Mass spectrometry: measuring molecular weight

The basic idea: weighing by charging

You cannot place a single molecule on an ordinary scale. Mass spectrometry solves this with a clever detour. The molecule first receives an electric charge, then it is sent through a field. How heavy a charged particle is determines how strongly it is deflected by that field or how fast it moves. From that motion the instrument calculates the mass back.

What a mass spectrometer actually measures is not the mass alone but the ratio of mass to charge. That ratio is called the m/z value. From the m/z value and the number of charges the molecular weight is derived. The result is a spectrum: a graph with peaks, where each peak represents a particle of a particular mass.

The three steps in every instrument

Every mass spectrometer works in three steps. First ionisation, in which the molecule receives a charge. For peptides one often uses soft methods that keep the molecule intact, such as electrospray ionisation or MALDI. Soft here means the peptide does not break into pieces during charging.

Then separation, in which the charged particles are sorted by mass in the analyser. And finally detection, in which a sensor counts how many particles of each mass arrive. That count becomes the spectrum. The height of a peak says something about how much of that particle was present.

What it tells you about a peptide

A peptide has a known amino acid sequence, and from that sequence you can add up the expected molecular weight. Mass spectrometry measures the real weight. If the measured value matches the calculated value, that is strong evidence the correct molecule is in the tube. If it differs, something is wrong: a wrong sequence, a missing piece, or an unwanted change to the molecule.

Modern instruments measure so accurately that they can see the difference of a single hydrogen atom. As a result they also reveal small changes, such as oxidation or a wrongly linked amino acid. That makes the method a fine-grained check on the identity and purity of a peptide.

Working together with HPLC

Mass spectrometry is often coupled to HPLC, a technique that first separates a mixture into individual components. The combination is called LC-MS. HPLC separates the substances in time, so they enter the mass spectrometer one by one. This gives you a separate mass measurement for each component.

This coupling is useful because a sample is rarely completely pure. With LC-MS you see not only the main product but also any by-products and their mass. It therefore gives a more complete picture than either technique alone.

Limits of the method

Mass spectrometry tells you the mass but not directly the three-dimensional shape of a molecule. Two molecules with the same atomic composition can have the same mass and yet be folded differently. The measurement also says nothing about biological activity. It confirms molecular identity, not function or safety.

For a full picture, laboratories therefore combine the mass measurement with other tests, such as HPLC for purity and sometimes extra analyses for structure. Together these form the analysis package on a certificate of analysis.