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您使用浏览器不支持直接复制的功能，建议您使用Ctrl+C或右键全选进行地址复制xThunder和flashgot哪个好用？【firefox吧】_百度贴吧
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xThunder和flashgot哪个好用？收藏
如题：那个更好用一些？ 在这个贴子里面说xThunder不好用啊，说点击链接无反应，是这样的吗？（据说xThunder能下专用链，有点想换）
firefox更好的浏览器品牌,傲游5,,欧美地区更多人都在用的国产双核浏览器,专注浏览器十余年,快速!安全!简单!专业品质,全球优选,多国齐推,立即免费下载.
flashgot- -
反正我用了那么久，没有任何问题就是有些小网站的迅雷专用链接调用不了迅雷，这个没办法，IE only,flashgot拿这种链接就更没辙
我现在正用这个，感觉还可以。
flashgot可以下载流媒体哦！很棒的。
如果两个都安装体验一段时间，会不会出现问题？比如不兼容导致崩溃之类的？
flashgot就有一点，下优酷视频什么的蛮方便。。但是我不需要，因为下下来也是分好多段的
不会吧。。我以前两个都装了，最近才把flashgot卸掉的
因为我有IDM，很强大，下载流媒体是小菜，不过，flashgot确实很不错，我用它来调用外部播放器。
优酷的视频本来就是一段一段的，大概7分半钟一段，甚至是看的时候都能感到有停顿。
有几个人还下载youku视频。。oabt是个好同学
xthunder吧, 很小很强大,况且作者一直在更新嘛
5.0不兼容IDM，貌似还没看到更新。。装flashgot是为了调用IDM。。
刚回复完，就看到兼容5.0的idmmzcc-7.3.1.xpi。。。
xthunder也可以调用IDM
不兼容更牛只是没法下载流媒体了其他下载一样接管,兼容的时候下载会卡住浏览器,不兼容了反倒不卡了,一直都在用IDM,深有体会
中间有个Internet Download Manager 7.3.1.xpi，就是这个
有吗？没注意到，就看到迅雷，旋风和快车，选项里也没idm啊。。。。原先没用过xthunder
个人觉得如果不下载流媒体的话 不要IDM扩展还好点
版本问题，没有那个下载器选项，去找个新的看看~~
刚才安装更新了，但是这个貌似和我自己修改的效果是一样的。
没注意看，Mozilla网上扩展版本还是0.9.1的，去blog换最新的1.0.0
呃~估计是版本问题，我用的这个里没有&img src="data:image/base64,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n7hwQAkCJClIBpvf2/Fp8EqFom47zImqZpquy6TUZTspqW1STnDSRFcGZZ39EH/rfUNKskAV99UiTbEV
登录百度帐号推荐应用QUANTIFICATION OF ENZYME ACTIVITY BY MASS SPECTROMETRY
United States Patent Application
The disclosure relates to methods of quantitatively analyzing the enzymatic activity of enzymes in samples containing a plurality of enzymes, using mass spectrometry. Isotopically labeled standards are employed. Purified enzymes and enzymes from crude cell lysates may be analyzed using the disclosed methods. As little as 0.02 pg of cell lysate may be detected. Also disclosed are kits for providing compositions so as to practice the disclosed methods.
Inventors:
Cutillas, Pedro Rodriguez (Surrey, GB)
Vanhaesebroeck, Bart (London, GB)
Application Number:
Publication Date:
08/27/2009
Filing Date:
04/25/2007
Export Citation:
UCL BUSINESS PLC. (LONDON, GB)
LUDWIG INSTITUTE FOR CANCER RESEARCH (NEW YORK, NY, US)
Primary Class:
Other Classes:
International Classes:
C12Q1/48; C12Q1/00
View Patent Images:
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Related US Applications:
July, 2006Hillebrand et al.November, 2005Kurtz et al.March, 2006Kriksunov et al.July, 2004Desai et al.August, 2007Candussio et al.July, 2008Moore et al.March, 2010Kim et al.February, 2007Lentz et al.September, 2009Ideno et al.September, 2003Sheppard et al.October, 2004Rai
Attorney, Agent or Firm:
MARSHALL, GERSTEIN & BORUN LLP (233 SOUTH WACKER DRIVE, 6300 SEARS TOWER, CHICAGO, IL, , US)
1. (canceled)
2. A quantitative method of measuring the activity of an enzyme in a sample that contains a plurality of biologically active enzymes, the method comprising: a) incubating the sample with a substrate composition to start an enzymatic reaction, wherein the substrate composition comprises a first substrate that is specific for a first enzyme that is known or suspected of being in the sample, and wherein the incubating is under conditions effective to permit a first reaction between the first enzyme and the first substrate to pro b) combining an aliquot from the enzymatic reaction with a measured quantity of a first standard of known molecular weight to form a first
and c) analyzing the first mixture by liquid chromatography-mass spectrometry (LC-MS) to determine the quantity of the first product that is present in the first mixture, wherein the quantity of the first product provides a quantitative measurement of the activity of the first enzyme in the sample.
