Mass 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.

IONIZATION
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.

MASS ANALYZER
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

Time-of-flight: separates with time.   Heavier molecules take longer to fly down a tube than lighter molecules

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.

DETECTOR
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.

WHICH IONIZATION METHOD TO CHOOSE?

Currently, there are two primary ionization methods for doing hydrogen exchange analysis:

Electrospray (ESI)

MALDI

In the past, FAB was also used.

Electrospray

• HPLC step washes away all the labile hydrogens at side chain positions
• Temperature and pH are easily controlled
• When coupled to HPLC, large, complex digests can be analyzed
• Samples must be desalted and free of impurities
• Multiple charge states can be complicating

MALDI

• 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 interfaces
(Protein Science 9, 2497)