Biological Mass Spectrometry

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1 Biochemistry 412 Biological Mass Spectrometry February 13 th, 2007

2 Proteomics The study of the complete complement of proteins found in an organism

3 Degrees of Freedom for Protein Variability Covalent Modifications in Proteins Post-translational modifications (e.g., phosphorylation, glycosylation, etc.) - more than 200 such modifications are known, and they can occur at multiple sites in a single protein Alternative splicing of a primary transcript - in extreme cases, a single gene can produce tens of thousands of different mrnas! Proteolytic processing Protein aging Thus, there are probably many millions of different proteins in our bodies!!

4 More Reality Therapy re Proteins They have personalities : each behaves differently They exist in different concentrations, ranging over a million-fold It will be extremely difficult to even identify them all (see previous slide) Take-home message: Proteomics presents challenges that are orders-of-magnitude more difficult than those presented by genomics!

5 Divide and Conquer Proteomics Identification by fractionation, fragmentation, mass analysis, & comparison with database(s) of virtual peptides * *Note: MudPIT stands for multidimensional protein identification technology. Lin et al (2003) Biochim. Biophys. Acta 1646, 1.

6 There are two major types of soft ionization methods of mass spectrometry for use in analyzing samples of biological origin: Matrix-assisted laser desorption Electrospray

7 Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Mann et al (2001) Annu. Rev. Biochem. 70, 437.

8 Electrospray Ionization (ESI) Mass Spectrometry Griffiths et al (2001) Biochem. J. 355, 545.

9 Attractive Features of Mass Spectrometry High resolution (can measure mass differences of ~0.1 Da) High sensitivity (10-13 to [!] moles) Ability to work with complex samples (MALDI-MS) Ability to interface with other separation techniques (e.g., LC) Reasonably affordable

10 Example of an ESI Mass Spectrum Griffiths et al (2001) Biochem. J. 355, 545. Note: mass-charge ratio

11 Simple summary of mass spec-based proteomics Domon & Aebersold (2006) Science 312, 212.

12 Mass spectrometry-based proteomics strategies Note: this is non-trivial! Domon & Aebersold (2006) Science 312, 212.

13 2-stage ( tandem ) mass spectrometry with CID ( collisioninduced dissociation ) between MS1 and MS2 Combines mass determination with amino acid sequence determination => unique peptide identification Domon & Aebersold (2006) Science 312, 212.

14 Quantitation in mass spectrometry is a challenge! Domon & Aebersold (2006) Science 312, 212.

15 Now for some interesting recent applications of biological mass spectrometry...!!

16 A classic proteomics use of mass spectrometry: Inventorying the complete complement of proteins coded for by a genome Example: The proteome of Plasmodium falciparum (the malaria parasite) Methodology: Multidimensional Protein Identification Technology ( MudPIT *) *see Washburn et al (2001) Nature Biotechnol. 19,

17 MudPIT (Washburn et al [2001] Nature Biotechnol. 19, ) is a highly parallel, automated method for carrying out proteomics inventories of expressed proteins in a complex sample. It is comparable to microarray experiments that are used to inventory expressed mrnas in a tissue. MudPIT involves digesting complex mixtures of proteins isolated from whole organisms with proteases, followed by automated liquid chromatography, tandem mass spectrometry, and look up searches of databases of predicted proteins (inferred from DNA sequencing data) to identify peptides predicted to have originated from the expressed proteins contained in the sample.

18

19 Vaccine Targets in Malaria Susan Okie, M.D. N. Engl. J. Med. 357, November 3, 2005

20 Florens et al (2002) Nature 419, 520.

21 Generation of Distance Constraints for Protein Structure Determination Using a Combination of Protein Cross-Links and Mass Spectrometry

22

23 Young et al (2000)

24 Young et al (2000)

25 Young et al (2000)

26 Hydrogen-Deuterium Exchange and Mass Spectrometry to Probe Protein Dynamics, Solvent-Accessibility, and Protein-Protein Interactions

27

28 Hamuro et al (2002)

29 Hamuro et al (2002)

30 Hydrogen - Deuterium Exchange and Protein Conformational Fluctuations Hoofnagle et al (2003) Annu. Rev. Biophys. Biomol. Struct. 32, 1.

31 Hydrogen - Deuterium Exchange: Schematic Experimental Design Hoofnagle et al (2003) Annu. Rev. Biophys. Biomol. Struct. 32, 1.

32 Hamuro et al (2002)

33 Hamuro et al (2002)

34 Fragment-Based Drug Discovery

35

36 High local concentration of bound monophore (molecule shown in red) stabilizes the disulfide bond. HS-CH 2 CH 2 -OH Erlanson et al (2000)

37 Mass spect data Erlanson et al (2000)

38 Erlanson et al (2000)

39 The Monophore Binds in the Same Relative Orientation, Regardless of Attachment Site Erlanson et al (2000)

40 Erlanson et al (2000)

41 Pharmacology Using Imaging Mass Spectrometry

42

43 Chaurand et al (2002)

for the Novice Mass Spectrometry (^>, John Greaves and John Roboz yc**' CRC Press J Taylor & Francis Group Boca Raton London New York

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