3. The method of claim 2, wherein the enzyme is a kinase and the conditions comprise including adenosine triphosphate (ATP) in the first reaction.
4. The method of claim 2, wherein the enzyme is a protein kinase.
5. (canceled)
6. The method of claim 2 wherein, in the analyzing step, the quantity of the first product is calculated by comparing mass spectrometric measurements of the first product and the first standard in the first mixture.
7. (canceled)
8. (canceled)
9. The method of claim 2, wherein the sample comprises a cell lysate that comprises enzymes from a cell.
10. The method of claim 2, wherein the analyzing of the first mixture by mass spectrometry comprises: performing a first mass spectrometry analysis to isolate a fraction of the first mixture that contains the first prod fragmenting the first product and the first stan and performing a second mass spectrometry analysis after the fragmenting to quantitatively measure at least one fragment from the first product and the first standard, wherein the fragment measurements indicate the quantities of the first product and the first standard in the first mixture.
11. (canceled)
12. (canceled)
13. (canceled)
14. The method of claim 2, wherein the enzyme participates in a cellular signaling pathway, and the pathway is selected from the group consisting of P13K/AKT Ras/Raf/MEK/E MAP JAK/STAT mTOR/TSC heterotrimeric G PKA PLC/PKC NK-kappaB cell cycle pathways (cell cycle kinases); TGF- TLR N W Nutrient signaling pathways (AMPK signaling); cell-cell and cell-substratum adhesion pathways (such as cadherin, integrins); stress signaling pathways (high/low salt, heat, radiation); cytokin antigen recepto and co-stimulatory immune signaling pathways.
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. The method of claim 2 wherein the first substrate comprises a first peptide.
20. (canceled)
21. (canceled)
22. The method of claim 2, wherein the first standard is identical to the first product, with the proviso that the mass of the first standard differs from the mass of the first product due to incorporation of at least one isotopic label.
23. (canceled)
24. The method of claim 2, wherein the first substrate consists essentially of the amino acid residues of the first peptide.
25. The method of claim 2, wherein the sample comprises a lysate of cells from a human or animal subject.
26. The method of claim 25 wherein the sample comprises a lysate from 100 or fewer cells.
27. (canceled)
28. (canceled)
29. The method of claim 25, wherein the human or animal subject is suspected of having a disease characterized by changes in the activity of an enzyme involved in a cellular process, and wherein the enzyme involved in the cellular process is the first enzyme.
30. The method of claim 2, further comprising quantitatively detecting the activity of a second enzyme, wherein the incubating further comprises simultaneously incubating the sample with a second substrate that is specific for a second enzyme that is known or suspected of being in the sample and that differs from the first enzyme, wherein the second enzyme modifies the second substrate in a second reaction under said conditions to f and wherein the analyzing further comprises measuring, by mass spectrometry, the quantity of the second product produced during the incubating step, wherein the quantity of the second product provides a quantitative measurement of the activity of the second enzyme in the sample.
31. The method of claim 30, comprising mixing an aliquot from the enzymatic reaction with a measured quantity of a second standard of known molecular weight to form a second mixture for analysis.
32. The method of claim 31, wherein the first and second standards are combined with the same aliquot, whereby the first and second mixtures are the same, to permit simultaneous mass spectrometric analysis of the first and second products.
33. The method of claim 31, wherein the analyzing of the second mixture comprises comparing mass spectrometric measurements of the second product and the second standard that are present in the second mixture, to calculate the quantity of the second product that is present in the second mixture, wherein the quantity of the second product in the second mixture provides a quantitative measurement of the activity of the second enzyme in the sample.
34. The method of claim 30, wherein the second enzyme is a protein kinase.
35. A method of screening compounds to identify a drug candidate comprising: measuring the activity of at least one enzyme according to the method of claim 2, in the presence and absence of at lea and comparing the activity of the at least one enzyme in the presence and absence of the at least one test compound, wherein the method identifies an inhibitor or agonist drug candidate from reduced or increased activity, respectively, of the at least one enzyme in the presence of the at least one compound.
36. A method of screening compounds to identify a drug candidate comprising simultaneously measuring the activity of two or more enzymes according to the method of claim 2, in the presence and absence of at least one test compound and comparing the activity of the at least two enzymes in the presence and absence of the at least one test compound, wherein the method identifies an inhibitor or agonist drug candidate from reduced or increased activity, respectively, of the at least two enzymes in the presence of the at least one test compound.
