17:793-797; Minshull and Stemmer (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3:284-290; Chnstians et al. (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17:259-264; Cramenet al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291; Cramen et al. (1997) "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotechnology 15-436-438; Zhang et al. (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proceedings of the National Academy of Sciences, U.S.A. 94:4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Naccines" Current Opinion in Biotechnology 8:724-733; Cramen et al. (1996) "Construction and evolution of antibody-phage libranes by DΝA shuffling" Nature Medicine 2:100-103; Cramen et al. (1996) "Improved green fluorescent protein by molecular evolution using DNA shuffling" Nature Biotechnology 14-315-319; Gates et al. (1996) "Affinity selective isolation of ligands from peptide libranes through display on a lac repressor 'headpiece dimer"' Journal of Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology NCH Publishers, New York, pp 447-457; Cramen and Stemmer ( 1995) "Combmatonal multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioTechniques 18:194-195; Stemmer et al., (1995) "Single-step assembly of a gene and entire plasmid form large numbers of ohgodeoxynbonucleotides" Gene, 164-49-53; Stemmer (1995) "The Evolution of Molecular Computation" Science 270. 1510; Stemmer (1995) "Searching Sequence Space" Bio/Technology 13-549-553, Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370.389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly In vitro recombination for molecular evolution " Proceedings of the National Academy of Sciences, U.S A 91 : 10747-10751.

Additional details regarding DNA shuffling and other diversity generating methods are found in U.S Patents by the inventors and their co-workers, including United States Patent 5,605,793 to Stemmer (February 25, 1997), "METHODS FOR IN VITRO RECOMBINATION;" United States Patent 5,811,238 to Stemmer et al. (September 22, 1998) "METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION," United States Patent 5,830,721 to Stemmer et al. (November 3, 1998), "DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY;" United States Patent 5,834,252 to Stemmer, et al. (November 10, 1998) "END-COMPLEMENTARY POLYMERASE REACTION," and United States Patent 5,837,458 to Mmshull, et al. (November 17, 1998), "METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING."

In addition, details and formats for DNA shuffling and other diversity generating protocols are found in a vanety of PCT and foreign patent application publications, including: Stemmer and Cramen, "DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASEMBLY" WO 95/22625; Stemmer and Lipschutz "END COMPLEMENTARY POLYMERASE CHAIN REACTION" WO 96/33207; Stemmer and Cramen "METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION" WO 97/0078; Mmshul and Stemmer, "METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING" WO 97/35966; Punnonen et al. "TARGETING OF GENETIC VACCINE VECTORS" WO 99/41402; Punnonen et al. "ANTIGEN LIBRARY IMMUNIZATION" WO 99/41383; Punnonen et al "GENETIC VACCINE VECTOR ENGINEERING" WO 99/41369, Punnonen et al. OPTIMIZATION OF IMMUNOMODULATORY PROPERTIES OF GENETIC VACCINES WO 9941368; Stemmer and Cramen, "DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY" EP 0934999; Stemmer "EVOLVING CELLULAR DNA UPTAKE BY RECURSIVE SEQUENCE RECOMBINATION" EP 0932670, Stemmer et al., "MODIFICATION OF VIRUS TROPISM AND HOST RANGE BY VIRAL GENOME SHUFFLING" WO 9923107, Apt et al., "HUMAN PAPILLOMA VIRUS VECTORS" WO 9921979; Del Cardayre et al "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE

SEQUENCE RECOMBINATION" WO 9831837; Patten and Stemmer, "METHODS AND COMPOSITIONS FOR POLYPEPTIDE ENGINEERING" WO 9827230, Stemmer et al., and "METHODS FOR OPTIMIZATION OF GENE THERAPY BY RECURSIVE SEQUENCE SHUFFLING AND SELECTION" WO9813487.

Certain U.S. Applications provide additional details regarding DNA shuffling and related techniques, as well as other diversity generating methods, including "SHUFFLING OF CODON ALTERED GENES" by Patten et al. filed September 29,

1998, (USSN 60/102,362), January 29, 1999 (USSN 60/117,729), and September 28,

1999, USSN09/407,800 (Attorney Docket Number 20-28520US/PCT); "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION", by del Cardyre et al. filed July 15, 1998 (USSN 09/166,188), and July 15, 1999 (USSN 09/354,922); "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACTD RECOMBINATION" by Cramen et al., filed February 5, 1999 (USSN 60/118,813) and filed June 24, 1999 (USSN 60/141,049) and filed September 28, 1999 (USSN 09/408,392, Attorney Docket Number 02-29620US): and "USE OF CODON- BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al., filed September 28, 1999 (USSN 09/408,393, Attorney Docket Number 02- 010070US); and "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Sehfonov and Stemmer, filed February 5, 1999 (USSN 60/118854) and USSN 09/416,375 filed October 12, 1999. As review of the foregoing publications, patents, published applications and U.S. patent applications reveals, recursive recombination of nucleic acids to provide new nucleic acids with desired properties can be earned out by a number of established methods and these procedures can be combined with any of a vanety of other diversity generating methods, e.g., to produce libranes that are optionally screened as descπbed herein.

In bπef, at least 5 different general classes of recombination methods are applicable to the present invention and set forth in the references above. First, nucleic acids can be recombined in vitro by any of a vanety of techniques discussed in the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by hgation and/or PCR reassembly of the nucleic acids. Second, nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells Third, whole genome recombination methods can be used in which whole genomes of cells or other organisms are recombined, optionally including spiking of the genomic recombination mixtures with desired library components. Fourth, synthetic recombination methods can be used, in which oligonucleotides corresponding to targets of interest are synthesized and reassembled in PCR or hgation reactions which include oligonucleotides which correspond to more than one parental nucleic acid, thereby generating new recombined nucleic acids. Oligonucleotides can be made by standard nucleotide addition methods, or can be made by tri-nucleotide synthetic approaches. Fifth, in silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which correspond to nucleic acid homologues (or even non-homologous sequences). The resulting recombined sequence strings are optionally converted into nucleic acids by synthesis of nucleic acids which correspond to the recombined sequences, e.g., in concert with oligonucleotide synthesis/ gene reassembly techniques. Any of the preceding general recombination formats can be practiced in a reiterative fashion to generate a more diverse set of recombinant nucleic acids, which are optionally screened using the purification and MS methods provided herein.

The above references provide these and other basic recombination formats as well as many modifications of these formats. Regardless of the format which is used, the nucleic acids of the invention can be recombined (with each other or with related (or even unrelated) to produce a diverse set of recombinant nucleic acids, including, e.g., sets of homologous nucleic acids.

DNA shuffling provides a robust, widely applicable, means of generating diversity useful for the engineering of proteins, pathways, cells and organisms with improved characteristics. In addition to the basic formats described above, it is sometimes desirable to combine shuffling methodologies with other techniques for generating diversity. In conjunction with (or separately from) shuffling methods, a variety of diversity generation methods can be practiced and the results (i.e., diverse populations of nucleic acids) screened for in the systems of the invention. Additional diversity can be introduced by methods which result in the alteration of individual nucleotides or groups of contiguous or non-contiguous nucleotides, i.e., mutagenesis methods. Mutagenesis methods include, for example, recombination (PCT/US98/05223; Publ. No. WO98/42727); oligonucleotide-directed mutagenesis (for review see, Smith, Ann. Rev. Genet. 19: 423-462 (1985); Botstein and Shortle, Science 229: 1193-1201 (1985); Carter. Biochem. J. 237: 1-7 (1986); Kunkel. "The efficiency of oligonucleotide directed mutagenesis" in Nucleic acids & Molecular Biology, Eckstein and Lilley, eds., Spπnger Verlag, Berlin (1987)). Included among these methods are oligonucleotide - directed mutagenesis (Zoller and Smith, Nucl. Acids Res. 10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983), and Methods in Enzymol. 154: 329-350 (1987)) phosphothioate-modified DNA mutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749- 8764 (1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl. Acids Res. 16:791- 802 (1988); Sayers et al., Nucl. Acids Res. 16: 803-814 (1988)), mutagenesis using uracil-containmg templates (Kunkel, Proc. Nat'l Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al.. Methods in Enzymol. 154:367-382)); mutagenesis using gapped duplex

