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Ming F. Tam Spring ‘06 Mass Spectrometric Analysis of Proteins and Peptides.

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Presentation on theme: "Ming F. Tam Spring ‘06 Mass Spectrometric Analysis of Proteins and Peptides."— Presentation transcript:

1 Ming F. Tam Spring ‘06 Mass Spectrometric Analysis of Proteins and Peptides

2 Ming F. Tam Spring ‘06 Outline 1The fundamentals—brief overview of hardwares and data acquisition. 2Protein characterization, i.e., sequencing. 3Protein identification. 4Protein quantification. 5Other applications.

3 Ming F. Tam Spring ‘06 Tell me, my dear, what is proteome and it’s origin??

4 Ming F. Tam Spring ‘06 Proteome The PROTEin complement of a genOME “Progress with Proteome Projects: Why all Proteins Expressed by a Genome Should be Identified and How to Do It” Biotech. Gen. Eng. Rev. (1995) 13, 19-50. Wilkins, MR; Sanchez, JC; Gooley, AA; Appel, RD; Humphrey-Smith, I; Hochstrazzer, DF and Williams, KL.

5 Ming F. Tam Spring ‘06 “Tryptic Mapping of Recombinant Proteins by Matrix- Assisted Laser Desorption/Ionization Mass Spectrometry” Billeci & Stults (1993) Anal. Chem. 65, 1709 “Identifying Proteins from Two-Dimensional Gels by Molecular Mass Searching of Peptide Fragments in Protein Sequence Databases” Henzel et al. (1993) Proc. Natl. Acad. Sci. USA 90, 5011

6 Ming F. Tam Spring ‘06 Mass Spectrometric analysis Data analysis Partial fractionation of crude extracts Chromatographic/electrophoretic separation General procedure IP/Affinity/ ion Xchange + RP Gel/capillary electrophoresis With/without Digestion

7 Ming F. Tam Spring ‘06 Gel separation pH 3 - 10 NL, 18 cm strip IEF/SDS PAGE Angelika Gorg’s manuals http://www.weihenstephan.de/blm/deg/

8 Ming F. Tam Spring ‘06 Do I need to go through IEF? Read O’Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021. IEF in tube gels vs IPG Equilibrium vs non-equilibrium IEF

9 Ming F. Tam Spring ‘06 Usual Problems: Sample load volume vs concentration 3 mm tube, 1 cm = 70 ul With 1 mg protein loading, conc = 14.3 mg/ml Solubility, cytosolic vs membrane proteins, interfering materials salt, nucleic acids, lipids

10 Ming F. Tam Spring ‘06 Avoid pH extremes

11 Ming F. Tam Spring ‘06 Same sample, different range! You see part of the total at all time!

12 Ming F. Tam Spring ‘06 With 3-10, 18 cm strip, 2.57 cm per pI unit. With 6-11, 18 cm strip, 3 cm per pI unit. Short Range (around neutral pH), 24 cm gel strips. 18 cm 2 nd -D, assume 500 daltons separated by 2 mm, from top to bottom, covers only 45,000 daltons. Gradient vs linear polyacrylamide gel. 2D gel cannot see all the proteins!

13 Ming F. Tam Spring ‘06 2D gel cannot see all the proteins! Corthals et al. Electrophoresis (2000) 21, 1104-1115 E. Coli proteins N=878 N=1029

14 Ming F. Tam Spring ‘06 Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis Roncada et al. (2005) Proteomics 5, 2331- 2339. An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes Suh et al. (2005) Anal. Biochem. 343, 166-175.

15 Ming F. Tam Spring ‘06 Degraded/modified samples (multiple spots/protein) N= 2700, 500 ug N=852 Yeast proteins 2D gel see too many proteins!