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. A method for screening an organism for a disease, disorder, or abnormality characterized by aberrant enzymatic activity, comprising: (a) quantitatively measuring the activity of the first enzyme from a cell lysate from at least one cell of the organism, according to the method of claim 2; and (b) comparing the measurement to a reference measurement of the activity of the first enzyme, wherein the presence or absence of the abnormality is identified from the comparison.
42. A method of characterizing a disease, disorder, or abnormality comprising: quantitatively measuring the activity of at least one enzyme from a sample according to the method of claim 2, wherein the sample comprises at least one diseased cell isolated from a mammalian subject, or comprises a lysate of t comparing the measurement(s) to a reference measurement of the activity of the and characterizing the disease or disorder by identifying an enzyme with elevated activity in the at least one cell known or suspected of being diseased compared to activity of the enzyme in non-diseased cells of the same type as the diseased cell.
43. The method of claim 42, wherein the disease is a neoplastic disease.
44. (canceled)
45. (canceled)
46. (canceled)
47. The method of claim 42, wherein the cell or cell lysate is obtained from cells from a medical biopsy obtained from a human and snap frozen to preserve enzymatic activity.
48. The method of claim 41, wherein the reference measurement (a) is obtained from cells obtained from the same organism at a different time or from a different location in the organism, (b) is obtained from cells of the same cell type, from a different organism of the same species, or (c) is a statistical measurement calculated from measurements of samples of cells of the same cell type, from multiple organisms of the same species.
49. The method of claim 42 wherein the reference measurement (a) is obtained from cells of the same cell type, from a different organism of the same species, (b) is obtained from cells obtained from the same organism at a different time or from a different location in the organism, or (c) is a statistical measurement calculated from measurements of samples of cells of the same cell type, from multiple organisms of the same species.
50. (canceled)
51. (canceled)
52. The method of claim 41 wherein the disease, disorder, or abnormality is cancer.
53. (canceled)
54. (canceled)
55. A quantitative method of detecting the activity of a signaling pathway in a sample having a plurality of biologically active enzymes comprising a) incubating the sample with a substrate composition to start an enzymatic reaction, wherein the substrate composition comprises a first substrate that is specific for the signaling pathway, and wherein the incubating is under conditions effective to permit a first reaction between at least one enzyme of the signaling pathway and the first substrate to pro b) combining an aliquot from the reaction with a measured quantity of a first standard of known molecular weight to form a first
and c) analyzing the first mixture by mass spectrometry to determine the quantity of the first product that is present in the first mixture, wherein the quantity of the first product provides a quantitative measurement of the activity of the signaling pathway in the sample.
56. A kit comprising (a) a plurality of substrate containers, wherein each substrate container contains at least one enzymatic peptide substrate that an enzyme modifi and (b) a plurality of standard containers, wherein each standard container contains at least one mass labeled standard of a known concentration, and wherein each mass labeled standard is identical to one of the products, with the proviso that the product and the standard have different molecular weights due to isotopic labeling of the standard or the product.
57. (canceled)
58. (canceled)
59. (canceled)
60. A composition comprising a mixture of two or more peptide standards of known molecular weight and concentration, wherein each of the standards comprises a chemical structure identical to an enzyme product and a molecular weight different than the enzyme product due to incorporation of at least one isotopic label in the standard.
61. (canceled)
62. (canceled)
63. (canceled)
Description:
CROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of priority of U.S. Provisional Application No. 60/796,168, filed Apr. 28, 2006, the disclosures of which is expressly incorporated by reference in its entirety.BACKGROUND OF THE DISCLOSURE1. Field of the InventionThe present invention relates to materials and methods for quantification of enzymes or enzyme activity in a sample. In particular, the present invention relates to methods of quantifying enzyme activity using spectroscopy such as mass spectroscopy. The information obtained is valuable for pharmaceutical rese medical diagnosis, prophylaxis, and many other practical applications.2. Related TechnologyBecause many enzymes act aberrantly in a variety of disease states, including cancer, it is valuable to have a means of quantifying enzymatic activity of samples. Quantitative measurements of specific enzymatic activity may lead to rapid diagnosis of patients' disease states and may also lead to swift evaluation of targeted therapies for specific disease states. The means for accomplishing this quantitative analysis has not been proposed in a manner that would allow for rapid and systematic analysis of samples.The detection and effective therapeutic blockade of signal transduction pathways in cancer is seriously hampered by the lack of simple tools to quantify changes in pathway activation status. Techniques currently available involve purification, or semi-purification, of samples or enzymes of interest from other enzymes (see, e.g., Cutillas et al, Mol Cell Proteomics 4(8):05), Gerber et al., Proc Natl Acad Sci USA 100(12): 03), Ballif et al., Proc Natl Acad Sci USA 102(3): 667-72 (2005), Loog, et al., J Biomolecular Screening 10(4): 320-8 (2005), Beausoleil et al., Proc Natl Acad Sci USA 101(33): 04), Rush et al., Nature Biotechnol 23(1): 94-101 (2005), Sonoda et al., Bioorg Med Chem Lett 14:847-50 (2004), Kratchmarova et al., Science 308:05), Luo et al., Endocrinology 146(10):05), Smolka et al., Mol Cell Proteomics 1(1):19-29 (2002), Goshe et al., Curr Opin Biotechnol 14(1):101-9 (2003), and Ducret et al., Protein Sci 7:706-19 (1998)). Often these other methods cannot give absolute quantification of enzyme activity, only rel and these other methods require large amounts of cells for meaningful measurements.Purification of the enzymes of interest prior to analysis of their activity can hamper the rapid assessment of a sample. Complexities in sample preparation or in analysis slow down a clinician's ability to assess a patient's diagnosis cost-effectively, rapidly, and accurately. The current means for using mass spectrometry for enzyme activity do not allow for rapid or multi-faceted analysis of enzymes.SUMMARY OF THE INVENTIONThe present disclosure addresses the need for materials and methods for analyzing enzyme activities of samples to yield quantitative data that may be compared across samples.One aspect of the invention is a quantitative method for detecting the activity of an enzyme in a sample that contains a plurality of enzymes. For example, in one variation, the method comprises: incubating the sample with a substrate composition that comprises a first substrate which is specific for a first enzyme that is known or suspected of being in the sample, wherein the first enzyme is a kinase and wherein the incubating is under conditions effective to permit a first reaction between the first enzyme and the first substrate to pro combining an aliquot from the first reaction with a measured quantity of a first standard of a known molecular weight to form a first
and analyzing the first mixture by mass spectrometry to determine the quantity of the first product that is present in the first mixture, wherein the quantity of the first product provides a quantitative measurement of the activity of the first enzyme in the sample. Although many embodiments of the enzyme are described in the context of kinases, the invention can be used to assay other classes of enzymes, too.In another variation, the method comprises: incubating the sample with a substrate composition to start an enzymatic reaction, wherein the substrate composition comprises a first substrate that is specific for a first enzyme that is known or suspected of being in the sample, and wherein the incubating is under conditions effective to permit a first reaction between the first enzyme and the first substrate to pro combining an aliquot from the enzymatic reaction with a measured quantity of a first standard of known molecular weight to form a first
and analyzing the first mixture by liquid chromatography-mass spectrometry (LC-MS) to determine the quantity of the first product that is present in the first mixture, wherein the quantity of the first product provides a quantitative measurement of the activity of the first enzyme in the sample.The term “enzyme” refers to any protein that has a biological activity of modifying, or catalyzing the modification of, a molecule referred to as a “substrate” into another molecule or molecules referred to as a “product.” For example, a kinase is an enzyme that modifies a substrate molecule by adding a phosphate moiety, to create a phosphorylated product molecule. Kinases can be protein kinases, lipid kinases, carbohydrate kinases such as phosphofructokinase, or small molecule kinases such as pyruvate kinase. Specific protein kinases which may be used in the disclosed methods are listed below in Table 1. An enzyme may include one or more polypeptide chains as well as modifications (e.g., glycosylation, phosphorylation, methylation, etc.) or co-factors (e.g., metal ions).The term “an enzyme” in the preceding description of the method refers to one or more enzymes. As described in greater detail below, the method can be practiced in a multiplex fashion to analyze the activity of multiple enzymes at once. Each enzyme modifies (e.g., catalyzes the modification of) a substrate to form a product. The use of ordinals (e.g., “first” or “second” or “third” and so forth) to refer to elements such as an enzyme, a substrate, a standard, or a product is for clarity purposes only, to identify which enzyme, substrate, product, and standard are related to each other and to distinguish the substrate, standard, and product of one enzyme from the substrate, product, and standard of another enzyme that is assayed. The ordinals are not meant to imply any particular relationship or required order between the multiple enzymes that are to be assayed.In some cases, the enzyme participates in a cellular signaling pathway. Cellular signaling pathways are the biochemical mechanisms by which cells convert extracellular signals into the required cellular response. Cellular signaling pathways are generally discussed in Hunter, “Signaling—2000 and Beyond,” Cell 100:113-117 (2000), the entirety of which is incorporated by reference herein. These signaling pathways involve a multitude of different enzymes and the methods disclosed herein can provide a measurement of the signaling pathway as a whole, not just of specific enzymes within the pathway. Some examples of signaling pathways, the activity of which can be measured using the methods disclosed herein, include P13K/AKT Ras/Raf/MEK/E MAP JAK/STAT mTOR/TSC heterotrimeric G PKA PLC/PKC NK-kappaB cell cycle pathways (cell cycle kinases); TGF- TLR N W Nutrient signaling pathways (AMPK signaling); cell-cell and cell:substratum adhesion pathways (such as cadherin or integrins); stress signaling pathways (e.g., high/low salt, heat, radiation); cytokin antigen recepto and co-stimulatory immune signaling pathways. In some cases when the enzyme is involved in a cellular signaling pathway, the