DNA (Kiamer et al., Nucl Acids Res. 12- 9441-9456 (1984); Kramer and Fπtz, Methods in Enzymol 154:350-367 (1987); Kramer et al.. Nucl. Acids Res. 16. 7207 (1988)); and Fntz et al., Nucl. Acids Res 16: 6987-6999 (1988)). Additional suitable methods include point mismatch repair (Kramer et al., Cell 38: 879-887 (1984)), mutagenesis using repair- deficient host strains (Carter et al., Nucl. Acids Res. 13: 4431-4443 (1985); Carter,

Methods in Enzymol. 154: 382-403 (1987)), deletion mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res. 14: 5115 (1986)), restπction-selection and restnction- puπfication (Wells et al., Phil. Trans. R. Soc Lond. A 317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar et al., Science 223: 1299-1301 (1984); Sakamar and Khorana. Nucl. Acids Res 14: 6361-6372 (1988); Wells et al., Gene 34:315-323 (1985); and Grundstrόm et al.. Nucl Acids Res. 13: 3305-3316 (1985). Kits for mutagenesis are commercially available (e g., Bio-Rad, Amersham International, Anglian Biotechnology).

Following recombination, any nucleic acids which are produced are optionally selected for a desired activity. In the context of the present invention, this can include testing for and identifying any activity that can be detected in an automatable format, by any of the assays in the art. A vanety of related (or even unrelated) properties can be assayed using any available assay and then screened, e.g., using high throughput MS.

In addition, any of the descnbed shuffling techniques can be used in conjunction with procedures which introduce additional diversity into a genome or library. Example methods are descπbed in Schellenberger U.S. Patent No. 5,756,316, descnbing chimenc nucleic acid multimers. and in U.S Patent No 5,965,408 descπbing chain termination methods of diversity generation In addition, diversity can be further

~> increased by using methods which are not homology based. For example, incremental truncation for the creation of hybπd enzymes (ITCHY) descnbed in Ostermeier et al. (1999) "A combinatonal approach to hybnd enzymes independent of DNA homology" Nature Biotech 17:1205, can be used to generate an initial recombinant library which serves as a substrate for one or more rounds of in vitro or in vivo shuffling methods. Methods for generating and using multispecies expression libraries have been descπbed, e.g., in U.S. Patent Nos. 5,783,431; 5,824,485.

Any of these diversity generating methods can be combined with each other or with shuffling reactions, in any combination selected by the user, to produce nucleic acid diversity, which may be screened for using any available screening method. For example, a library of diverse nucleic acids is optionally expressed and the components of interest puπfied and screened by high throughput MS as descnbed herein

Cell growth plates The cell growth plates of the invention are optionally 1536, 384 or 96-well microtiter plates, or the like. For example cell colonies containing gene hbranes are picked directly from transformation plates into 1536, 384 or 96-well microtiter plates containing appropπate growth media using, for example, a Q-bot from Genetix. The maximum speed of the Q-bot is about 4000 colonies per hour. The microtiter plates are typically incubated in a plate shaker for cell growth, e.g., typically for 1 day to about 2 weeks depending on the organism. Media and cell growth conditions are appropnate to the particular cells which are incubated. The cell growth plate is also used for product generation when, for example, enzyme reactions are being studied. Products of reactions between enzymes and substrates are of interest when evolving new functional enzymes These products and or the reactants should be analyzed in a high-throughput method so that many members of the enzyme gene library can be analyzed in a short penod of time. To allow high- throughput measurement of the products and reactants, the products are optionally generated as part of the automated system of the invention Therefore, any product generation steps that must be undertaken in the assay are optionally performed on the cell growth plate After generation of products, the samples, e.g., the products and/or reactants, are optionally puπfied for injection into a mass spectrometer for analysis Off-line punfication system

The off-line parallel puπfication system of the invention allows high- throughput mass spectrometry analysis because it allows the samples to be puπfied in a system that is not sequentially tied to and slowing down the mass spectrometry analysis The system allows for off-line parallel punfication of the products and/or reactants with no time-consuming column separation

The off-line parallel punfication of the invention is performed as part of the product generation on the cell growth plate In this way the system allows all samples to be sufficiently punfied for mass spectrometry analysis without a column separation that is performed sequentially and in-line with the mass spectrometer To do this the system provides a chemical puπfication step that is selected based on the type of sample, e g , reactant and/or product, analyzed Furthermore, this chemical puπfication step can be performed in the wells of the cell growth plate in the off-line system of the invention For example, the off-line chemical punfication step optionally compπses the use of a different or additional buffer when generating the products and/or reactants of interest Alternatively, the off-line parallel puπfication system compnses the use of an ion exchange resm when generating the reactants and/or products of interest By thus prepaπng the sample as it is produced, the system of the invention takes no additional time for puπfying and/or separating the components to be analyzed Alternatively, the punfication system compnses a component reactor, e g , an enzyme reactor, that produces puπfied products for direct injection into a mass spectrometer A component reactor, as used herein, refers to a solid support which is used to remove components of interest from a cell lysate or to remove a cell lysate from the components of interest, e g , by attaching the components to the solid support Components of interest, include, but are not limited to, nucleic acids, polynucleotides, proteins, polypeptides, enzymes, carbohydrates, lipids, and the like For example, proteins, enzymes, peptides, or the like that have been tagged, e g , by fusing a sequence for a specific tag to the gene that encodes, e g , the enzyme, peptide, or protein, are optionally puπfied and immobilized on the solid support, e g , m a specific and stable manner, thus forming, e g , an enzyme reactor Typically, the enzymes, proteins, or peptides are removed from a cell lysate by binding the tagged enzymes to a tag binding moiety immobilized on the solid support For example enzymes or other proteins are expressed in cells and the cells are lysed , e g . using EDTA, lysozyme, DTT, PMBS heat, sonication, or the like If secreted proteins are the component of interest, no lysis is necessary Other library components are also optionally tagged with a molecule that will bind the solid support. For example, biotin is optionally added chemically or enzymatically to any library component of interest, e.g., a nucleic acid, carbohydrate or small organic molecule.

The tagged components are then exposed to a tag binding matnx or solid support compnsing a tag binding moiety. Examples of tag binding molecules and corresponding tags are provided below. The tag binding matnx or solid support typically compπses a tag binding moiety, e.g., a molecule that binds to the specific tag on the enzyme, and a solid matnx matenal Optional solid supports include, but are not limited to, dispensable beads or particles, e.g., agarose, polystyrene, or magnetic beads, membranes, microwell plates or pins. The tagged enzymes or proteins bind to the tag binding moiety on the solid support. The unbound matenal is either dispensed or centπfuged or sucked away, e.g., in the case of beads or membranes. Magnetic beads are optionally separated from the unbound fraction by magnets, e.g., that remove the beads and the tagged enzymes from the cell lysate. Pms are typically lifted in and out of the lysate wells, e.g., in the cell growth plate. The use of pms optionally provides especially high throughput because the puπfication takes so little time. Washing is optionally performed after remo al of the unbound matenal, in an analogous fashion. The solid support is washed with, e.g., a buffer, before performing reactions.

