SPECTROMETRY - INTRODUCTION
spectrometry is invaluable for measuring the molecular weight of proteins. Typical
analysis can identify the molecular weight of a 20,000 kD protein to within +/- 2.0
daltons while analysis of peptides can be even more accurate. It is for these
reasons that we wish to use mass spectrometry to investigate the incorporation of hydrogen
into proteins during hydrogen exchange.
Because deuterium weights 2 Da and hydrogen weighs 1 Da, we can easily distinguish between
deuterated and non-deuterated proteins and/or peptides.
This is a simple diagram of
a mass spectrometer. It can be divided into three parts: ionization, mass
analyzer, and detector.
Proteins and peptides must first be converted to the gas phase via ionization.
Ionization of proteins/peptides can be done in several ways. MALDI (matrix assisted laser
desorption mass spectrometry) uses a laser beam to zap crystallized protein/matrix
mixtures into the gas phase. ESI (electrospray ionization) converts a liquid-protein
solution into fine droplets. The solvent is evaporated from the fine droplets and
charge is deposited on the protein molecules. ESI can be coupled to HPLC. As
peptides/proteins elute from an HPLC column they are sent directly into the mass
spectrometer where they are ionized.
Once proteins/peptides are ionized, they must be separated according to their molecular
weight. There are several types of mass analyzers: magnetic sector,
quadrupole, ion trap, time-of flight.
Magnetic sector: uses
a magnetic field to separate
Quadrupole: uses a
combination of RF fields and voltage to separate
Ion trap: a 3D
quadrupole, uses RF and electric fields to separate
separates with time. Heavier molecules take longer to fly down a tube than lighter
All mass analysis is done in
a vacuum so that molecules do not collide with each other. Sometimes, molecular
collisions are desirable and can be used to fragment larger molecules into smaller
ones. This procedure is called MS/MS.
After a collection of proteins/peptides have been
separated according to mass, they must be detected. Ions can be detected with
electron multipliers or with diode array detectors.
SPECTROMETRY - DETAILS
METHOD TO CHOOSE?
Currently, there are two primary ionization methods for
doing hydrogen exchange analysis:
In the past, FAB was also used.
- HPLC step washes away all the labile hydrogens at side
- Temperature and pH are easily controlled
- When coupled to HPLC, large, complex digests can be
- Samples must be desalted and free of impurities
- Multiple charge states can be complicating
- Potential for higher throughput
- Deuterium losses can be harder to control
- Generally easier to do than ESI
- All data are aquired in a single spectra
- Sample salts and impurities are much more tolerated
- Lack of multiple charge states make spectra simple
People using MALDI:
Komives Lab, UCSD
Use of MALDI to look for binding surfaces
(PNAS 95, 14705-14710)
Fitzgerald Lab, Duke
SUPREX technique to analyze protein stability
(PNAS 97, 8296-8301)
K. Takio, Japan
(Protein Science 9, 2497)