16 Ming F. Tam Spring ‘06 Boss! How much protein should I load?

17 Ming F. Tam Spring ‘06 Gygi et al. Proc. Natl. Acad. Sci. USA 2000, 97: 9390-9395. “Evaluation of two-dimensional gel electrophoresis based proteome analysis technology”

18 Ming F. Tam Spring ‘06 Standard procedure Gel separation Isolate spots

19 Ming F. Tam Spring ‘06 You have to see the spots first! Visualization : Coomassie stain, Colloidal blue stain, Neuhoff et.al., Electrophoresis, 1985, 6, 427-448. Blue-silver stain, Electrophoresis (2004) 25, 1327-1333. Silver stain, www.narrador.emble-heidelberg.de/GroupPages/PageLink/activities/protocols.html Sypro dyes If U have the money!

20 Ming F. Tam Spring ‘06 Negative stain for not too complicated patterns. C. Fernandez-Patron et al. (1995) Anal. Biochem. 224, 203-211. Gillespie & Elliott (2005) Anal. Biochem. 345, 158-160. “Comparative advantage of imidazole—sodium dodecyl sulfate-Zinc reverse staining in polyacrylamide gel”

21 Ming F. Tam Spring ‘06 Standard procedure Gel separation Isolate spots Enzyme digestion

22 Ming F. Tam Spring ‘06 Many steps are no longer needed! Optimized Sample-Processing Time and Peptide Recovery For the Mass Spectrometric Analysis of Protein Digests Terry, DE, Umstot, E. and Desiderio, DM J Am Soc Mass Spectrom 2004, 15, 784-794

23 Ming F. Tam Spring ‘06 Most of the time use modified trypsin. NH 4 HCO 3 is the usual buffer. Why? Don’t need too much trypsin, 0.5 uM. Need Ca ++ for the reaction, ~500-fold of the enzyme or 250 uM. Excess Ca ++, what would happen? Temperature? Depends on whether you want internal standards. Additives in the buffer? Usual considerations:

24 Ming F. Tam Spring ‘06 Peptide extraction Can the peptides diffuse out? Solvents used? Microwave digestion & extraction Zhong et al. (2005) J Am Soc Mass Spectrom 16, 471-481. “Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification”

25 Ming F. Tam Spring ‘06 Sample preparation

26 Ming F. Tam Spring ‘06 It is not as the manufacturers claimed! Salts are bad!

27 Ming F. Tam Spring ‘06 On target washing

28 Ming F. Tam Spring ‘06 Zip-Tipped

29 Ming F. Tam Spring ‘06 This is a money game! Was NT$57 Now NT$66

30 Ming F. Tam Spring ‘06 Alternative approach Anal Chem (2003) 75, 663-666 Stop and Go Extraction Tips Or StageTips

31 Ming F. Tam Spring ‘06 CC  /acetone Nitrocellulose/acetone isopropanol Different matrix, different results

32 Ming F. Tam Spring ‘06 CC  Acetonitrile/TFA

33 Ming F. Tam Spring ‘06 SA/Acetonitrile/TFA

34 Ming F. Tam Spring ‘06 Ydj1p, Cog5p NC/CCA From #15 Ydj1p, Cog5p pyridine/CCA

35 Ming F. Tam Spring ‘06 Data acquisition 1.Clean samples! 2.Different matrix, different results 3.Calibration—linear for unknowns beyond the range, quadratic for unknowns within range

36 Ming F. Tam Spring ‘06 Linear vs. Quadratic MASS CalculatedLinear Obs. Quadratic Obs. 1115.601115.47 (117 ppm) 1115.52 (71 ppm) 1398.711398.54 (122 ppm) 1398.63 (58 ppm) 1446.731446.57 (111 ppm) 1446.64 (62 ppm) 1646.961646.78 (109 ppm) 1646.87 (55 ppm) 1746.751746.53 (126 ppm) 1746.64 (63 ppm) 1889.001889.76 (402 ppm) 1888.87 (69 ppm) Linear Standards: 1046.54, 2465.20 Quadratic standards: 1046.54, 1677.77, 2710.39

37 Ming F. Tam Spring ‘06 External vs. Internal RealExt. Obs.Int. Obs. 955.57955.70 (136 ppm)955.58 (10 ppm) 1231.691231.79 (81 ppm)1231.70 (8 ppm) 1928.941929.10 (83 ppm)1929.00 (31 ppm) 2043.872044.03 (78 ppm)2043.92 (24 ppm)