The tagged component immobilized on the solid support, e.g., in a puπfied and stable format, thereby provides a reactor, e g., an enzyme reactor Reactions are optionallv earned out on the solid support and the tagged components, e.g., tagged enzymes, aie easily removed after the reaction, e.g., by lifting the set of pins, to which the tagged components are bound, out of a reaction well. The removal of the tagged components leaves behind a punfied product, e.g., that is optionally injected directly into a mass spectrometer, IR or NMR spectrometer, or the like without further punfication or decontamination Alternative methods of detection of the results include measurement of chromogemc or fluorogenic substrates and/or products One extremely stable interaction that is optionally used to provide a reactor as descnbed above utilizes the binding of biotin to avidm or biotm to streptavidin Avidin and streptavidin are optionally immobilized on a vanety of solid supports available from a variety of suppliers, e g , magnetic beads, agarose beads, or membranes An enzyme is typically biotinylated in vivo by genetically fusing a special peptide tag to the N- or C- terminus of the enzyme while expressing the protein. See, e.g., Schatz (1993) Biotechnology 11, 1138-1143. The biotin-holoenzyme ligase recognizes those N- or C- terminal peptides as substrates and biotmylates a lysine residue in that peptide. The level of expression of these new substrates for the biotm-holoenzyme ligase is so high typically that not all molecules are biotinylated. Overexpression of the birA gene and addition of small amounts of biotm to the expression medium circumvents this problem. See, e.g., Smith et al. (1998) Nucleic Acids Res. 26, 1414-1420. Because of overexpression of the recombinant enzyme the amount of BCCP bound in the reactor is neglected or BCCP knockouts are optionally constructed for expression of the enzyme bio-tag fusions Many vaπations on the theme of punfication and immobilization of components of interest, e.g., enzymes, proteins, nucleic acids, or the like, will be evident upon further review by those of skill of the art.

Additional pairs of compounds useful for tagging include, but are not limited to, biotin and streptavidin, biotin and avidin, maltose binding protein and amylose; His-Tag Ohgo-his at the N- or C-termmus using immobilized metal chelate chromatography with NTA, IDA, TED, and the like as chelators; glutathione-S- transferase and reduced glutathione; strep-tag short artificial streptavidin binding tag and streptavidin, epitope tags, such as E-tag, myc-tag, HAG-tag, His-tag, and the like with monoclonal antibodies; chitm binding domain and chitin; S-tag and RNAse minus S- peptide mutant; cellulose binding proteins with cellulose domains; thioredoxin and DsbA with Thiobond; hexa-arginine poly-cation-tag with a polyanion column matenal, IGg and other IGg denved peptides with ProteinA or ProteinG minimized peptides; calmoduhn binding peptide with calmoduhn; and histactophilm with IMAC (immobilized metal chelate chromatography).

In one embodiment, a library of genes is provided, which genes encode one or more tagged enzymes. For example, a sequence for biotin is fused to an enzyme sequence to express a tagged enzyme, e g., in cells. The cells are optionally lysed and the enzymes are typically bound to a tag-bindmg moiety on a solid support, e g., a reactor The enzymes are then optionally removed and reacted with substrates, e.g., punfied substrates. The products produced in this manner are then pure enough for analysis, e.g., by mass spectroscopy or another high throughput system Alternatively, the enzymes are reacted with substrates in the cell lysate and then removed In another embodiment, the component of interest is a secreted protein. In this case, the protein is optionally removed from the cell supernatant, e.g , using a solid support reactor as descnbed herein, for further reaction or analysis. In addition, the cell supernatant is optionally removed for use in further reactions. The reactor as descnbed above is optionally used multiple times, e.g., using the same or different substrates or reaction conditions, because it is optionally removed from the reaction upon completion, e.g., washed, and reused. This is especially useful when enzyme libranes are screened for novel activities and matching substrates are identified The reactors or solid supports of the present invention enable the use of puπfied enzymes, e.g., in activity assays, and results in a reusable system that is optionally used with multiple different substrates at different times, thereby providing an enzyme reactor, e.g., for chemical processing and engineeπng. Alternatively, the reactor is used with multiple different substrates at the same time because the reacted sample does not have to be puπfied before injection into a mass spectrometer Additional details regarding solid support reactors is found in USSN 60/148,848, "Evolution and Use of Enzymes for Combinatonal and Medicinal Chemistry," by Liu et al., filed August 12, 1999.

Autosampler

An autosampler is coupled with the apparatus of the invention to transport samples between the cell growth plate, where cells are grown and reactants and/or products of interest are generated and punfied, to the mass spectrometer for injection and analysis. Autosamplers can be purchased from standard laboratory equipment suppliers such as Gilson and CTC Analytics Such samplers function at rates of about 10 seconds/sample to about 1 mm/sample

In addition, robotic sampler handlers are optionally used to pick cell colonies into the cell growth plate and add reagents in the off-line parallel punfication system For the generation of common arrangements involving fluid transfer to or from microtiter plates, a fluid handling station is used. Such robotic handlers include but are not limited to those produced by Beckman instruments and Genetix (e.g., the Q-bot). In addition, several "off the shelf fluid handling stations for performing such transfers are commercially available, including e g.. the Zymate systems from Zymark Corporation (Zymark Center, Hopkinton, MA; http://www.zymark.com/) and other stations which utilize automatic pipettors, e.g., in conjunction with the robotics for plate movement (e.g., the ORCA® robot, which is used in a vanety of laboratory systems available, e.g., from Beckman Coulter, Inc. (Fullerton, CA).

Robotic sampler handlers are also optionally used to remove enzymes from a cell growth plate or enzyme reactor as descπbed above. For example, a robotic handler is optionally used to lift a set of pins from a reaction well or to position a magnet to lift a set of magnetic beads from a cell growth beads, e.g., beads compπsing a tagged enzyme.

Mass spectrometer

A vanety of mass spectrometer instruments are commercially available For example, Micromass (U.K.) produces a vanety of suitable instruments such as the Quattro LC (a compact tπple stage quadrupole system optimized e.g., for API LC-MS- MS) which utilizes a dual stage orthogonal "Z" spray sampling technique. Other suitable tnple stage quadrupole mass spectrometers (e.g., the "TSQ" spectrometer) are produced by the Fmnigan Corporation.

II. Transforming cells In one embodiment of the present invention a cell is provided that has been transformed with a plasmid containing one or more members of a library of related gene sequences. The library of related gene sequences is optionally created by a general method for recursive sequence recombination. For example, the method can begin with a gene encoding an enzyme or enzyme subunit and evolved for the ability to act on a new substrate, or for enhanced catalytic properties with an old substrate, either alone or in combination with other genes in a multistep pathway.

The term "gene" is used herein broadly to refer to any segment or sequence of DNA associated with a biological function. Genes are optionally obtained from a vanety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. The ability to use a new substrate can be assayed in some instances by the ability to grow on a substrate as a nutnent source. In other circumstances such ability can be assayed by decreased toxicity of a substrate for a host cell, hence allowing the host to grow in the presence of that substrate Biosynthesis of new compounds, such as antibiotics, can be assayed similarly by growth of an indicator organism in the presence of the host expressing the evolved genes For example, when an indicator organism used in an overlay of the host expressing the evolved gene(s), wherein the indicator organism is sensitive or expected to be sensitive to the desired antibiotic, growth of the indicator organism would be inhibited in a zone around the host cell or colony expressing the evolved gene(s).

The library can vary widely in size from 10 to more than 10⁵, 10⁹, 10¹² members or mote In some embodiments, the starting segments and the recombinant hbranes generated will include full length coding sequences and any essential regulatory sequences such as a promoter and polyadenylation sequence, for enhanced expression In othei embodiments, the recombinant DNA segments in the library can be inserted into a common vector providing sequences necessary for expression before performing screening or selection. A library containing related genes that encode enzymes is optionally produced, e.g., by recombination of a plurality of related genes The library is optionally an in vitro set of molecules or contained in a phage, cell or the like. In other embodiments, the library compnses a library of enzyme genes that have been fused to provide tagged enzymes. The library is then screened, for example by the high throughput mass spectrometry of the present invention, e g., to detect an improved enzyme or a desired product

Cells are then transfected or transformed with one or more of the above librarv members using standard technology well known to those of skill in the art Basic texts disclosing the geneial methods of use in this invention include Sambrook, Ausubel and Bergei . all supra

