chapter 9 mass spectrometry (ms) -microbial functional genomics 조광평 cbbl

Chapter 9 Mass Spectrometry (MS)

-Microbial Functional Genomics

조광평CBBL

OutlineOutline

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

9.1 Introduction9.1 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 chapter① 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

9.2 Fundamentals of mass spectrometry (1/3)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)


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 analyzersI. Linear quadrupoles (Q)

II. Time of flight (TOF)

III. Sectors

IV. Quadrupole ion traps (QIT)

V. Fourier transform ion cyclotron resonance (FTICR)


9.2.4 Coupling separation methods with mass spectrometry 9.2.5 Ion structural characterizing

Two basic types of MS experiments to investigate structureI. Ion fragmentation studies

II. Ion reaction studies

9.3 Fundamentals of protein and peptide MS (1/5)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


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.


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.


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.)


9.3.2 Peptide measurements Two important points:

The tryptic peptides The sequence coverage

9.4 Mass spectrometry for protein and proteome characterization (1/3) 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

9.4 Mass spectrometry for protein and proteome characterization (2/3)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

9.4 Mass spectrometry for protein and proteome characterization (3/3)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.

9.5 Summary9.5 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 the slower, labor-intensive gel electrophoresis technologies with higher-throughput, wider dynamic range gel-less methods

chapter 9 mass spectrometry (ms) -microbial functional genomics 조광평 cbbl

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