Table 1.

Current alternative or complementary experimental approaches and their pros and cons regarding each HDX-MS experimental limitation.

HDX-MS limitations	Alternative/complementary approaches	Short description	Advantages	Disadvantages
Peptide-level resolution	Gas-phase fragmentation: electron transfer dissociation (ETD) or electron capture dissociation (ECD) (24, 39, 44–47)	-Peptide ions are fragmented by ETD/ECD instead of CID. The ck and zk fragment ions report the nascent D content of the associated fraction of the parent peptide ion	-With gentle ESI conditions, fragments from ETD/ECD are accompanied by little to no H/D scrambling and can be solved to determine the D occupancy with resolution approaching residue level	-H/D scrambling can still occur without the optimized ion optics and gentle ESI conditions -Lack of easy-to-use and reliable software for ETD/ECD data processing -Impractical for routine implementation
Insufficient peptide coverage (specifically with large complexes, highly glycosylated Ag, and disulfide-bonded Ab)	Ion mobility spectrometry (IMS) (38, 45)	-Incorporation of IMS after the chromatography step deconvolutes unresolved overlapping peptides in chromatographic separation	-Increases the resolving power for overlapping mass-spectra and allows for identification of more peptides	-Challenge in routinely incorporating IMS in HDX-MS experiment (complicated experimental setup)
	Enzymatic deglycosylation of the glycoprotein (48–50)	-PNGase F prior to HDX-MS labeling	-Easy to implement -Reduces complexity of resulting peptides -Enhances detectability of glycosylated regions of the protein	-Risk destabilizing native structure of the Ag and can lead to aggregation -May misinterpret Ab-Ag interacting site
		-PNGase A or PNGase H+ after HDX-MS labeling	-Allows characterization of the native conformational dynamics and interaction at the glycosylation sites	-Requires offline pepsin digestion and manual sample injection into the LC-MS system
	Disulfide bond reduction for Ab (19, 38, 51)	-The chemical reductant TCEP is commonly added to the quench buffer at high concentration	-Protein becomes more protease susceptible and increases sequence coverage	-Can deteriorate both LC and MS performance
	Improve digestion efficiency (52–54)	-Multiple replicate pepsin digestions -Alter digestion conditions (e.g., off-line digestion, denaturants, etc) -Change to or supplement with another protease	-Produces more reproducible peptides -Generates many new short and overlapping peptide fragments due to different cleavage specificities of different digestive enzymes	-Material and time cost of experiment increases
Discern the difference between direct binding interface and allosteric conformational change	Complementary experiments and assays (10, 11, 13, 20, 25, 32, 55, 56)	-Site-directed mutagenesis followed by functional assays -Disulfide trapping in cells -Chemical crosslinking with MS -Kinetic millisecond HDX-MS (TRESI-HDX)	-Provides additional information to increase the certainty and better define the directly contacting regions	-Might need to try multiple approaches to reach a conclusion