III. Growing cells

In general, any type of cell is optionally used as a recipient of evolved genes Cells of particular interest include many bactenal cell types, both gram-negative and gram-positive, such as Rhodococcus, Streptomyces, Actinomycetes, Corynebactenum, Penicilhum, Bacillus. Escheπchia coli, Pseudomonas, Salmonella, and Erwinia Cells of interest also include eukaryotic cells, particularly mammalian cells (e.g , mouse, hamster, pnmate, human), both cell lines and pnmary cultures Such cells include stem cells, including embryonic stem cells, zygotes, fibroblasts, lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts (NIH3T3), kidney, liver, muscle, and skm cells Other eukaryotic cells of interest include plant cells, such as maize, πce, wheat, cotton, soybean, sugarcane, tobacco, and arabidopsis, fish, algae, fungi (Penicilhum, Fusaπum, Aspergillus, Podospora, Neurospora), insects, yeasts (Picchia and Saccharomyces), and the like

The choice of host will depend on a number of factors, depending on the intended use of the engineered host, including pathogenicity, substrate range, environmental hardiness, presence of key intermediates, ease of genetic manipulation, and likelihood of promiscuous transfer of genetic information to other organisms Particulai antageous hosts are E coli, lactobacilh, Streptomyces, Actinomycetes, and filamentous fungi

In the piesent invention, single colonies of cells are picked directly from transformation plates into 1536, 384 or 96-well microtiter plates or cell growth plates with appropnate growth media, such as LB, using, e g , a Q-bot from Genetix The maximum speed of the Q-bot is about 4000 colonies per hour The microtiter plates are typically incubated in a special plate shaker for cell growth

Each single colony is grown up to uniformity (this is optionally achieved by automating the piocess, e g , inoculum size and culture conditions, and providing temperature and humiditv controlled incubators) in a single microtiter well on the cell growth plate In one aspect library members, e g , cells, viral plaques, spores or the like, are separated on solid media to produce individual colonies or plaques Using an automated colon} picker (e g , the Q-bot, Genetix, U K ), colonies are identified, picked and 10 000 different mutants inoculated into 96 or 384 well microtiter dishes, that optionally contain about 2 oi 3 glass balls/well, e g , 3 mm glass balls The Q-bot does not pick an entire colony, but rather inserts a pin through the center of the colony and exits with a small sampling of cells, (or myceha) and spores (or viruses in plaque applications) The time the pin is in the colony, the number of dips to inoculate the culture medium, and the time the p is in the medium each affect inoculum size, and each can be controlled and optimized The uniform process of the Q-bot decreases human handling error and increases the rate of establishing cultures (roughly 10,000/4 hours) These cultures are then shaken in a temperature and humidity controlled incubator The glass balls in the microtitre plates, if used, act to promote uniform aeration of cells and the dispersal of mycehal fragments similar to the blades of a fermenter. For example, Streptomyces tend to clump together dunng culture, but remain relatively homogenous in culture if glass beads are added duπng mixing.

IV. Generating cell components of interest. In one embodiment of the invention, one or more cells or clones, or a cell colony is then treated in one of several ways to initiate product formation from, for example, enzyme reaction pathways of the cells. If enzyme or protein expression was purposely suppressed duπng cell growth, expression can be induced by removing the suppressor or by adding activator molecules. Cells that contain active enzymes can be lysed and treated with permeabihzing agents to enable bulky and/or strongly ionic substrates to penetrate cell walls. This is especially cπtical for gram-negative bactena like E. coli. Some cell components, e.g., enzymes or proteins, are secreted into the media (i.e., if expressed in gram-positive bactena like bacillus with an appropπate signal sequence) in which case no extra treatment is necessary.

In some cases, the components of interest, e.g., enzymes, proteins, or nucleic acids, are optionally puπfied on a punfication resin. Reagents, e.g., enzyme substrates, are added to the puπfied components of interest, thus providing puπfied components or products. The protein puπfication step eliminates a lot of sample preparation steps to follow. In some embodiments, a component of interest is puπfied using a component or enzyme reactor as descnbed above Reactions are optionally performed in such a reactor and the enzymes or components removed, e.g.. by centπfugation or magnetization, to provide a puπfied product for analysis, e.g., by MS. Initiation of product formation is optionally achieved by inoculating the bactenal culture into a different medium In all cases, the initiation of product formation is performed in a parallel 96 or 384-well format on the cell growth plate of the invention

V. Sample preparation by off-line parallel purification.

Two factors influence the quantitative detection of the analyte in a mass spectrometer First, the impuπties in the matnx can suppress or mask the signal. Second, mass spectrometers are highly sophisticated instruments that are not designed for handling crude samples Strongly ionic buffers and macromolecules like DNA or proteins m the matnx will lead to reduction in signal and in the worst case to clogging of the machine Therefore, sample cleanup is of the utmost importance

The present invention provides high-throughput methods for assays, e g , enzyme assays, with whole cells or partially or completely lysed cells Instead of a chromatographic separation step, the samples are cleaned up with extraction methods to get nd of proteins, nucleic acids, general cell junk, and debns, such as by solid phase extractions or ethanol/methanol precipitation The methods used are viable for many components, including but not limited to sugars, peptides, polynucleotides, small inorganic molecules, polyketides, beta-lactam antibiotics, tnazme deπvatives, and the

Traditionally, crude samples were cleaned on a liquid phase chromatography column pnor to introducing them into a mass spectrometer Liquid chromatography mass spectrometry (LC/MS) was probably the most common way to clean crude samples However, each column run is time consuming (10-30 minutes per sample), limiting the speed of the analysis

Flow injection analysis (FIA) is generally only limited by the speed of the autosampler, which ranges from about 30 to about 40 seconds per injection and getting faster as new models of autosamplers are manufactured Sample preparation for FIA takes into account every step from cell growth to reaction or product formation to introduction into the mass spectrometer One important factor is to adjust reaction conditions for product formation to accommodate MS compatibility as much as possible without compromising screening quality Reaction or assay conditions are as close as possible to the real envnonmental conditions under which the products and/or reactants of interest will be used For example, when enzyme pathways are at issue, the reaction conditions are as close as possible to the conditions under which the enzymes are used, e g , to ensure that directed evolution of the enzymes leads to the desired mutant vanants For example, production media of polyketides in Streptomyces contains inexpensive components typically used in fermentors In general, the conditions chosen are project dependent One skilled in the art will understand both the relevant biology and the appropπate form of analytic measurement, and thus can select reaction conditions Once these conditions are defined, further sample cleanup is often unnecessary Effective sample cleanup is dependent on the physico-chemical nature of the analyte as well as the matπx. However, all sample cleanup is optionally done on the cell growth plate in an off-line system in parallel with the MS analysis.

Several strategies are optionally employed to accommodate a vanety of different analytes in biological matnces. For example, small molecule substrates of interest with hydrophobic moieties like atrazine can penetrate into E.coh. cytoplasma without lysis of the cells. Using a volatile buffer like ammonium acetate allows a very simple cleanup. In one aspect, cells are centnfuged and the buffer added to the supernatant. Substrate is added and cell debns is filtered off in a parallel fashion.

In another embodiment, small inorganic ion analytes are often masked by coordinating metal ions. Reaction buffers for enzyme reactions with these analytes are optionally chosen to reduce the concentration of ionic species to a minimum, and the remaining cations are removed by cationic exchange resin

In another aspect, an ohgosacchande is the analyte of interest. Ohgosacchaπdes are cleaned up by removing all ionic species using a mixed ion exchange resin. E. coh. cells are partially lysed, and all cell debns, DNA and protein impuπties are precipitated with ethanol and removed by filtration.

In another aspect, the product or reactant of interest is a hydrophobic molecule, such as a polyketide. Hydrophobic molecules are extracted from the aqueous phase by organic solvents that also remove ionic impuπties. In another aspect, cells are lysed and enzymes or other components of interest, such as peptides, nucleic acids, and the like, are attached to a solid support, e.g., an enzyme reactor as descπbed above. The enzymes are optionally contacted by substrates on the solid support and then removed from the reaction upon completion, resulting in products that are sufficiently pure to be used directly in mass spectrometry without further puπfication such as liquid chromatography

Another example of offline sample preparation compπses 96-well parallel solid phase extraction (SPE), in which a plurality of samples, e.g., about 96 or about 384 samples, are simultaneously loaded on to a solid phase extraction plate, e.g., a 96-well plate, e.g , from Waters Corp Milford MA. Unwanted components are washed from the plate, e.g , using one or more buffers or solvents Components of interest are retained inside a column of the SPE plate and optionally eluted by a relative high strength solvent into a corresponding microwell plate, e.g., a 96- ell plate Samples prepared in this manner are sufficiently punfied for injection into a mass spectrometer. In all of the above cases, sample preparation was adopted to process 96 samples in parallel in a highly automated fashion, thereby ensuπng that screening was only rate dependent on the speed of sequential analysis of the mass spectrometer. Additionally, these adjustments to growing conditions or generation solvents provide sufficient purification of the sample for injection into a mass spectrometer.