38 Ming F. Tam Spring ‘06 But watch out for Signal suppression!!!

39 Ming F. Tam Spring ‘06 You can never be too clean! MALDI spectrum of a silver stained empty gel spot

40 Ming F. Tam Spring ‘06

41

42 http://prospector.ucsf.edu/ucsfhtml4.0/misc/trypsin.htm

43 Ming F. Tam Spring ‘06 Peptide digests separated with cation exchanger/ reverse-phase on line with an ESI/ion trap. LC-LC/MS-MS (MUDPIT) to replace Gel/MS-MS Washburn et al. Nature Biotech 2001, 19: 242-247 “Large scale analysis of the yeast proteome by MultiDimensional Protein Identification Technology” Alternatives to 2-D gel Multidimensional protein identification technology

44 Ming F. Tam Spring ‘06 5 micron particles, 0.1 mm ID, 4 and 10 cm long

45 Ming F. Tam Spring ‘06 6212 ORF, 1484 proteins identified Predicted Observed Average # of peptides identified for each protein in a particular CAI range

46 Ming F. Tam Spring ‘06

47 Search the Databases

48 Ming F. Tam Spring ‘06 General free programs PAWS From Rockefeller U.  Genomic Solutions http://65.219.84.5/paws.html

49 Ming F. Tam Spring ‘06 Moverzhttp://65.219.84.5/moverz.htmlhttp://65.219.84.5/moverz.html http://bioinformatics.genomicsolutions.com/MoverZDL.html

50 Ming F. Tam Spring ‘06

51 Two types of data: Peptide mapping/peptide mass fingerprinting

52 Ming F. Tam Spring ‘06 Peptide mapping

53 Ming F. Tam Spring ‘06 Sbp1/R1-3

54 Ming F. Tam Spring ‘06 Fenyo (2000) Curr Opinion Biotech 11, 391-395 E coli ~ 4000 ORFs Yeast ~ 6000 ORFs Human ~ 100,000 ORFs Single tryptic peptide at a mass accuracy of 0.5 dalton

55 Ming F. Tam Spring ‘06 2nd type: MS/MS data

56 Ming F. Tam Spring ‘06 Another free program

57 Ming F. Tam Spring ‘06 On Web resources: Prowl at Rockefeller U http://prowl.rockefeller.edu/PROWL/prowl.ht ml

58 Ming F. Tam Spring ‘06

59

60

61 Genomic Solutions http://www.genomicsolutions.com/showPage.php http://www.genomicsolutions.com/showPage.php

62 Ming F. Tam Spring ‘06

63 Mascot at Matrix-Science http://www.matrixscience.com/ http://www.matrixscience.com/

64 Ming F. Tam Spring ‘06

65 EMBL http://www.narrador.embl-heidelberg.de/GroupPages/PageLink/peptidesearchpage.html http://www.narrador.embl-heidelberg.de/GroupPages/PageLink/peptidesearchpage.html

66 Ming F. Tam Spring ‘06

67 Blast2p http://dove.embl-heidelberg.de/Blast2/msblast.html Homology search with partial sequence from ms/ms data http://dove.embl-heidelberg.de/Blast2/msblast.html

68 Ming F. Tam Spring ‘06 http://magpie.ucalgary.ca/

69 Ming F. Tam Spring ‘06 Prospector at UCSF http://128.40.158.151/mshome3.4.htm http://128.40.158.151/mshome3.4.htm

70 Ming F. Tam Spring ‘06

71 EXPASY at SIB http://us.expasy.org/ http://us.expasy.org/

72 Ming F. Tam Spring ‘06

73 Guidelines for “protein identification” Taylor, GK & Goodlett, DR (2005) RCM 19, 3420

74 Ming F. Tam Spring ‘06

75 Write a paper on tandem MS. Approximately 3 pages, single spaced. Should cover the basic principal of the instrument and include an application. Due date: Thursday, April 12, noon. Send to: mftam@gate.sinica.edu.twmftam@gate.sinica.edu.tw

76 Ming F. Tam Spring ‘06

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