enzyme is an intracellular enzyme, i.e., an enzyme found only within a cell.As applied to this method, the term “quantitative” refers to the method's ability to provide an absolute measurement of enzymatic activity that can be compared to measurements taken at a different time or place. Quantitative measurements are more valuable for many purposes than relative measurements that can only be compared to other measurements taken at the same time that may yield information such as a ratio. As described below in greater detail, the use of a measured quantity of the standard permits quantitative calculation of the activity of an enzyme in a sample.The term “enzyme composition” reflects the fact that the method can be practiced with impure samples that contain a plurality (two or more) of enzymes as well as other materials. For example, any biological sample or extract that contains biologically active enzymes can be used as an enzyme composition to practice methods of the invention. As described below in greater detail, whole cells or tissue samples, cell lysates, bodily fluids or secretions or excretions, plant extracts, are examples of enzyme compositions. In these contexts, plurality may refer to, tens, hundreds, thousands, or more enzymes.The incubating step involves placing the enzyme composition and the substrate composition together under conditions wherein the enzyme is biologically active, to permit the enzyme to modify the substrate. For an enzyme composition that comprises one or more whole cells, the incubating may involve adding the substrate to the culture media of the cell, for example. For an enzyme composition that is a cell lysate, the incubating may involve mixing the enzyme and the substrate together. Factors required for enzymatic activity, such as a particular temperature or pH, salt concentration, co-factors, ATP, GTP, and the like, will generally be known for enzymes, and even when unknown, would be expected to be similar to the physiological microenvironment where the enzyme is active in vivo.In some variations, the enzyme composition is a mixture of purified enzymes. The enzyme composition can also be all or a fraction of a cell lysate which contains enzymes from the cell. In certain cases, the lysate comes from a human or animal subject. The lysate may be of fewer than 100 cells, or fewer than 25 cells, or even fewer than 10 cells. In certain cases, the first enzyme is a kinase and, in specific embodiments, is a protein kinase or lipid kinase. In some cases, the first enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase.In one embodiment, the analysis occurs by tandem mass spectrometry, which involves a first mass spectrometry analysis to isolate a fraction of the ionized sample that contains the first product an fragmenting the first product and the first stan and performing a second mass spectrometry analysis after the fragmenting to quantitatively measure at least one fragment from the first product and the first standard, wherein the fragment measurements indicate the quantities of the first product and the first standard. The analysis may also be performed by conventional mass spectrometry, in which matrix assisted laser desorption ionization (MALDI) or electrospray ionization is coupled with single mass analyzers such as time of flight (TOF), quadrupoles, sectors, or ion traps. In some variations, the measurement is performed by quantitative evaluation of the unfragmented molecular ions. In a typical variation, the quantity of the first product of the enzymatic reaction is calculated by comparing mass spectrometric measurements of the first product and the first standard in the first mixture.In some cases, the methods further include purifying the first product and first standard before the determining step to provide a purified sample for analysis. Any techniques that are useful for chemical or biochemical separation may be used for the purifying step, including the use of chromatographic techniques, affinity purification materials and methods, electrophoresis techniques, and the like. In certain cases, the purification is done by high pressure liquid chromatography (HPLC).In some cases, the enzyme composition further includes protease inhibitors added prior to or contemporaneous to starting the enzymatic reaction. Protease inhibitors serve to inhibit degradation of the enzyme or degradation of protein substrates, products, and standards. More generally, in some variations of the invention, the method includes the addition of factors that are necessary for the enzymatic reaction, or that improve the enzymatic reaction, or that prevent degradation of the product.In one embodiment, the first enzyme is a protein kinase such as Akt/PKB or a phosphoinosotide kinase. Kinase activity may require the availability of a phosphate donor. Thus, in some cases, the methods include addition of adenosine triphosphate (ATP) to the enzymatic reaction. In some cases, phosphatase inhibitors are included prior to or contemporaneous to starting the enzymatic reaction, to prevent degradation (dephosphorylation) of the reaction product.In one embodiment, the substrate comprises a peptide. The peptide may be any size that is recognized and modified by the target enzyme to be assayed. Smaller peptides are preferred due to ease of manufacture and manipulation and because they may present fewer sites for modification by non-target enzymes, i.e., they may have greater enzyme specificity. In some cases, the peptide has 5 to 45 amino acid residues. A number of specific peptide sequences that are useful as substrates for certain specific enzymes are set forth below in greater detail. In certain cases, the peptide is a peptide having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. or SEQ ID NO: 32. Numerous enzyme-substrate combinations have been described in the literature and the invention is not limited to this set of examples.In some cases, the standard is identical to the product of the enzymatic