Mass Spectrometers

Mass spectrometers measure the mass of charged molecules. A charged molecule moves through an electrical or magnetic field in a precise way determined by its mass. Mass spectrometry became important to proteomics when researchers discovered how to gently put a charge on proteins without destroying them.

Mass Spectrometry Tutorials

A number of good mass spectrometry tutorials are available on the web. There are also several animations which demonstate the principles and operation of mass spectrometers. You may need Flash to see the animations. In addition there are a number of tutorials dedicated to specific types of mass spectrometers. See the related links to the left.

Tandem mass spectrometers are built out of two mass spectrometers. The first one selects the peptides by mass one by one and the second mass spectrometer reads out the intensities of the fragment ions of each peptide.

Many different combinations of mass spectrometers have been tried. A popular configuration today is a quadrupole linked to a time-of-flight (called a Q-TOF). Another common combination is one time-of-flight linked to another time-of-flight (called a TOF-TOF).

Ion-trap mass spectrometers are unique in that they can trap the peptide ions. The ion trap holds the peptides until they fragment, then it switches mode and measures the fragment ions. Thus the ion trap acts as both the first and second mass spectrometer. Because this is the functional equivalent of a tandem mass spectrometer for the price of a single one, ion traps are popular with those who have limited budgets.

Mass spectrometers for proteomics are expensive. A used ion trap costs $50,000 to $100,000. A new Q-TOF or TOF-TOF costs $300,000 to $500,000. A new FTICR costs around $800,000.

Tandem Mass Spectrometer Components

To identify proteins using a tandem mass spectrometrer, the instrument needs:

1. a way to ionize the peptides,
2. a first mass spectrometer that selects a peptide by its mass,
3. a means of fragmenting this peptide, and
4. a second mass spectrometer to read out the fragments.

Mass Spectrometry Principles

• Charged particles curve when traveling though a magnetic field.
• Heavier particles accelerate more slowly.

Many different mass spectrometry designs — quadrupole, ion-trap, Fourier transform ion-cyclotron resonance — use magnets.

Time-of-flight mass spectrometers measure the time it takes a given peptide to travel a given distance.

Ionization of Peptides

Proteins in living organisms are in liquid and are usually not charged. Mass spectrometers can measure only charged particles in a gas. Mass spectrometry for proteomics became possible only when this problem was solved. Two people got Nobel prizes for their solutions:

• One solution is electrospray ionization (ESI). Peptides in an acidic liquid are sprayed in a fine mist. The liquid in the droplets evaporates, leaving the peptide plus positively charged hydrogen ions from the acid.
• The second solution is Matrix-Assisted Laser Dissociation Ionization (MALDI). Peptides are dried onto a solid surface (the matrix), which is then hit with a laser beam. The peptides float free as the matrix disintegrates.

Fragmenting Peptides

After being selected in the first component of the tandem MS/MS, the peptides are dissociated into fragments that the second component can analyze.

The detectors in a mass spectrometer count charged particles — ions. To be detectable, therefore, the fragments must have an associated charged hydrogen ion — a proton. This "mobile proton" determines the peaks in the mass spectrum.

You can try fragmenting proteins yourself (in cyberspace) with MS-Product - a program that predicts the way that peptides fragment.

Interpreting Spectra of Fragmented Peptides

Peptides are most likely to break at their weakest link. Mass spectrometers are tuned so that the weakest link is between the amino acids. When the peptide breaks, both ends can be detected (if charged), so each break gives two daughter ions. See the figure above. These daughter ions form the MS/MS spectrum.

In the figure above the peptide above consists of the amino acid chain QAMHW. If it fragments perfectly between the amino acids, it will create the daughter b-ions on the left (b1=Q, b2=QA, b3=QAM …) and the daughter y-ions on the right (y1=W, y2=HW, y3=MHW…). These daughter ions yield the MS/MS spectrum seen here.

Interpretation of mass spectra is dealt with in much more detail in the Proteomics section of this web site on the page entitled "Manual Verification of Peptide Identifications".