大学精品课件：生物工程制药课件：lec4 Proteomics.ppt

1、Lecture 4 ProteomicsnBecause both cell function and its Because both cell function and its biochemical regulation depend on biochemical regulation depend on protein activity,and because the protein activity,and because the correlation between message level and correlation between message level and p

2、rotein activity is low,the protein activity is low,the measurement of expression has proven measurement of expression has proven to be inadequate.Consequently,theto be inadequate.Consequently,the development of drug-discovery development of drug-discovery technologies has begun technologies has begu

3、n to shift from to shift from genomics to proteomics.genomics to proteomics.Proteome in drug developmentnDiscovery of validation of drug targetsnScreening and optimization of drug leadsnPrediction of drug toxicitynMechanism of actionProteomicsProteomicsnProteomics,as a scientific Proteomics,as a sci

4、entific field,is defined as the study field,is defined as the study of the protein products of the of the protein products of the genome,and their interactions genome,and their interactions and functions.Similarly,the and functions.Similarly,the proteins expressed at a given proteins expressed at a

5、given time in a given environment time in a given environment constitute a proteome.constitute a proteome.For drug discovery,the ideal For drug discovery,the ideal proteomics method would beproteomics method would beone that is:one that is:nSensitive enough to detect low-Sensitive enough to detect l

6、ow-abundance proteins.abundance proteins.nAble to detect activity over in Able to detect activity over in addition to abundance.addition to abundance.nAble to detect proteinproteinAble to detect proteinprotein and proteinsmall molecule and proteinsmall molecule interactions.interactions.nEasily impl

7、emented and performed Easily implemented and performed quickly.quickly.ProteomicsMass spectrometrynTechniques that enable the transfer and Techniques that enable the transfer and charging of large molecules such as proteins charging of large molecules such as proteins and peptides,as well as transfe

8、r into a and peptides,as well as transfer into a gaseous phase(e.g.electrospray ionization gaseous phase(e.g.electrospray ionization ESI and matrix-assisted laser desorptionESI and matrix-assisted laser desorption ionization MALDI),have allowed proteins ionization MALDI),have allowed proteins to be

9、analyzed by MS.ES ionization ESI to be analyzed by MS.ES ionization ESI produces a fine spray of charged particles produces a fine spray of charged particles through a charged needle,whereas MALDI through a charged needle,whereas MALDI involves crystallizing the sample of involves crystallizing the

10、sample of interest within a matrix that can be interest within a matrix that can be vaporized quickly using a laser pulse.vaporized quickly using a laser pulse.Two general MS methodsare employed for protein identification using MS.nThe first,peptide-mass fingerprinting,The first,peptide-mass fingerp

11、rinting,compares the pattern of molecular weights compares the pattern of molecular weights of peptides generated from a proteolytic of peptides generated from a proteolytic digestion to theoretical fingerprints digestion to theoretical fingerprints derived from protein databases.The derived from pr

12、otein databases.The second method,tandem mass spectrometry second method,tandem mass spectrometry(MSn(MSn),selects peptides of interest and),selects peptides of interest and uses a secondary fragmentation process to uses a secondary fragmentation process to determine the peptide sequence,which is de

13、termine the peptide sequence,which is then identified using protein sequence then identified using protein sequence databases.databases.The central importance of MS in proteomics is attributable tonthe large amounts of structural the large amounts of structural data that the method can create data t

14、hat the method can create at great speed,and is at great speed,and is demonstrated by the fact that demonstrated by the fact that most separation and detection most separation and detection methods rely on MS for protein methods rely on MS for protein identification.identification.nProteomicsnDrug l

15、eadsBioinformaticsnBioinformatics methods not only Bioinformatics methods not only catalog experiments,but also catalog experiments,but also provide algorithms for data provide algorithms for data analysis and comparisons in analysis and comparisons in numerous contexts,including numerous contexts,i

16、ncluding protein and gene identification,protein and gene identification,protein structurefunction protein structurefunction relationship predictions,and relationship predictions,and functional connections between functional connections between proteins.proteins.BioinformaticsnThis type of organizat

17、ion and This type of organization and analysis is crucial to all areas analysis is crucial to all areas of the drug-discovery process from of the drug-discovery process from the entificationthe entification of novel drug of novel drug targets,in which informatics targets,in which informatics technol