VI. Mass spectrometry

Mass spectrometry is a genenc method that allows detection of a large vanety of different small molecule metabolites. Ionspray and electrospray mass spectrometr) ha e been used n many different fields for the analysis of organic compounds and for charactenzation of biomacromolecules. It is however, usually coupled to a separation technique, such as high performance liquid chromatography or capillary zone electrophoresis, which is performed in-line with the mass spectrometry analysis This slows down the rate of mass spectrometry and limits its use as a high- throughput method. For a general discussion of mass spectrometry theory and techniques, see, e.g., Kirk-Othmer Encyclopedia of Chemical Technology, Volume 15, Forth Edition, pages 1071-1094, and all references therein. See, also. Mass Spectrometry for Biotechnology, G Siuzdak, Academic Press, San Diego, CA, 1996; Electrospray lonization Mass Spectrometry: Fundamentals, Instrumentation, and Applications, R. Cole (Ed.), Wiley and Sons, 1997; Mass Spectrometry for Chemists and Biochemists, Johnstone et al.. Cambndge University Press, 1996; Mass Spectrometry: Principles and Applications, Hoffman ct al., Wiley and Sons, 1996; Quadrupole Mass Spectrometry and its Applications, Dawson (ed.), Spnnger Verlag, 1995; and Advances in Mass Spectrometry, Karjala en et al. (eds.), Elsevier Science, 1998). Electrospray methods are used instead of gas chromatography procedures because no pπor denvatization is required to inject the sample Flow injection analysis methods (FIA) with lonspray-ionization and tandem mass spectrometry further the ability of the present invention to perform high-throughput mass spectrometry analysis The ionspray method allows the samples to be injected without pπor denvatization and the tandem mass spectrometry (MS-MS) allows extremely high efficiency in the analysis Therefore, no column separation is needed

Electrospray lonization is a very mild lonization method that allows detection of molecules that are polar and large which are typically difficult to detect in GC-MS without pπor denvatization Modern electrospray mass spectrometers detect samples in femtomole quantities Since a couple of microliters are injected, samples are optionally injected in nanomolar concentrations, attomolar concentrations or lower Quantitation is very reproducible with standard errors ranging from 2% - 5% Tandem mass spectrometry uses the fragmentation of precursor ions to fragment ions within a tπple quadrupole MS The separation of compounds with different molecular weights occurs in the first quadrupole by the selection of a precursor ion The identification is performed by the isolation of a fragment ion after collision induced dissociation of the precursor ion in the second quadrupole Reviews of this technique can be found in Kenneth, L et al (1988) "Techniques and Applications of Tandem Mass Spectrometry" VCH publishers, Inc

Tnple quadrupole mass spectrometers allow MS/MS analysis of samples For example, a tπple quadrupole mass spectrometer with electrospray and atmosphenc pressure chemical lonization sources, such as a Finnigan TSQ 7000, is optionally used The machine is optionally set to allow one particular parent ion through the first quadrupole which undergoes fragmentation reactions with an inert gas The most prominent daughter ion can then be singled out in the third quadrupole This method creates two checkpoints for analyte identification The particle must have the correct molecular mass to charge ratio of both parent and daughter ion Tandem mass spectrometry thus leads to higher specificity and often also to higher signal to noise ratio It also introduces further separation by distinguishing analyte from impuπties with same mass to charge ratio

Other techniques of use in the present invention include, but are not limited to, neutral loss and parent ion scanning Neutral loss is a method of mass spectrometry scanning in which all compounds that lose a neutral molecular fragment, l e , a specific neutral fragment, duπng collision induced dissociation (CID) are detected Parent ion mode detects all compounds that produce a common daughter ion fragment dunng CID These techniques are optionally used, e g to quantitate the amount of product and starting matenal simultaneously For systems in which the expected product is not known, e g , a standard is not available, the neutral loss and/or parent ion method allows backtracking or deconv olution based on fragmentation patterns to determine the structuie and/or identity of the starting matenal For example, the parent mass is determined based on the various fiagments pioduced This is especially useful for detecting novel enzyme activity when the product of the enzyme reaction is not known, but is predictable.

In neutral loss methods, components of interest are allowed to pass the first quadrupole, e.g., in a tπple quadrupole spectrometer, one at a time by scanning the first quadrupole in a certain mass range. The components, e.g., ions, are fragmented in the second mass filter by CID. If a specific neutral fragment is lost from a parent ion dunng the CID process, a daughter ion is formed, which daughter ion has a mass equal to the mass of the parent ion minus the mass of the neutral molecule. The daughter ion will pass the third filter and be detected. In this way, any ion or components losing a neutral fragment, e.g., a constant neutral fragment (No) duπng the CID process in the second quadrupole is optionally detected by scanning the first and third quadrupoles simultaneously with a mass offset equal to the mass N₀.

In the parent ion method, ions or components of interest are allowed to pass the first quadrupole one at a time. These ions are fragmented in a second mass filter by CID. The third quadrupole is then set to allow only specific ions to pass. Thus, all components, e.g., products or reactants, producing a specific fragment ion as set in the second quadrupole are detected by scanning the first quadrupole mass filters in the range of interest while setting the third quadrupole mass filter on that specific ion.

The speed of the analysis is limited only by the motonc movements of the autosampler used to inject the samples, such as a CTC Analytics and Gilson, Inc.

Middleton, Wisconsin. The speed for example, is optionally set at 30 seconds without wash and 40 seconds with wash of the injection needle Such a sampling rate allows 2880 samples per day to be analyzed by MS if automated overnight runs are used. Thus, an entire 96-well microtitre plate of samples is run in less than an hour. Preferably, the speed of the autosampler is set at about 15 seconds per sample, allowing about 5000 samples to be screened in one day or about 200 per hour Autosampler companies are currently working to increase the throughput to one plate in 10 minutes including the washing, which would then allow for about 8500 MS samples to be run in a day.

With the above mass spectrometry system and the off-line puπfication of the samples of interest, sample introduction to the machine is typically the most rate controlling step. The present invention provides a high-throughput screening method for use with mass spectrometry by providing faster sample puπfication steps The rate of screening is optionally increased beyond that of the autosampler by using pooling strategies, e.g., with the neutral loss, parent ion screening methods descπbed above. A plurality of samples, e.g., similar or related samples, are optionally pooled or mixed together and injected into the mass spectrometer as one sample. The data is then deconvoluted to provide identification or analysis for each of the pooled samples. For example, five different substrates are reacted with an enzyme and the results pooled. The five different substrates may produce five related or similar compounds as products. The products are pooled and analyzed. Neutral loss analysis is then optionally performed on the pooled samples. For example, a specified neutral fragment is removed from all the samples, e.g., in the second quadrupole, and then the data is deconvoluted to determine the parent ion as detected in the first quadrupole to provide results for each of the individual samples

About 2 to about 1000 samples are optionally pooled, thus increasing the throughput to about 400 samples per hour to about 240,000 samples per hour, e.g., at one injection every 15 seconds. If the speed of the autosampler is increased beyond 1 injection every 15 seconds, even greater screening rates are obtained. Optionally, more samples are pooled to provide greater screening rates. Typically about 5 to about 500 samples are pooled. More typically about 5 to about 100 samples are pooled or about 10 to about 20 samples At 15 seconds per injection MS rate, the screening rate for pools compπsing 100 samples each is about 24,000 samples per hour or about 576,000 samples per day. Typically at least about 500 samples, e.g., cell colonies or library members, at least about 1000 samples, at least about 5000 samples, at least about 10,000 samples, at least about 25,000 samples, or at least about 100,000 samples are screened, e.g., for presence, absence, or activity of one or more component, e.g.. non-column-separated components, in less than an hour In other words, at least about 1000 samples, at least about 25,000 samples, at least about 100,000 samples, or at least about 500,000 samples or more are screened in about 1 day

