. 2020 Mar 25;29(6):1285–1301. doi: 10.1002/pro.3853

Table 1.

Summary of the different parameters that can be modulated to improve HDX‐MS analysis

Modulation of experimental conditions for improved sequence coverage and data quality
Quench and digestion	Standard: pH: 2.5 Temperature: 0°C Protease: Porc pepsin √ Other proteases: Rhizopuspepsin9 Nepethesin I Nepenthesin II10 √ On‐column digestion yields more peptides than in‐solution digestion.11 It is worth making its own column with its favorite protease.12 √ Increased pressure on‐column by increasing flow13 or using a back‐pressure regulator.14 So far only possible on BEH pepsin columns. √ Increased protein concentration 15, 16 √ Useful additives: Urea, TCEP, Gu‐HCl,13 and detergent
Liquid chromatography‐mass spectrometry	Standard: LC: Reverse‐phase chromatography on C18 column with prior trapping for desalting. MS: Often Synapt or Xevo—G2 in MS^E mode √ Drift‐time aligned MS^E (HDMS^E)17 √ LC column with shorter alkyl chain—for example, BEH C4 or C813 √ Gradient optimization (8–30%, 8–50%)15 √ Saw‐tooth gradient after peptides elution to prevent carryover16
Miscellaneous observations	Use of PEG‐based detergents (e.g., Triton X‐100) complicates peptides identification. Good practice to start with a benchmark condition—for example, locked protein.16, 18, 19 Adding TCEP helps but too much (>1 M) reduces spectral quality.15

Modulation of experimental conditions for improved sequence coverage and data quality

Quench and digestion

Standard: pH: 2.5 Temperature: 0°C Protease: Porc pepsin

√ Other proteases:

Rhizopuspepsin9

Nepethesin I

Nepenthesin II10

√ On‐column digestion yields more peptides than in‐solution digestion.11 It is worth making its own column with its favorite protease.12

√ Increased pressure on‐column by increasing flow13 or using a back‐pressure regulator.14 So far only possible on BEH pepsin columns.

√ Increased protein concentration 15, 16

√ Useful additives:

Urea, TCEP, Gu‐HCl,13 and detergent

Liquid chromatography‐mass spectrometry

Standard:

LC: Reverse‐phase chromatography on C18 column with prior trapping for desalting.

MS: Often Synapt or Xevo—G2 in MS^E mode

√ Drift‐time aligned MS^E (HDMS^E)17

√ LC column with shorter alkyl chain—for example, BEH C4 or C813

√ Gradient optimization (8–30%, 8–50%)15

√ Saw‐tooth gradient after peptides elution to prevent carryover16

Miscellaneous observations

Use of PEG‐based detergents (e.g., Triton X‐100) complicates peptides identification.

Good practice to start with a benchmark condition—for example, locked protein.16, 18, 19

Adding TCEP helps but too much (>1 M) reduces spectral quality.15

Note: The tick indicates parameters that have shown improvement compared with the standard conditions.

Abbreviations: BEH, Ethylene Bridged Hybrid; HDX‐MS, hydrogen–deuterium exchange coupled to mass spectrometry; LC, Liquid Chromatography; MS, Mass spectrometry; PEG, Polyethylene Glycol; TCEP, tris(2‐carboxyethyl)phosphine; HDMS, High Definition MS .