18、ogy enables the mining of technology enables the mining of DNA and protein sequence databases DNA and protein sequence databases for analysis of similarities,to for analysis of similarities,to screening of active compounds screening of active compounds in in silicosilico by virtual screening and/or

19、by virtual screening and/or docking of compound collections.docking of compound collections.Microfabrication and miniaturizationn Miniaturization has two advantages.Miniaturization has two advantages.First,it provides miniaturized First,it provides miniaturized instruments that can improve the instr

20、uments that can improve the development and automation of current development and automation of current techniques through reduction of sample techniques through reduction of sample amount,increased sample number throughput,amount,increased sample number throughput,and increased sensitivity.Second,i

21、t and increased sensitivity.Second,it provides miniaturized containers to provides miniaturized containers to segregate and identify samples.In the segregate and identify samples.In the broadest sense,such microfabricatedbroadest sense,such microfabricated containers encompass both protein-array con

22、tainers encompass both protein-array technology and lab-on-a-chip technology.technology and lab-on-a-chip technology.Lab-on-a-chip technologyLab-on-a-chip technologynIt refers to enclosed fluidics It refers to enclosed fluidics devices that create channels devices that create channels and reaction c

23、hambers on and reaction chambers on substrates such as glass.substrates such as glass.Types of information from proteomicsnThe methods for producing proteomic data can The methods for producing proteomic data can be divided into two major categories:the be divided into two major categories:the class

24、ical approach that strives to classical approach that strives to catalogue all proteins,and the functional catalogue all proteins,and the functional approach that seeks to classify proteins to approach that seeks to classify proteins to be studied by properties such as activity or be studied by prop

25、erties such as activity or affinity.The classical approach tends to affinity.The classical approach tends to provide information about identity and,inprovide information about identity and,in some cases,abundance.The functional some cases,abundance.The functional approach provides identity and abund

26、ance,as approach provides identity and abundance,as well as information about protein function well as information about protein function and,in some cases,protein interactions.and,in some cases,protein interactions.2DGE-MSnUntil recently,the tools of choice were Until recently,the tools of choice w

27、ere two-dimensional gel electrophoresis(2DGE)two-dimensional gel electrophoresis(2DGE)for separation,and mass spectrometry(MS)for separation,and mass spectrometry(MS)for protein identification.However,2DGE for protein identification.However,2DGE is limited because it fails to detect is limited becau

28、se it fails to detect proteins at the extremes of separation proteins at the extremes of separation either by size or by isoelectriceither by size or by isoelectric point,and point,and because it is insufficiently sensitive for because it is insufficiently sensitive for low-abundance proteins.2DGE i

29、s ineffective low-abundance proteins.2DGE is ineffective for the separation of membrane proteins,for the separation of membrane proteins,which represent nearly 50%of important which represent nearly 50%of important drug targets.It has been estimated that drug targets.It has been estimated that more

30、than 50%of proteins in cells are of more than 50%of proteins in cells are of low abundance.low abundance.The structures of the original ICAT reagentsnThe reagent consists of three moieties:an affinity tag(biotin),which is used to isolate ICAT-labelled peptides;a linker that can incorporates stable i

31、sotopes and a maleimide(马来酰亚胺)group which reacts specifically with the thiol group of cysteine.Two labelled forms of the reagent are used,the heavy containing eight deuteriums and the light with none.Schematic diagram of the ICAT approachn Proteins from two different cell states are extracted and la

32、belled with the light or heavy ICAT reagents.The samples are then combined and digested.The ICAT-labelled peptides are isolated by affinity chromatography using an avidin column and then analysed HPLC-MS(/MS)directly or by MALDI of the collected HPLC fractions.The ratio of the peaks areas for specif

33、ic ICAT-labelled pairs defines the relative abundance of its parent proteins between the two cell states.MudPIT(multidimensional protein identification technology)n MudPIT is a non-gel approach for the identification of proteins from complex mixtures.The technique consists of a 2-dimensional chromat

34、ography separation,prior to electrospray mass spectrometry.By exploiting a peptides unique physical properties of charge and hydrophobicity,complex mixtures can be separated prior to sequencing by tandem MS.The first dimension is normally a strong cation exchange(SCX)column,as these have high loadin

