蛋白质组学介绍

1、ProteomicsProteomicsandandmass sPectroscoPymass sPectroscoPyProteomicsProteomics The dream of having genomes completely sequenced is now a reality. The complete sequence of many genomes including the human one is known. However, the understanding of probably half a million human proteins encoded by

2、less than 30,000 genes is still a long way away and the hard work to unravel the complexity of biological systems is yet to come. A new fundamental concept called proteome (PROTEin complement to a genOME) has recently emerged.ProteomicsProteomics should drastically help to unravel biochemical and ph

3、ysiological mechanisms of complex multivariate diseases at the functional molecular level. The discipline of proteomics has been initiated to complement physical genomic research. The term “proteome” was coined in 1994 by an Australian graduate student (Mark Wilkins), it has come to be used and defi

4、ned in a variety of different waysProteomicsProteomics Definition-The identification, characterization and quantification of all proteins involved in a particular pathway, organelle, cell, tissue, organ or organism that can be studied in concert to provide accurate and comprehensive data about that

5、system. Or - A complete description of proteins expressed in any given cell at any given time ProteomicsProteomics A cellular proteome is the collection of proteins found in a particular cell type under a particular set of environmental conditions such as exposure to hormone stimulation It can also

6、be useful to consider an organisms complete proteome, which can be conceptualized as the complete set of proteins from all of the various cellular proteomes. This is very roughly the protein equivalent of the genome. The term proteome has also been used to refer to the collection of proteins in cert

7、ain sub-cellular biological systems. For example, all of the proteins in a virus can be called a viral proteome. ProteomicsProteomics So where are we in our understanding of the cell? 31-60 K total genes in the human genome with little difference between the fruit fly and us! Where does the diversit

8、y come from?Answer: Its the proteins! ProteomicsProteomics The proteome is larger than the genome, especially in eukaryotes, in the sense that there are more proteins than genes. This is due to: alternative splicing of genes post-translational modifications like glycosylation or Phosphorylation. alt

9、ernative sPlicing of genesalternative sPlicing of genes A given piece of pre-mRNA which has been transcribed from one gene can be chopped and reconnected in different ways to yield various new mRNAs which then exit the nucleus to be translated in the cytoplasm. When the pre-mRNA has been transcribed

10、 from the DNA, it includes several introns and exons. The regulation and selection of splice sites is done by Serine/Arginine-residue proteins alternative sPlicing of genesalternative sPlicing of genes Four known modes A - Alternative selection of promoters: this is the only method of splicing which

11、 can produce an alternative N-terminus domain in proteins. In this case, different sets of promoters can be spliced with certain sets of other exons. B - Alternative selection of cleavage/polyadenylation sites: this is the only method of splicing which can produce an alternative C-terminus domain in

12、 proteins. In this case, different sets of polyadenylation sites can be spliced with the other exons. alternative sPlicing of genesalternative sPlicing of genes Four known modes C - Intron retaining mode: Instead of splicing out an intron, the intron is retained in the mRNA transcript. However, the

13、intron must be properly encoding for amino acids. The introns code must be properly expressible, otherwise a stop codon or a shift in the reading frame will cause the protein to be non-functional. D -Exon cassette mode: Certain exons are spliced out to alter the sequence of amino acids in the expres

14、sed protein. Post-translational modificationsPost-translational modifications PTMs involving addition include: Acetylation - the addition of an acetyl group, usually at the N-terminus of the protein Alkylation - the addition of an alkyl group (e.g. methyl, ethyl) Methylation -the addition of a methy

15、l group, usually at lysine or arginine residues. (This is a type of alkylation.) Biotinylation - acylation of conserved lysine residues with a biotin appendage Glutamylation - covalent linkage of glutamic acid residues to tubulin and some other proteins. Post-translational modificationsPost-translat

16、ional modifications PTMs involving addition include: Glycylation - covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail Glycosylation - the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein Isoprenyl

17、ation - the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol) Lipoylation - attachment of a lipoate functionality Post-translational modificationsPost-translational modifications PTMs involving addition include: Phosphopantetheinylation - the addition of a 4-phosphopantetheinyl moi

18、ety from coenzyme A, as in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis Phosphorylation - the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine Post-translational Post-translational modificationsmodifications For instance, the peptide hormon

19、e insulin Cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; The resulting protein consists of two polypeptide chains connected by disulfide bonds. Proteomics - Proteomics - Bridging the genome to Bridging the genome to the functions of the cellthe

20、functions of the cellAreas of Proteomics Protein Analysis/Chemistry - Look at PT modifications, structure and function, enzyme behavior Expression - what why and when - 2D gels, MS, HPLC/protein chips, Cell Mapping - protein-protein interactions - affinity tags, two hybrid, antibody pull downProteom

21、icsProteomics A surprising finding of the Human Genome Project is that there are far fewer protein-coding genes in the human genome than proteins in the human proteome 20,000 to 25,000 genes coding for proteins. about 1,000,000 proteins. The human body may contain more than 2 million proteins, each

