Chapter 9 Mass Spectrometry (MS)
-Microbial Functional Genomics
조광평
CBBL
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
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 topdown approaches
9.2 Fundamentals of mass spectrometry (1/3)
 9.2.1 Basic components of any mass spectromete
 Three important fundamental components:
I.
II.
Ion source
Ion analyzer
III. Ion detector
 9.2.2 Ionization methods
 Two main methods:
I.
II.
Electrospray ionization (ESI or ES)
Matrix-assisted laser desorption / ionization (MALDI)
9.2 Fundamentals of mass spectrometry (2/3)
 9.2.3 Mass analyzers
 The figures of merit of analyzer  Mass analyzers
I.
II.
III.
IV.
V.
VI.
VII.
Mass resolving power
Mass accuracy
Mass range
Detection limits
Dynamic range
Scan speed
Tandem MS
I.
II.
III.
IV.
V.
Linear quadrupoles (Q)
Time of flight (TOF)
Sectors
Quadrupole ion traps (QIT)
Fourier transform ion cyclotron resonance (FTICR)
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.
II.
Ion fragmentation studies
Ion reaction studies
9.3 Fundamentals of protein and peptide MS (1/5)
 Some of basic terms:
I.
II.
III.
IV.
Average molecular mass
Monoisotopic molecular mass
Isotopic packet
Protonated
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.
9.3 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.
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.)
9.3 Fundamentals of protein and peptide MS (5/5)
 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)
 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)
 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)
 9.4.4 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 Summary
 MS is likely to provide new information that might not have been
easily achieved with the traditional molecular biology hypothesisdriven 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