VII. Kits The system descnbed herein is optionally packaged to include many, if not all, of the necessary reagents for performing the preferred function of high throughput mass spectrometry using an off-line parallel punfication system Such kits also typically include appropriate containers and instructions for using the devices and reagents, and in cases where reagents are not predisposed in the devices themselves, with appropriate instructions for introducing the reagents into the cell growth plate or mass spectrometer of the device. Such kits typically include a cell growth plate with necessary reagents predisposed in the wells or separately packaged. Generally, such reagents are provided in a stabilized form, so as to prevent degradation or other loss during prolonged storage, e.g., from leakage. A number of stabilizing processes are widely used for reagents that are to be stored, such as the inclusion of chemical stabilizers (i.e., enzymatic inhibitors, microcides/bacteriostats, anticoagulants), the physical stabilization of the material, e.g., through immobilization on a solid support, entrapment in a matrix (i.e., a gel), lyophilization, or the like.

The discussion above is generally applicable to the aspects and embodiments of the invention described above. Moreover, modifications can be made to the method and apparatus described herein without departing from the spirit and scope of the invention as claimed, and the invention can be put to a number of different uses including the following.

The use of a mass spectrometry system to perform high-throughput screening of enzyme reaction pathways.

The use of a mass spectrometry system as described herein to perform high throughput screening of reactants and or products of enzyme reactions. The use of a mass spectrometry system as described herein to perform high throughput screening of nucleic acid library.

The use of an off-line parallel purification as described herein to perform high throughput mass spectrometry screening.

The use of an off-line parallel purification as described herein to perform high throughput mass spectrometry screening of enzyme reaction pathways.

An assay utilizing a use of the mass spectrometry system described herein.

EXAMPLES

The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters which could be changed or modified to yield essentially similar results. Example 1: HTP-MS-- Atrazine Production

Atrazine is a member of the family of tnazine-deπved herbicides. Bactena from sites contaminated with this widely used herbicide were isolated that were able to metabolize and degrade atrazine. A Pseudomonas strain was found to contain a gene encoding atzA, a 473 ammo-acid protein that catalyzes the transformation of atrazine to hydroxy atrazine, the first step in the degradation pathway of atrazine (see also, De Souza, M., Sadowsky, M. J. & Wackett, L. P.: Atrazine Chlorohydrolase from Pseudomonas sp. Strain ADP: Gene Sequence, Enzyme Punfication, and Protein Characteπzation. J. Bactenol. 178:4894-4900 (1996)) (see also, Figure 1) The biochemical degradation of atrazine by the Pseudomanas strain sp.

ADP is an environmentally sound way of cleaning up contaminated sites. In order to be economically competitive, an increase of the wild type activity of the atzA gene was desirable. The atzA gene was cloned into a pUC-denved vector under the control of a lac promoter, and the vector transformed into E. coli TGI The expression of the gene was repressed in presence of glucose and induced with isopropyl thiogalactose (IPTG). The plasmid also contained the gene for Kanamycin resistance.

Library construction and cell growth

The atzA gene was shuffled, and the initial library plated onto Kanamycin / 2% glucose plates A robotic colony picker (Q-bot, Genetix) picked all colonies into a microtiter plate of 96 wells containing 2XYT (100 μL) medium with kanamycin and 2% glucose per well The cells were grown in a specially designed shaker for microtiter plates (Kuehner, Switzerland) at 37 °C overnight. The saturated cultures were diluted 20- fold into 2XYT (100 μL) with Kanamycin and IPTG to initiate expression and grown again overnight at 37 °C.

Atrazine degradation

Cells were harvested by centnfugation and resuspended into 100 μL ammonium acetate (10 mM, pH 7) 5 μL of resuspended cells were transferred into a reaction well containing 100 μL of reaction buffer with atrazine (100 μM) and ammonium acetate (10 mM, pH 7). The reaction proceeded for 6 hours at room temperature under constant shaking The reaction was quenched by adding an equal volume of methanol (100 μL) The entire reaction mixture was transferred onto a filter plate and any solid cell debns and precipitates removed by filtration. The samples were injected directly into the electrospray mass spectrometer by flow injection and analyzed by tandem mass spectrometry.

MS/MS method development

A solution of 1 mM atrazine in acetonitπle was prepared and used to develop a MS/MS method on a tnple quadruple mass spectrometer (Fmnigan TSQ 7000). The mobile phase was acetonitnle. The collision energy was set to -20eV.

The transition of m/z = 216 (parent ion) to m z =174 (daughter ion) was monitored (see Figure 2, panels A and B).

MS/MS analysis

Figure 3 shows results of a typical plate of 96 samples. Each row contains twelve different reaction conditions with vaπous mutants that were reproduced across the eight columns. A penodical pattern of 12 peaks is clearly visible. Bactenal cell growth, reaction and sample workup were performed in parallel fashion as descπbed above.

Matenal s

Ammonium acetate, glucose and IPTG and Kanamycin were purchased from Sigma. 2XYT medium was prepared according to Sambrook, J., Fntsch, E. F. & Maniatis, T.: Molecular Cloning. A Laboratory Manual Cold Spnng Harbor Laboratory Press 1989. Microtiter plates for cell growth were stenle flat-bottom shallow well plates from Nunc. Reactions were performed in 96 well Costar polystyrene V-bottom plates. Filter plates were from Milhpore HV 0 45 μm Durapore

Example 2: High throughput screening for directed evolution of enzymes and pathways using mass spectrometry

High throughput chemical screening of enzyme reactions involves quantitative detection of substrate(s) and product(s) The most universal detection method to date is mass spectrometry w hich allow s identification of a particular organic molecule, e.g., based on mass to charge ratio Electrospray lonization is a mild method of transfemng charged polar organic molecules into the gas phase and applicable for most biologically relevant organic molecules DNA shuffling technology is used to create a library of related gene sequences that encode enzyme(s) that catalyze chemical reactions. The library of related gene sequences are, e.g., on plasmids that are transformed into bactena. Typically, a single bactenal clone carnes a unique gene sequence representing a unique vaπant of a particular enzyme or enzyme pathway, although many other shuffling formats are also suitable.

Figure 4 descnbes the steps that are typically used to monitor enzyme reactions by mass spectrometry from a single bactenal colony.

A: Single colony picking and growth in 96 or 384 well format

Single colonies are picked directly from transformation plates into 384 or 96-well microtiter plates with appropnate growth media using the Q-bot from Genetix The maximum speed of the Q-bot is about 4000 colonies per hour. The microtiter plates are incubated in a special plate shaker for cell growth.

B: Product generation using whole cells, cell lysis or puπfied enzymes Each single colony was grown up in a single microtiter well to uniformity and then treated in several different ways to initiate product formation. If enzyme expression is purposely suppressed dunng cell growth, which is sometimes desirable, expression can be induced by removing the suppressor or adding activator molecules

Cells that contain active enzymes are lysed or treated with permeabihzing agents to enable for bulky and/or strongly ionic substrates to penetrate cell walls. This is especially useful for gram-negative bactena like E. co . Some enzymes are secreted into the media (i.e. if expressed in gram-positive bactena like bacillus with an appropπate signal sequence) in which case no extra treatment is necessary.

In some cases, the enzyme of interest is puπfied on a puπfication resin, and the substrate added to the puπfied proteins The protein punfication step eliminates sample preparation steps noted below (e.g., see C) However, protein punfication methods are typically used for single enzyme evolutions and are not as often for pathway evolution

Initiation of product formation can also be achieved by inoculating the bactenal culture into a different medium In the above cases, initiation of product formation is performed in a parallel fashion on microtiter plates, e.g., in a 96 or 384 well format.