reaction, with the proviso that the molecular weight or mass of the standard is different from the product due to an isotope incorporated into either the product or the standard. Stable isotopes (those that are not radioactive or not decaying over time) are preferred. In certain cases, the isotope is one or more of a 13C, 15N, and 2H.In some variations, both the substrate and the standard further comprise a tag (e.g., polyhistidine or other peptide or epitope tag, or biotin or streptavidin tag, etc.) for use in an optional purification step. In some embodiments, the substrate includes modifications to the amino acid sequence, whereas in other embodiments, it consists essentially of amino acids only.In certain cases, the sample is cell lysate from a human or animal subject and the human or animal subject is suspected of having a disease characterized by changes in the activity of an enzyme involved in a cellular process. In one embodiment, the disease suspected is cancer.In some cases, the methods disclosed herein may be used to quantify the enzymatic activity of second enzyme, wherein the incubating step further comprises simultaneously incubating the enzyme composition with a second substrate that is specific for a second enzyme that differs from the first enzyme, wherein the second enzyme modifies the second substrate to f and wherein the determining step further comprises determining the quantity of the second product produced during the incubating step. In certain cases, an aliquot from the reaction is mixed with a measured quantity of a second standard of a known molecular weight to form a sample for analysis. In some cases, the first and second standards are mixed with the same aliquot to permit simultaneous mass spectrometric analysis of the first and second products. In certain cases, the method comprises determining the quantity of the second product produced during the incubating step by analyzing the sample by mass spectrometry to measure quantities of the second product and the second standard in the sample, wherein the quantity of the second product provides a quantitative measurement of the activity of the second enzyme. In the same fashion, the method can be performed to assay a third enzyme, a fourth enzyme, a fifth enzyme, and so on.In some variations, all of the enzymes to be assayed fall within the same class (e.g., protein kinases), whereas in other variations, enzymes of different classes are assayed together.Another aspect of the invention is a method for screening compounds in order to identify a drug candidate comprising: measuring the activity of at least one enzyme from a biological sample, using a met and comparing the activity of the at least one enzyme in the presence and absence of the at least one test compound, wherein the method identifies an inhibitor or agonist drug candidate from reduced or increased activity, respectively, of the at least one enzyme in the presence of the at leaset one test compound. In certain cases, the method comprises measuring the activity of two or more enzymes in the presence or absence of a test compound. In various embodiments, the two or more enzymes are in the same signaling pathway, such as, for example, a pathway involved in cell growth, replication, differentiation, survival, or proliferation. Identification of a test compound as an inhibitor or an agonist of a particular enzyme or group of enzymes (as in the case of two or more enzymes being studied) can be accomplished by measuring the activity of a first enzyme or signaling pathway in the absence and presence of the test compound and comparing the activities as measured in order to assess the effect the test compound has. In certain cases, the methods can be used to assess the biological activity of the compound on non-target enzymes or pathways that may be relevant to drug metabolism/clearance, drug toxicity, and side-effects. This assessment may be useful for evaluating a compound as a potential drug candidate and/or its suitability for or efficacy in clinical trials. In some cases, the method comprises additional steps to further evaluate the compound. For example, the test compound is mixed with a pharmaceutically acceptable carrier to form a composition and the composition is administered to a subject to determine the effect of the composition in vivo. The subject can be a healthy subject for safety testing and/or a diseased subject and/or a model for a disease, for purpose of therapy or proving therapeutic efficacy. In one specific embodiment, the subject is a mammalian subject.Another aspect of the invention is a method for screening an organism for a disease, disorder, or abnormality characterized by aberrant enzymatic activity comprising: quantitatively measuring the activity of an enzyme from a biological sample from an organism (e.g., a cell lysate from at least one cell of the organism) as described herein, and comparing the measurement to a reference measurement of the activity of the enzyme, wherein the presence or absence of the abnormality is identified from the comparison. Numerous enzyme-disease associations have been described in the literature and some are summarized below. Enzymes involved in cell growth, replication, differentiation, survival, or proliferation are only the preferred enzymes for such screening. In one exemplary embodiment, the a the first enzyme is Akt/PKB or a pho and/or the first substrate is a first peptide which is SEQ ID NO: 7. In some cases, the cell lysate is obtained from a medical biopsy from a human and snap frozen to preserve enzymatic activity. In certain cases, the reference measurement is obtained from the same organism at a different time or from a different location in the organism. In other cases, the reference measurement is obtained from cells of the same cell type, from a different organism of the same species. In still other cases, the reference measurement is a statistical measurement calculated from measurements of samples of cells of