35、g capacities.The second dimension is reverse phase chromatography(RP),which complements the SCX as it is efficient at removing salts and has the added advantage of being compatible with electrospray mass spectrometry.MudPIT(multidimensional protein identification technology)nSample preparation is re

36、latively straightforward,the samples are denatured,the cysteines reduced and alkylated and the proteins digested with a protease such as trypsin.The samples are then acidified and loaded onto the SCX column(see diagram below).Charged peptides bind to the SCX column,whereas any uncharged peptides pas

37、s through and bind to a reverse phase trap column.The peptides are then eluted from the trap column onto an analytical RP column,using a reverse phase gradient,separated and eluted into a tandem mass spectrometer.Peptide fragmentation data is then obtained to identify the peptides and hence the prot

38、eins from which they are derived.MudPIT(multidimensional protein identification technology)nIn the next step,salt at a particular concentration is injected onto the SCX column,displacing further peptides from it onto the RP trap column.Salt is removed by washing and again an analytical RP separation

39、 is performed and the eluting peptides analysed by mass spectrometry.Incremental increases of salt are used(salt step gradient from around 0-200 mM).The end result is multiple protein identifications from each salt step.Functional proteomic analysisnThere are two basic designs for protein There are

40、two basic designs for protein arrays.In the first,a surface is arrays.In the first,a surface is arrayed with a non-specific affinity arrayed with a non-specific affinity substrate that binds a subset of the substrate that binds a subset of the proteome.Ciphergen Biosystemsproteome.Ciphergen Biosyste

41、ms arrays,arrays,for example,interact with proteins for example,interact with proteins using a number of such weak affinity using a number of such weak affinity associations,followed by MS analysis.associations,followed by MS analysis.In the second type of design,a surface In the second type of desi

42、gn,a surface is arrayed with specific antibodies,is arrayed with specific antibodies,proteins,peptides,nucleic acids or proteins,peptides,nucleic acids or other small molecules to the surface of other small molecules to the surface of the chip,which select specific proteinsthe chip,which select spec

43、ific proteins.Two other assays that rely on an ability Two other assays that rely on an ability to segregate proteins by affinity areto segregate proteins by affinity aren phage display,and bar-coded nanoparticlesphage display,and bar-coded nanoparticles.Phage display identifies interacting Phage di

44、splay identifies interacting proteins by DNA sequencing of phage that bind proteins by DNA sequencing of phage that bind to a selective surface of phage,while bar-to a selective surface of phage,while bar-coded nanoparticles are created such that coded nanoparticles are created such that individual

45、particles cover a single small individual particles cover a single small molecule,peptide or protein,and provide the molecule,peptide or protein,and provide the same sort of encoding through a nonbiological same sort of encoding through a nonbiological means.The barcodes identify the attached means.

46、The barcodes identify the attached molecules.Like protein arrays and the two-molecules.Like protein arrays and the two-hybrid system,phage display and bar-coded hybrid system,phage display and bar-coded nanoparticlesnanoparticles cannot quantify activity,but can cannot quantify activity,but can crea

47、te protein profiles for different create protein profiles for different environments.environments.Reverse transfectionnA novel type of array that interrogates A novel type of array that interrogates both proteinprotein interactions and both proteinprotein interactions and transcriptional regulation

48、utilizes a transcriptional regulation utilizes a process called reverse transfection.In process called reverse transfection.In this process,cDNA is arrayed on a glass this process,cDNA is arrayed on a glass slide,and cells that attach to the slide slide,and cells that attach to the slide are locally

49、 transfected with the cDNAare locally transfected with the cDNA.After fixing,the expressed protein is After fixing,the expressed protein is visualized either by incubation with visualized either by incubation with fluorescently-labeled antibodies against fluorescently-labeled antibodies against the

50、protein,or by fluorescent microscopy the protein,or by fluorescent microscopy if the protein of interest is fused to a if the protein of interest is fused to a fluorescent tag.fluorescent tag.The Yeast Two-Hybrid The Yeast Two-Hybrid SystemSystemnThe two-hybrid system is a more The two-hybrid system