C: Off-line parallel punfication of analyte from biological matnx There are at least two factors which influence quantitative detection of an analyte in a mass spectrometer. The signal can be suppressed or masked by impunties in the matπx. Also, mass spectrometers are sensitive instruments that are not typically designed for handling crude samples. Strongly ionic buffers and macromolecules like DNA or proteins in the matnx can lead to a reduction in signal and in some cases to clogging of the machine. Therefore, sample cleanup is often beneficial.

Traditionally, crude samples were cleaned on a liquid phase chromatography column pnor to introducing them into the machine. Liquid chromatography combined with mass spectrometry (LC/MS) is a useful way to clean crude samples. However, each column run is time consuming, limiting the speed of sample analysis. Flow injection analysis (FIA) is typically rate dependent on the speed of the autosampler, which in current formats ranges from about 30 to about 40 seconds per injection and which is getting faster as newer models of autosamplers are manufactured.

Sample preparation for FIA takes into account steps from reaction with cells to introduction to the mass spectrometer. One factor is to adjust reaction conditions for product formation to accommodate MS compatibility without compromising screening quality Reaction conditions are typically as close as possible to the target environmental conditions under which these enzymes are used, in order to ensure that the screen is meaningful These conditions are project dependent. Once the conditions are defined, further sample cleanup is often beneficial Effective sample cleanup is dependent on the physico-chemical nature of the analyte as well as the matnx

Several strategies are used to accommodate a vanety of different analytes in vanous biological matπces. A few of these strategies are provided below

As noted above in Example 1, small molecule substrates with hydrophobic moieties like atrazine penetrate into the E. coh cytoplasm without lysis Using a volatile buffer such as ammonium acetate allowed very simple cleanup. Substrate was added and cell debns filtered off in a parallel fashion. Small inorganic ion analytes were often masked by coordinating metal ions. Reaction buffers were chosen to reduce the concentration of ionic species to a minimum, and the remaining cations removed by cationic exchange resm.

Ohgosacchande analytes were cleaned by removing all ionic species using a mixed ion exchange resm. Since the cells (E. coh) were partially lysed, cell debns, DNA and protein impunties were precipitated with ethanol and removed by filtration.

Hydrophobic molecules like polyketides were extracted from the aqueous phase by organic solvents, which also was an efficient method to remove all ionic impunties. Sample preparation was adopted to process 96 samples in parallel in a highly automated fashion, thereby ensunng that the screening rate was only dependent on the speed of sequential analysis in the mass spectrometer

D: Flow-iniection analysis on electrospray tandem mass spectrometer Tnple quadrupole mass spectrometers allow MS/MS analysis of samples.

The machine can be set to let one particular parent ion through the first quadrupole which undergoes fragmentation reactions with an inert gas. The most prominent daughter ion can then be singled out in the third quadrupole. This method creates two checkpoints for analyte identification. The particle detected has the correct molecular mass to charge ratio for both the parent and daughter ion. Tandem mass spectrometry thus leads to higher specificity and often also to a higher signal: noise ratio. It also introduced further separation by distinguishing analyte from impuπties with same mass to charge ratio.

Flow injection analysis of off-line puπfied samples using tandem mass spectrometry allowed sample analysis of about 100 samples or more in less than one hour The throughput limitations were set by the nature of sequential analysis of the mass spectrometer as opposed to parallel analysis of other detection methods (i.e. UV/VIS spectrometers) Sample introduction to the machine was the rate limiting step.

Although the foregoing invention has been descπbed in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spiπt or scope of the appended claims All patents, patent applications, and publications cited herein are incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS-

1. A method of performing high throughput mass spectrometry screening, the method compπsing-

(1) growing one or more cell; (n) punfying one or more non-column-separated component from the one or more cell, the puπfying compπsing an off-line parallel adjustment of cell growing conditions; and,

(m) performing flow- jection analysis using electrospray tandem mass spectrometry, thereby obtaining mass-to-charge ratio data and providing high throughput mass spectrometry screening of the one or more non-column-separated component.

2. The method of claim 1, wherein step (I) occurs simultaneously with step (n)

3. The method of claim 1, wherein at least about 100 cell colonies are screened for presence or activity of the one or more non-column-separated component in less than an hour

4. The method of claim 1, wherein at least about 200 cell colonies are screened for presence or activity of the one or more non-column-separated component in less than an hour

5. The method of claim 1, wherein at least about 500 cell colonies, at least about 1000 cell colonies, at least about 5000 cell colonies, at least about 10,000 cell colonies, at least about 25,000 cell colonies, or at least about 100,000 cell colonies are screened for presence or activity of the one or more non-column-separated component in less than an hour

6. The method of claim 1, wherein at least about 200 cell colonies, at least about 1000 cell colonies, at least about 25,000 cell colonies, at least about 100,000 cell colonies, or at least about 500,000 cell colonies or more are screened for the presence or activity of the one or more non-column-separated component in about 1 day

7. The method of claim 5, compπsing concurrently performing flow injection analysis on a plurality of cell colonies

8. The method of claim 7, wherein the plurality of cell colonies compπses about 2 to about 1000 cell colonies.

9. The method of claim 8, wherein the plurality of cell colonies compπses about 5 to about 500 cell colonies.

10. The method of claim 9, wherein the plurality of cell colonies compnses about 5 to about 100 cell colonies.

11. The method of claim 10, wherein the plurality of cell colonies compnses about 5 to about 20 cell colonies

12. The method of claim 1, wherein said puπfying one or more non- column-separated component compπses performing step (n) in a volatile buffer, a buffer that reduces concentration of ionic species, an ion exchange resin, or an organic solvent.

13. The method of claim 1, wherein the non-column-separated components are produced from whole cells, cell lysate, cell supernatant, or from reactions of punfied cell enzymes with added substrates

14. The method of claim 1, wherein the one or more non-column- separated component is selected from a protein, a protein binding molecule, a carbohydrate, a carbohydrate binding molecule, a product of an enzyme catalyzed reaction, a nucleic acid, and a product of a nucleic acid catalyzed reaction.

15. The method of claim 1, wherein the one or more non-column- separated component is selected from an enzyme, an enzyme substrate, and an enzyme product

16. The method of claim 1, wherein the one or more non-column- separated component is selected from a substrate with one or more hydrophobic moieties, an inorganic ion. an ohgosacchande. a hydrophobic molecule, atrazine, and a polyketide

17. The method of claim 1, wherein punfymg the one or more non- column-separated component compnses attaching the one or more non-column separated components to a solid support.

18. The method of claim 17, wherein the solid support compnses one or more magnetic beads, one or more agarose beads, one or more polystyrene beads, one or more pins, a microwell plate,or a membrane.

19. The method of claim 17, wherein the one or more non-separated column component compnses a library of enzymes, which enzymes each compnses a tag moiety, and wherein the solid support compnses a tag binding moiety

20. The method of claim 19, wherein the tag moiety compnses biotin, avidm, or streptavidin and the tag binding moiety compnses biotin, avidm, or streptavidin.

21. The method of claim 19, further compnsmg contacting the library of enzymes with one or more enzyme substrate to produce one or more product, wherein performing flow injection analysis compnses performing flow-injection analysis on the one or more product.

22. The method of claim 1, wherein the one or more non-column- separated component compπses one or more enzyme substrate and one or more product of an enzymatic reaction, the method further compnsing simultaneously quantifying the amount of the one or more product of an enzyme reaction and the one or more enzyme substrate

23. The method of claim 1, wherein performing flow injection analysis using electrospray tandem mass spectrometry compπses performing or more of: neutral loss mass spectrometry and parent ion mass spectrometry.

24. The method of claim 23, compπsing performing the neutral loss mass spectrometry or the parent ion mass spectrometry on a tnple quadrupole mass spectrometer

25. The method of claim 24, wherein performing the neutral loss mass spectrometry comprises:

(a) scanning the one or more non column-separated-component in a first quadrupole at a specified mass range, (b) fragmenting the one or more non-column-separated component in a second quadrupole by collision induced dissociation, thereby producing one or more neutral fragments and one or more daughter ion; and, (c) detecting the one or more daughter ion.