the same cell type, from multiple organisms of the same species.In some cases, the methods disclosed herein further comprise quantitatively measuring activity of at least one positive control enzyme from the biological sample. A positive control provides assurance that the sample was not handled in a manner that caused unacceptable enzyme degradation or denaturization.One continuing need in medicine, especially oncology and infectious diseases, is to be able to better characterize a disease in an individual patient to permit better selection of a medicament that is more likely to be therapeutically effective and/or have fewer side effects. Therefore, another aspect of the invention is a method of characterizing a disease, disorder, or abnormality comprising: quantitatively measuring the activity of at least one enzyme from a sample using any of the methods disclosed herein, wherein the sample comprises at least one cell known or suspected of being diseased isolated from a mammalian subject, or comprises a lysate of t comparing the measurement(s) to a reference measurement of the activity of the and characterizing the disease or disorder by identifying an enzyme with elevated activity in the at least one diseased cell compared to activity of the enzyme in non-diseased cells of the same type as the diseased cell. In certain cases, the disease is a neoplastic disease. In some embodiments, the method further comprises selecting a composition or compound for administration to the mammalian subject, wherein the composition or compound inhibits the activity of the enzyme with the elevated activity in the at least one diseased or neoplastic cell. In some cases, the method further comprises administering a composition or compound that inhibits the activity of the enzyme with the elevated activity in the at least one diseased or neoplastic cell. In certain cases, the method further comprises prescribing a medicament to the mammalian subject, wherein the medicament inhibits the activity of the enzyme with the elevated activity in the at least one diseased or neoplastic cell. In one specific embodiment, the mammalian subject is a human.In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.In some variations of the invention, the method is a method for screening for or diagnosing a disease state and the method includes a step of measuring enzyme activity as described herein in a biological sample from an organism, and a step of diagnosing the absence or the presence of the disease, or predisposition for the disease, by the measurement of enzyme activity. For example, a comparison of the measurement for a particular subject to measurements from other healthy subjects, or diseased subjects, of the same subject at an earlier point in time, indicates the proper conclusion about the disease state in the subject.Another aspect of the invention is a quantitative method of detecting the activity of a signaling pathway in a sample having a plurality of biologically active enzymes comprising: incubating the sample with a substrate composition which comprises a first substrate that is specific for the signaling pathway, and wherein the incubating is under conditions effective to permit a first reaction between at least one enzyme of the signaling pathway and the first substrate to pro combining an aliquot from the reaction with a measured quantity of a first standard of known molecular weight to form a first
and analyzing the first mixture by mass spectrometry to determine the quantity of the first product that is present in the first mixture, wherein the quantity of the first product provides a quantitative measurement of the activity of the signaling pathway in the sample. A substrate that is specific for a signaling pathway may be converted into a product by one or more enzymes involved in the pathway, but should be unmodified by other enzymes that may be presented in the sample but that do not participate in the pathway.Another aspect of the invention is a kit comprising two or more items useful for practicing a method of the invention, packaged together. For example, in one variation, the kit comprises a plurality of substrate containers, wherein each substrate container contains at least one enzymatic substrate that an enzyme modifies to form a product and a plurality of standard containers, wherein each standard container contains at least one mass labeled standard of a known concentration, wherein the mass labeled standard is identical to one of the products, with the proviso that the product and the standard have different molecular weights due to isotopic labeling of the standard or the product. In some cases, the kit further comprises a container having protease inhibitors such as Na-p-tosyl-L-lysine chlormethyl ketone hydrochloride (TLCK), phenylmethylsulphonylfluoride (PMSF), leupeptin, pepstatin A, aprotinin, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF), 6-aminohexanoic acid, antipain hydrochloride {[(S)-1-carboxy-2-phenylethyl]-carbamoyl-L-arginyl-L-valyl-arginal-phenylalanine}, benzamidine hydrochloride hydrate, bestatin hydrochloride, chymostatin, epoxysuccinyl-L-leucyl-amido-(4-guanidino)butane, ethylenediamine tetraacetic acid disodium salt, N-ethylmaleimide, and Kunitz trypsin inhibitor. In certain cases, the kit further includes a container of phosphatase inhibitors. Exemplary phosphatase inhibitors include, but are not limited to, sodium fluoride, sodium orthovanadate, ocadaic acid, Vphen, microcystin, b-glycerophosphate, lacineurin, cantharidic acid, cyclosporin A, delamethrin, dephostatin, endothall, fenvalerate, fostriecin, phenylarsine oxide, and resmethrin.In certain cases, the kit comprises substrate which are peptide having 6 to 250 amino acid residues. In some cases, the substrates are peptides having 5 to 45 residues.Another aspect of the invention is a composition comprising a mixture of two or more standards of known molecular weight and concentration, wherein each of the standards comprises a chemical structure identical to an enzyme product and a molecular weight different than the enzyme product due to incorporation of at least one isotopic label in the standards. In some cases, the standards comprise peptides having 5 to 45 amino acids residues. In certain cases, the composition further includes protease inhibitors and/or phosphatase inhibitors. In one embodiment, the composition is packaged in a kit further including at least one container having at least one of the enzyme substrates.Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the drawing and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention shou variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. For example, although aspects of the invention may have been described by reference to a genus or a range of values for brevity, it should be understood that each member of the genus and each value or sub-range within the range is intended as an aspect of the invention. Likewise, various aspects and features of the invention can be combined, creating additional aspects which are intended to be within the scope of the invention. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows (a) a schematic of the steps for determining the enzymatic activity of a protein kinase and (b) a standard plot of the correlation between ratio of enzymatic product to internal standard (a mass labeled enzymatic product) and known concentration of the enzymatic product, wherein the bottom table shows the recalculated concentrations based upon the ratios measured and the known concentration FIG. 2. shows (a) at top, a plot of the enzymatic activity of Akt measured for various enzyme amounts, at bottom, a matrix assisted laser desorbtion ionization—time of flight mass spectroscopy (MALDI-TOF MS) spectrum using 0.02 pg of Akt/PKB (roughly 500 zmol) protein and (b) at top, representative chromatograms from LC-MS analyses obtained using different amount of cell lysate and measuring amount of product produced, at bottom, a plot of the quantitative data derived frFIG. 3. shows (a) MS quantification of kinase activity for B lymphoma cells treated with PI3K inhibitors WM or IC87114, (b) MS quantification of kinase activity for B lymphoma total cell lysates and Akt immunoprecipitates in the presence (right) or absence (absence) of the PI3K inhibitor WM, and (c) kinase activity quantification in B lymphoma cell lysates in absence (top graph) or presence (middle and bottom graph) of PI3KFIG. 4. shows (a) MS quantification of B16/B16 solid tumor cell kinase activity in absence (left) or presence (right) of the PI3K inhibitor, LY294002 and (b) kinase activity quantification of CD34+ CD38- stem cells and CD34+ CD38+ bulk tumor fracti andFIG. 5 shows a multiplex analysis wherein 3 different enzymes—(a) and (d) PKC, (b) S6 p70 kinase, and (c) Erk—with four different substrates—(a) SEQ ID NO: 12; (b) SEQ ID NO: 5; (c) SEQ ID NO: 10; and (d) SEQ ID NO: 23—in the same sample were analyzed
where the first four columns correspond to reaction times 0, 10, 30, and 60 minutes, respectively, and the last column reflects all four time points in one graph for each enzyme/substrate analysis.DETAILED DESCRIPTIONThe detection and effective therapeutic modulation (stimulation, up-regulation, inhibition, or blockade) of signal transduction pathways in human diseases, including, but not limited to, cancer, diabetes, allergies, inflammation, and neurodegenerative diseases, is seriously hampered by inadequate tools to quantify changes in pathway activation status. The techniques described here, in one embodiment, enable the measurement of signal transduction pathway activity in a biological sample (such as a tissue, fluid, or cell sample) with the sensitivity, specificity, and precision needed for providing clinically useful information. This analytical strategy may be applied to any protein or enzyme whose product or substrate is amenable to mass spectrometric detection. In preferred variations, at lease one selective substrate of the target enzyme is available. Enzymes and substrates/products involved in a signal transduction pathway provide clinically useful information about the pathway. Because this method is based upon a biochemical (e.g., enzymatic) reaction that amplifies the signal of the target molecule, it could be described as a proteomic analytical equivalent the polymerase chain reaction (PCR) used to amplify nucleic acid sequences.In addition, the specificity of mass spectrometry as used in methods of the invention offers the opportunity of measuring several reaction products simultaneously in a fast “multiplex” format that can be automated for clinical implementation.The mechanism of action of many pharmaceutical agents (as well as lead, pre-clinical, and clinical candidate compounds) is to modulate enzymatic activity, which is a major factor in controlling cellular and tissue biochemistry. By providing a rapid, sensitive, specific, and optionally multiplex means for analyzing enzyme activities involved in signal transduction, metabolism, and related biochemical processes, the materials and methods of the invention are useful for both drug research and development and drug prescription, administration, and patient monitoring. For example, in the field of drug development, the materials and methods of the invention are useful for assessing the biological activity of a compound on a target pathway, and also for assessing the biological activity of the compound on non-target pathways that may be relevant to drug metabolism/clearance, drug toxicity, drug-drug interactions, and side-effects.In a typical drug screening, the activity of a system is independently measured in the absence and presence of a test compound. The affect of that test compound is evaluated as a comparison between the measured activity in the absence of the compound and the activity in the presence of the compound. The methods disclosed herein are a means of measuring the effect of a potential drug candidat}