sequences. Mass spectrometry Hydrogen–deuterium exchange mass spectrometry can determine the overall deuterium content of molecules which have undergone H/D exchange. Because of the sample preparation required, it is typically considered to provide an accurate measurement of non-exchangeable hydrogen atoms only. It can also involve H/D exchange in the gas phase or solution phase exchange prior to ionization. It has several advantages in NMR spectroscopy with respect to analysis of H–D exchange reactions: much less material is needed, the concentration of sample can be very low (as low as 0.1 uM), the size limit is much greater, and data can usually be collected and interpreted much more quickly. The deuterium nucleus is twice as heavy as the hydrogen nucleus because it contains a neutron as well as a proton. Thus a molecule that contains some deuterium will be heavier than one that contains all hydrogen. As a protein is increasingly deuterated, the molecular mass increases correspondingly. Detecting the change in the mass of a protein upon deuteration was made possible by modern protein mass spectrometry, first reported in 1991 by Katta and Chait. Determining site specific deuteration via mass spectrometry is more complicated than using NMR spectroscopy. For example, the location and relative amount of deuterium exchange along the peptide backbone can be determined roughly by subjecting the protein to proteolysis after the exchange reaction has been quenched. Individual peptides are then analyzed for overall deuteration of each peptide fragment. Using this technique the resolution of deuterium exchange is determined by the size of the peptides produced during digestion. Pepsin, an acid protease, is commonly used for proteolysis, as the quench pH must be maintained during the proteolytic reaction. To minimize the back-exchange, proteolysis and subsequent mass spectrometry analysis must be done as quickly as possible. HPLC separation of the peptic digest is often carried out at low temperature just prior to electrospray mass spectrometry to minimize back-exchange. More recently, UPLC has been used due to its superior separation capabilities. It was proposed in 1999 that it might be possible to achieve single-residue resolution by using collision-induced dissociation (CID) fragmentation of deuterated peptides in conjunction with tandem mass spectrometry. It was soon discovered that CID causes "scrambling" of the deuterium position within the peptides. However, fragmentation produced by MALDI in-source decay (ISD), electron capture dissociation (ECD), and electron transfer dissociation (ETD) proceed with little or no scrambling under the correct experimental