26. The method of claim 24, wherein performing the parent ion mass spectrometry compπses

(a) scanning the one or more non column-separated-component in a first quadrupole,

(b) fragmenting the one or more non-column-separated component in a second quadrupole by collision induced dissociation; and, (c) scanning a third quadrupole at a specified mass.

27. A method for monitoπng one or more product or reactant by high throughput mass spectrometry, the method compnsing.

(I) providing a cell that has been transformed with a plasmid containing one or more member of a library of related gene sequences; (n) growing a cell colony or culture from the cell,

(in) producing the one or more product or reactant from the cell colony or culture in a biological matπx, thereby producing a non-column-separated sample,

(IV) punfying the non-column separated sample from the biological matnx, the punfying compnsing an off-line parallel adjustment of the biological matnx used for producing the non-column separated sample; and,

(v) mo tonng the non-column separated sample by flow-mjection analysis using electrospray tandem mass spectrometry, thereby monitoπng the one or more product or reactant

28. The method of claim 27, wherein the products or reactants are selected from a protein, a product of a protein reaction, a nucleic acid, and a product of a nucleic acid catalyzed reaction

29. The method of claim 27, wherein the products or reactants are selected from an enzyme, and a product of an enzyme catalyzed reaction.

30. The method of claim 27, wherein step (in) occurs simultaneously

31. The method of claim 27, wherein punfying compπses alteπng or adding a buffer to the biological matnx in which the non-column-separated sample is produced, thereby producing a sample that can be injected directly into a mass spectrometer for analysis of the sample

32. The method of claim 27, wherein at least about 200 library members, at least about 1000 library members, at least about 5000 library members, at least about

10,000 library membets, at least about 25,000 library members, or at least about 100,000 library members are screened for presence or absence of products or reactants in less than about 1 hour.

33. The method of claim 27, wherein at lest about 200 library members, at least about 1000 library members, at least about 25,000 library members, at least about

100,000 library members, at least about 500,000 library members, or least about 1,000,000 samples or more are screened for the presence or absence of products or reactant^ in about 1 day

34. The method of claim 27, wherein the reaction is an enzyme reaction

35. The method of claim 27, wherein the gene sequences encode enzymes.

36. The method of claim 35, wherein the one or more product or reactant compnses an enzyme substrate and a product of an enzymatic reaction, the method further compnsing quantifying an amount of the enzyme substrate and an amount of the product of the enzymatic reaction

37. The method of claim 27, wherein the cell is a bactena

38. The method of claim 27, wherein the purifying step occurs in reaction conditions that substantially mimic environmental cellular conditions

39. The method of claim 27, wherein said punfymg compπses performing step (iv) in a volatile buffer, a buffer that reduces concentration of ionic species, an ion exchange resm, or an organic solvent.

40. The method of claim 27, wherein, the non-column-separated sample is produced using whole cells, cell lysate, cell supernatant, or a reaction of at least one puπfied cell enzyme with at least one substrate for the at least one cell enzyme.

41. The method of claim 27, wherein the non-column separated sample is selected from: a substrate with one or more hydrophobic moieties, an inorganic ion, an ohgosacchande, a hydrophobic molecule, atrazine, a hpid molecule, and a secondary metabolite

42. The method of claim 41, wherein the secondary metabolite is a polyketide.

43. The method of claim 27, step (v) compnsing performing neutral loss/parent ion mass spectrometry, thereby quantifying an amount of the one or more product or reactant

44. An apparatus for high throughput mass spectrometry screening, the apparatus compπsing:

(I) a cell growth plate for growing cell samples and reacting one or more of an enzyme, an enzyme substrate, and a enzyme product; (π) an off-line parallel puπfication system coupled to or within the cell growth plate, for punfying the samples;

(in) an automatic sampler operably coupled to the off-line parallel punfication system; and

(in) a mass spectrometer operably coupled to the automatic sampler, said automatic sampler compπsing a sample handler that transports samples from the offline parallel punfication system to the mass spectrometer for injection and analysis

45. The apparatus of claim 44. wherein the automatic sampler transports at least about 100 samples in about an hour.

46. The apparatus of claim 44, wherein the automatic sampler transports at least about 200 samples in about an hour

47. The apparatus of claim 44, wherein the automatic sampler combines two or more samples and simultaneously injects the two or more samples into the mass spectrometer

48. The apparatus of claim 47, wherein the mass spectrometer screens at least about 200 samples, at least about 1000 samples, at least about 5000 samples, at least about 10000 samples, at least about 25,000 samples, or at least about 100,000 samples in about an hour

49. The apparatus of claim 47, wherein the mass spectrometer screens at least about 200 samples, at least about 1000 samples, at least about 25000 samples, at least about 100,000 samples, at least about 500,000 samples, or at least about 1,000,000 samples in about 1 day

50. The apparatus of claim 44, wherein the rate of screening is determined by the maximum rate at which the automatic sampler transports samples between the off-line punfication system and the mass spectrometer

51. The apparatus of claim 44, wherein the offline puπfication system compπses a volatile buffer, a buffer that reduces concentration of ionic species, an ion exchan "Cge resin or an organic solvent

52. The apparatus of claim 44, wherein the offline punfication system compnses a component reactor

53. The apparatus of claim 52, wheiein the component reactor compπses an enzyme reactor

54. The apparatus of claim 52. wherein the enzyme reactor compnses a solid support for immobilizing one or more components

55. The apparatus of claim 54, wherein the one or more components comprise one or more enzyme, protein, nucleic acid, carbohydrate, hpid, sugar. oligosaccharide, peptide, polynucleotide, small organic molecule, or secondary metabolite.

56. The apparatus of claim 54, wherein the solid support comprises one or more magnetic beads, one or more agarose beads, one or more polystyrene beads, one or more pins, or a membrane.

57. The apparatus of claim 44, wherein the cell growth plate comprises a library of related genes, which genes encode proteins or enzymes, and wherein each gene comprises a specific tag moiety.

58. The apparatus of claim 57, wherein the offline parallel purification system is within the cell growth plate and comprises a solid support, which solid support comprises a tag binding moiety, which tag bonding moiety binds to the specific tag.

59. The apparatus of claim 58, wherein the automatic sampler removes the solid support from the cell growth plate.

60. The apparatus of claim 44, wherein the mass spectrometer is an electrospray tandem mass spectrometer.

61. The apparatus of claim 44, wherein the mass spectrometer is a triple quadropole mass spectrometer.

62. The apparatus of claim 44, further comprising a computer and software operably coupled to the apparatus for recording and analyzing data from the mass spectrometer.

63. The apparatus of claim 62, wherein the computer further comprises software for controlling the automatic sampler.

64. A method for analyzing a plurality of components, the method comprising: (i) providing a plurality of components; which components comprise tagged components; (ii) binding the tagged components to a tag binding moiety on a solid support; (iii) reacting the tagged components with one or more reagents in a reaction mixture, thereby producing one or more products; (iv) removing the tagged components from the reaction mixture or washing the reaction mixture from the solid support; and, (v) analyzing the tagged components, the one or more reagents, or the one or more products in a high throughput system.

65. The method of claim 64, wherein the solid support comprises one or more magnetic beads, one or more agarose beads, one or more polystyrene beads, one or more pins, a microwell plate, or a membrane.

66. The method of claim 64, wherein the tagged components comprises biotin, avidin or streptavidin and wherein the tag binding moiety comprises biotin, avidin, or streptavidin.

67. The method of claim 64, wherein the components comprise enzymes, peptides, proteins, polynucleotides, carbohydrates, lipids, sugars, oligosaccharides, small organic molecules, secondary metabolites, or nucleic acids.

68. The method of claim 64, the method further comprising providing a library of genes, which genes encode one or more enzymes, which enzymes comprise the plurality of tagged components.

69. The method of claim 68, further comprising expressing the one or more tagged enzymes in one or more cells, which cells or cell supernatant comprises the reaction mixture.

70. The method of claim 69, further comprising lysing the one or more cells.

71. The method of claim 64, wherein the high throughput system comprises a mass spectrometer.