22、having different functions. The discrepancy implies that protein diversity cannot be fully characterized by gene expression analysis, thus proteomics is useful for characterizing cells and tissues. so how does it work?so how does it work? Most proteins function in collaboration with other proteins,

23、and one goal of proteomics is to identify which proteins interact. This often gives important clues about the functions of newly discovered proteins so how does it work?so how does it work? Proteins are resolved, sometimes on a massive scale. Protein separation can be performed using 2-D gel electro

24、phoresis, usually separates proteins first by isoelectric point and then by molecular weight. Once proteins are separated and quantified, they are identified Individual spots are cut out of the gel and cleaved into peptides with proteolytic enzymesso how does it work?so how does it work? These pepti

25、des can then be identified by mass spectrometry, Specifically: matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. In this procedure, a peptide is placed on a matrix, which causes the peptide to form crystals. so how does it work?so how does it work? Then the pe

26、ptide on the matrix is ionized with a laser beam and an increase in voltage at the matrix is used to shoot the ions toward a detector in which the time it takes an ion to reach the detector depends on its mass. The higher the mass, the longer the time of flight of the ion. so how does it work?so how

27、 does it work? In a MALDI-TOF mass spectrometer, the ions can also be deflected with an electrostatic reflector that also focuses the ion beam. Thus, the masses of the ions reaching the second detector can be determined with high precision and these masses can reveal the exact chemical compositions

28、of the peptides, and therefore their identities! so how does it work?so how does it work? Protein mixtures can also be analyzed without prior separation. These procedures begin with proteolytic digestion of the proteins in a complex mixture The resulting peptides are often injected onto a high press

29、ure liquid chromatography column (HPLC) that separates peptides based on hydrophobicity. HPLC can be coupled directly to a time-of-flight mass spectrometer using electrospray ionization so how does it work?so how does it work? e l e c t r o s p r a y i o n i z a t i o n : A technique used in mass sp

30、ectrometry to produce ions. It is especially useful in producing ions from macromolecules because i t o v e r c o m e s t h e p r o p e n s i t y o f t h e s e molecules to fragment when ionized so how does it work?so how does it work? Peptides eluting from the column can be identified by tandem mas

31、s spectrometry (MS/MS). The first stage of tandem MS/MS isolates individual peptide ions, and the second b r e a k s t he pe pt i d e s i n t o f r a g m e n t s a n d u s e s t h e fragmentation pattern to determine their amino acid sequences. Labeling with isotope tags can be used to quantitativel

32、y c o m p a r e p r o t e i n s concentration among two or more protein samples.so how does it work?so how does it work? Finally, use databases. Computer compares sequences to other sequences stored in an internationally accessible database. Determines the identity of the isolated protein As the ent

33、ire human genome is known, computers are able to determine nearly every potential protein. New proteins are “discovered” when they match sequences predicted by the computer that have not previously been found.medical aPPlicationsmedical aPPlicationsAlzheimers disease:Elevations in beta secretase cre

34、ates amyloid/beta-protein, which causes plaque to build up in the patients brain, which causes dementia. Targeting this enzyme decreases the amyloid/beta-protein and so slows the progression of the disease. A procedure to test for the increase in amyloid/beta-protein is immunohistochemical staining,

35、 in which antibodies bind to specific antigens or biological tissue of amyloid/beta-protein. medical aPPlicationsmedical aPPlicationsHeart disease: Commonly assessed using several key protein based biomarkers. Standard protein biomarkers for CVD include interleukin-6, interleukin-8, serum amyloid A

36、protein, fibrinogen, and troponins. cTnI cardiac troponin I increases in concentration within 3 to 12 hours of initial cardiac injury and can be found elevated days after an acute myocardial infarction. A number of commercial antibody based assays as well as other methods are used in hospitals as pr

37、imary tests for acute myocardial infarction.medical aPPlicationsmedical aPPlicationsRenal cell carcinoma:Proteomic analysis of kidney cells and cancerous kidney cells is producing promising leads for biomarkers and developing assays to test for this disease. In kidney-related diseases, urine is a po

38、tential source for such biomarkers. Recently, it has been shown that the identification of urinary polypeptides as biomarkers of kidney-related diseases allows to diagnose the severity of the disease several months before the appearance of the pathology. medical aPPlicationsmedical aPPlications . Ph

39、enylketonuria (PKU) Affects in in 5,000 newborns Most common nervous system disorder Allele is on chromosome 12 Lack the enzyme needed for the metabolism of the amino acid phenylalanine A build up of abnormal breakdown pathwayPhenylketone Accumulates in urine. If diet is not checked, can lead to sev

40、ere mental retardationoverview of overview of ProteomicsProteomics Nucleus DNA RNA Cytoplasm Protein expression and modification Protein isolation Mass spectroscopy Protein sequence Identificationoverview of Proteomicsoverview of Proteomics Proteomics research is highly interdisciplinary, bringing t

