1. Chapter 9 Mass Spectrometry (MS) -Microbial Functional Genomics 조광평 CBBL

2. Outline  9.1 Introduction  9.2 Fundamentals of mass spectrometry  9.3 Fundamentals of protein and peptide mass spectrometry  9.4 Mass spectrometry of protein and proteome characterization  9.5 Summary

3. 9.1 Introduction  Mass spectrometry is a structural biological tool  Measurement of molecular ion of intact and fragmented biomolecules  The objective of this chapter is to illustrate how MS is becoming an essential tool for characterizing complex mixtures of protein  Two sections of this chapter ① Fundamentals of biological MS, major ionization methods, different types of mass analyzers, interrogating ion structure, characterizing proteins and peptide ② Status of MS for complex protein measurements, bottom-up approaches and top- down approaches

4. 9.2 Fundamentals of mass spectrometry (1/3)  9.2.1 Basic components of any mass spectromete  Three important fundamental components: I.Ion source II.Ion analyzer III.Ion detector  9.2.2 Ionization methods  Two main methods: I.Electrospray ionization (ESI or ES) II.Matrix-assisted laser desorption / ionization (MALDI)

5. 9.2 Fundamentals of mass spectrometry (2/3)  9.2.3 Mass analyzers  The figures of merit of analyzer I.Mass resolving power II.Mass accuracy III.Mass range IV.Detection limits V.Dynamic range VI.Scan speed VII.Tandem MS  Mass analyzers I.Linear quadrupoles (Q) II.Time of flight (TOF) III.Sectors IV.Quadrupole ion traps (QIT) V.Fourier transform ion cyclotron resonance (FTICR)

6. 9.2 Fundamentals of mass spectrometry (3/3)  9.2.4 Coupling separation methods with mass spectrometry  9.2.5 Ion structural characterizing  Two basic types of MS experiments to investigate structure I.Ion fragmentation studies II.Ion reaction studies

7. 9.3 Fundamentals of protein and peptide MS (1/5)  Some of basic terms: I.Average molecular mass II.Monoisotopic molecular mass III.Isotopic packet IV.Protonated

8. 9.3 Fundamentals of protein and peptide MS (2/5)  9.3.1 Protein measurements Figure 9.1 Electrospray Fourier transform ion cyclotron resonance (ES-FTICR) mass spectra of the protein ubiquitin, illustrating the multiply charged ions observed in the positive ion mass spectra (a), and the deconvoluted view (b) showing the isotopic molecular region.

9. 9.3 Fundamentals of protein and peptide MS (3/5) Figure 9.2 Deconvoluted mass spectrum of the collisional dissociation (MS/MS) of the 10 + charge state (with an m/z of 857) of ubiquitin. Inset reveals the fragment ion identities and sequence locations.

10. 9.3 Fundamentals of protein and peptide MS (4/5) Figure 9.3 Alphabetic code used to designate fragment ion types and locations from a generic peptide. (Data from Roepstorff and Fohlman, 1984; Biemann, 1988.)

11. 9.3 Fundamentals of protein and peptide MS (5/5)  9.3.2 Peptide measurements  Two important points:  The tryptic peptides  The sequence coverage

12. 9.4 Mass spectrometry for protein and proteome characterization (1/3)  While the genome is static, the proteome is dynamic  9.4.1 Overview of MS approaches for protein studies  Protein analysis by MS-based methodologies can be broken down into 3 general areas  Individual protein analysis  Protein complex analysis  Whole-proteome analysis  Mass spectrometry analysis techniques  Bottom-up proteomics  Top-down proteomics

13. 9.4 Mass spectrometry for protein and proteome characterization (2/3)  Data processing and bioinoformatics  Peptide mass fingerprinting (PMF)  MS/MS spectral searching  9.4.2 Bottom-up Mass spectrometry proteomics  Two main approaches for bottom-up MS proteomic measurements  More traditional method employs conventional gel electrophoresis as the first step to separate and visualize proteins  More recent technique exploits the capabilities of high-resolution liquid chromatography (either in a one- or two-dimensional mode) as an online interface with MS  9.4.3 Top-down MS proteomics  Sample preparation  Molecular mass measurement  Structural interrogation

14. 9.4 Mass spectrometry for protein and proteome characterization (3/3)  9.4.4 Relating mass spectrometry proteomic data to biological information Figure 9.8 Functional category piechart of protein classes identified by shotgun proteomics LC-MS/MS methodology for the microbe Shewanella oneidensis. (Reprinted from VerBerkmoes et al., 2002.)Reprinted with permission from J. Proteome Res., 2002, vol. 1, pp. 239–252. Copyright (2002) Aerican Chemical Society.

15. 9.5 Summary  MS is likely to provide new information that might not have been easily achieved with the traditional molecular biology hypothesis- driven approach  The future of this field is likely to be marked by a replacement of t he slower, labor-intensive gel electrophoresis technologies with hi gher-throughput, wider dynamic range gel-less methods