Hydrogen Exchange Mass Spectrometry: Are We Out of the Quicksand?

Abstract

Although the use of hydrogen exchange (HX) mass spectrometry (MS) to study proteins and protein conformation is now over 20 years old, the perception lingers that it still has “issues”. Is this method, in fact, still in the quicksand with many remaining obstacles to overcome? We do not think so. This critical insight addresses the “issues” and explores several broad questions including: have the limitations of HX MS been surmounted and has HX MS achieved “indispensable” status in the pantheon of protein structural analysis tools.

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

“…All experiments were performed in duplicate. The error of measuring the mass of each peptide was ± 0.20 Da in this experimental setup, consistent with previously obtained values [37][38][39]. Deuterium uptake was calculated by subtraction of the centroid of the isotopic distribution for peptide ions from undeuterated protein from the centroid of the isotopic distribution for peptide ions from the deuterium-labeled sample.…”

Replication in Bioanalytical Studies with Hdx MS: Aim as High as Possible

“…HDX MS is finding use in many areas [2] including in the biopharmaceutical industry [3] where protein comparability can be a central question. Just as measurement of HDL levels in a single patient can vary depending on the time of day, fasting status, age, genetics, etc., so too can the levels of deuterium in a sample according to parameters such as temperature, pH, time, analysis instrumentation and analyst [4]. An important question, therefore, is how reproducible are HDX MS measurements?…”

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

“…The number of D atoms carried on each peptide fragment is given by its measurable increase in mass, usually calculated as the increment in the peptide mass centroid. These results provide structural information resolved to the level of individual fragments and are broadly able to indicate where in the protein important behavior occurs (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level.…”

“…However, routine NMR analysis is limited to small highly soluble proteins that can be obtained in quantity (multi mgs), isotopically labeled ( 15 N, 13 C), and studied at millimolar concentration. A developing technology, HX measured and analyzed by mass spectrometry (HX MS) (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) can extend this proven capability to the much larger and more complex protein systems that make biology work. The method requires only picomoles of protein at submicromolar concentrations; it can be used to study the properties and functioning of proteins at any condition one chooses, and the experimental protein need not even be very pure.…”

“…Given this ideal dataset, simple subtraction of the D-dependent mass centroid increment of neighboring overhanging peptides would allow carried D label to be computed at amino acid resolution. These approaches avoid the H-D scrambling problem encountered in gas-phase cleavage by collision-induced dissociation (5,39,40) but they present some difficult technical challenges (7,21), especially for sequence regions in larger proteins that are distant from the chain termini. A promising alternative is to proceed initially with the proteolytic fragmentation approach (below) and then select particular peptides for further ECD/ETD fragmentation analysis (21,30) Significance This paper shows how hydrogen exchange-mass spectrometry data can be deconvolved to obtain direct protein structural information at amino acid resolution.…”