41、ogether: biology chemistry instrumentation Statistics computer scienceoverview of Proteomicsoverview of ProteomicsSummary time! Spend 15- 20 mins going over this as a group MAKE NOTES! /cfapps/research/data_admin/Site602/mainpageS602P0.htmlmass sPectroscoPymass sPectro

42、scoPymass sPectroscoPymass sPectroscoPy An analytical technique used to measure the mass-to-charge ratio of ions. It is most generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample components. The technique has several applications,

43、 including: 1) identifying unknown compounds by the mass of the compound molecules or their fragmentsmass sPectroscoPymass sPectroscoPy 2) determining the isotopic composition of elements in a compound. 3) determining the structure of a compound by observing its fragmentation 4) quantifying the amou

44、nt of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative) mass sPectroscoPymass sPectroscoPy 5) studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum) 6) determining other physical, chemical, or even

45、biological properties of compounds with a variety of other approaches. A mass spectrometer is a device that measures the mass-to-charge ratio of ions. This is achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of io

46、n flux. mass sPectroscoPymass sPectroscoPy All Mass specs consist of: A high vacuum system 10-6 torr A sample inlet GC, HPLC, electron impact, or direct chemical isolation An ion source Converts molecules to gas-phase ions MALDI, fast atom bombardmentmass sPectroscoPymass sPectroscoPy All Mass specs

47、 consist of: A mass filter/ analyzer Time of flight, magnetic sector, MALDI, or ion trap A detector Array detector, conversion dynode, or electron multiplyerionizationionization In mass spectrometry, a substance is bombarded with an electron beam having s u f f i c i e n t e n e r g y t o fragment t

48、he molecule The positive fragments which are produced (cations and radical cations) are accelerated in a vacuum through a magnetic field and are sorted on the basis of mass-to-charge ratio. ionizationionization Since the bulk of the ions p r o d u c e d i n t h e m a s s spectrometer carry a unit po

49、sitive charge, the value m/e is equivalent to the molecular weight of the fragment. T h e a n a l y s i s o f m a s s spectroscopy information involves the re-assembling o f f r a g m e n t s , w o r k i n g backwards to generate the original molecule. ionizationionization Electron Impact ionization

50、 (EI): It comprises an electron gun, a time-of-flight mass spectrometer with position-sensitive detector (PSD). Analyte must be in a vapor state, limiting biological material below 400Da Useful for metabolites, pollutants, and pharmaceutical compoundsionizationionization Chemical ionization: Especia

51、lly useful technique for organic chemists when no molecular ion is observed in EI mass spectrum Ionization of sample (analyte) is achieved by interaction of its molecules with reagents such as CH4 or NH3 Very good for determining molecular mass as high intensity molecular ions are produced owing to

52、less fragmentation.ionizationionization electrospray ionization: A technique used in mass spectrometry to produce ions. I t i s e s p e c i a l l y u s e f u l i n p r o d u c i n g i o n s f r o m macromolecules because it overcomes the propensity of these molecules to fragment when ionized Is the

53、primary ion source used in liquid chromatography-mass spec because its a liquid-gas interface that is capable of coupling liquid chomatography with mass spectrometry mass analyzersmass analyzers Once ions are created and leave the ion source they pass into a mass analyzer This separates the ions and

54、 measures their masses What is actually measured is the mass to charge ratio (m/z) of each ion At any given time, ions of a particular mass pass through and are counted by the detector In this way, the analyzer scans through a large range of massesmass analyzersmass analyzers Quadrupole mass spectro

55、metry: Essentially a mass filter that is capable of transmitting only the ion of choice. A mass spectrum is obtained by scanning through the mass range of interest over time. Two opposite rods have an opposite applied potential and affect the trajectory of ions traveling down the flight path centere

56、d between the four rodsmass analyzersmass analyzers Quadrupole mass spectrometry: T h e m a s s r a n g e o f t h e oscillating ions is scanned by changing the DC voltage and the frequency. T h e r e s o l u t i o n o f t h e spectrometer can be increased by either employing eight poles or by connec

57、ting two or three qaudupoles in series Excel at applications where particular ions of interest are being studied because they can stay tuned on a single ion for extended periodsmass analyzersmass analyzers Ion trap mass spectrometry: The ion trap consists of t h r e e e l e c t r o d e s w i t h hyp

58、erbolic surfaces, the central ring electrode, and t w o a d j a c e n t e n d c a p electrodes T h e d e v i c e i s r a d i a l l y symmetrical the electrodes are aligned and isolated using ceramic spacers and posts mass analyzersmass analyzers Ion trap mass spectrometry: Advantages: (i) high sensi

59、tivity (ii) compactness and mechanical simplicity in a device which is nevertheless capable of high performance (iii) tandem mass spectrometry experiments are available by performing sequential mass analysis measurements (iv) high resolution mass analyzersmass analyzers Magnetic sector analyzer: Gen

60、erated ions are accelerated a n d a r e p a s s e d a r o u n d a curved track (the sector) leading to a detector. By increasing the magnetic field applied to the ions, heavier ions with higher momentum can be induced to follow the curved track.mass analyzersmass analyzers Magnetic sector analyzer: