An Efficient and Inexpensive Refrigerated LC System for H/D Exchange Mass Spectrometry

Abstract

Loss of deuterium label during the LC step in amide hydrogen/deuterium exchange mass spectrometry (H/D-MS) is minimized by maintaining an acidic mobile phase pH and low temperature (pH 2.5, 0°C). Here we detail the construction and performance of a low-cost, thermoelectrically refrigerated enclosure to house high-performance liquid chromatography (HPLC) components and cool mobile phases. Small volume heat exchangers rapidly decrease mobile phase temperature and keep the temperature stable to ±0.2°C. Using a superficially porous reversed-phase column, we obtained excellent chromatographic performance in the separation of peptides with a median peak width of 4.4 s. Average deuterium recovery was 80.2% with an average relative precision of 0.91%.

Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody

“…The flow path for our online digestion system [19] at different stages in the analysis process is shown in Figure 1.…”

“…The immobilized pepsin column washing procedure described in this work is not easily implemented on single valve systems [19] because injection interrupts the elution step (see Figure 1). This setup requires that the pepsin column wash must be delayed until the end of the gradient run.…”

“…A single-valve, two-pump system for H/D exchange with a refrigerated valve and column compartment was used to maintain 0°C throughout the online digestion and separation, as described previously (see Figure 1) [19]. Following sample injection, a loading pump carried the sample through a 50 mm × 2.1 mm immobilized pepsin column, prepared inhouse [14], using 0.1 % formic acid at a flow rate of 200 μL min -1 .…”

“…This test was performed using an HDX PAL robot (LEAP Technologies, Carrboro, NC, USA) with a refrigerated compartment held at 0°C housing the trap, columns and valves. The HDX PAL system is comprised of two valves and features a back-flush of the pepsin column to The system is comprised of a two-position, 12 port valve [19]. The red regions of the flow path denote the location of sample at the different stages of analysis.…”

Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

“…Totally deuterated control samples were prepared manually by labeling for at least 14 h at 22°C using a 5-fold excess of labeling buffer and then quenched as described above. Immediately prior to LC-MS analysis on a time-of-flight mass spectrometer (Agilent 6220; Santa Clara, CA, USA), individual samples were rapidly thawed by hand and injected into a refrigerated column compartment constructed in-house [27]. Following rapid proteolysis of the quenched samples in an immobilized pepsin column [28] at 0°C, ACTR peptides were desalted using a C 12 trap and separated with a water/acetonitrile gradient, as described previously [11].…”