2.3 Description
of Mass Spectrometer
and Fluidics Setup
The processing of proteins and/or membranes of interest for HDX
analysis is carried out using an integrative fluidics system consisting
of the following three instruments: an HDx-3 PAL liquid handling
robot and climate-controlled chromatography system (LEAP Technologies), a Dionex UltiMate 3000 UHPLC system, and an Impact
HD QTOF mass spectrometer (Bruker). These components act in
unison to digest and desalt the deuterated proteins of interest,
separate the resulting peptides, and deliver the peptides to the
mass spectrometer for high-resolution mass analysis.
The HDx-3 PAL liquid handling robot is capable of fully
automating the setup of HDX-MS samples; however, we primarily
set these samples manually. This provides two major advantages:
First, we can setup extremely short pulses of deuterium (<10 s) that
is not possible with a robotic approach, and second, we can setup all
HDX samples within 1–2 h, limiting any possible problems with
protein instability over the many hours to days it would have to sit
in the liquid handling robot. For any samples performed with the
robot, it is essential that there is testing to verify the long-term
stability of the protein of interest and that it can handle storage at
2
C for the entire length of the HDX experiment without aggregation/degradation.
The major advantage of the HDx-3 PAL system is that it can
automate all injection, digestion, desalting, separation, and washing steps. This can also be performed with a manual valve system,
which is utilized to direct sample constituents throughout the
immobilized flowpath. A diagram of a representative fluidics flowpath, is shown in Fig. 1.
Protein is injected onto the system, and digested over two
immobilized pepsin columns (Poroszyme™ Immobilized Pepsin
Cartridge, 2.1 mm  30 mm; Thermo Fisher) held at 10
C and
2
C, respectively, at a flow of 200 μL/min for 3 minutes (see Note
1 for tips on optimal digestion). The resulting peptides are collected
and desalted on a C18 trap column [Acquity UPLC
Ⓡ BEH™ C18
1.7 μm column (2.1 Â 5 mm); Waters]. Once digestion and collection/desalting are complete, the C18 trap is put in line with the
analytical column. The UltiMate 3000 UHPLC controls the flow
and gradient of Mobile Phase A + B over the C18 trap and analytical
columns, eluting peptides onto the mass spectrometer, with separation occurring at 40 μL/min ~10,000 psi. Nonlinear-shaped
acetonitrile gradients are used to generate elution profiles with an
equal spread of peptides over time. The short gradient can lead to a
large number of peptides eluting per unit time, complicating the
tandem MS/MS analysis required for peptide identification of
non-deuterated samples. This is particularly true for proteins
>250 kDa. For this reason, any project studying proteins larger
than 250 kDa uses two different gradients for MS/MS analysis,
including a ~20-min short gradient which is the same as the one
used for MS analysis of deuterated samples and a ~60-min long
Probing Protein-Membrane Interactions Using HDX-MS
469
of Mass Spectrometer
and Fluidics Setup
The processing of proteins and/or membranes of interest for HDX
analysis is carried out using an integrative fluidics system consisting
of the following three instruments: an HDx-3 PAL liquid handling
robot and climate-controlled chromatography system (LEAP Technologies), a Dionex UltiMate 3000 UHPLC system, and an Impact
HD QTOF mass spectrometer (Bruker). These components act in
unison to digest and desalt the deuterated proteins of interest,
separate the resulting peptides, and deliver the peptides to the
mass spectrometer for high-resolution mass analysis.
The HDx-3 PAL liquid handling robot is capable of fully
automating the setup of HDX-MS samples; however, we primarily
set these samples manually. This provides two major advantages:
First, we can setup extremely short pulses of deuterium (<10 s) that
is not possible with a robotic approach, and second, we can setup all
HDX samples within 1–2 h, limiting any possible problems with
protein instability over the many hours to days it would have to sit
in the liquid handling robot. For any samples performed with the
robot, it is essential that there is testing to verify the long-term
stability of the protein of interest and that it can handle storage at
2
C for the entire length of the HDX experiment without aggregation/degradation.
The major advantage of the HDx-3 PAL system is that it can
automate all injection, digestion, desalting, separation, and washing steps. This can also be performed with a manual valve system,
which is utilized to direct sample constituents throughout the
immobilized flowpath. A diagram of a representative fluidics flowpath, is shown in Fig. 1.
Protein is injected onto the system, and digested over two
immobilized pepsin columns (Poroszyme™ Immobilized Pepsin
Cartridge, 2.1 mm  30 mm; Thermo Fisher) held at 10
C and
2
C, respectively, at a flow of 200 μL/min for 3 minutes (see Note
1 for tips on optimal digestion). The resulting peptides are collected
and desalted on a C18 trap column [Acquity UPLC
Ⓡ BEH™ C18
1.7 μm column (2.1 Â 5 mm); Waters]. Once digestion and collection/desalting are complete, the C18 trap is put in line with the
analytical column. The UltiMate 3000 UHPLC controls the flow
and gradient of Mobile Phase A + B over the C18 trap and analytical
columns, eluting peptides onto the mass spectrometer, with separation occurring at 40 μL/min ~10,000 psi. Nonlinear-shaped
acetonitrile gradients are used to generate elution profiles with an
equal spread of peptides over time. The short gradient can lead to a
large number of peptides eluting per unit time, complicating the
tandem MS/MS analysis required for peptide identification of
non-deuterated samples. This is particularly true for proteins
>250 kDa. For this reason, any project studying proteins larger
than 250 kDa uses two different gradients for MS/MS analysis,
including a ~20-min short gradient which is the same as the one
used for MS analysis of deuterated samples and a ~60-min long
Probing Protein-Membrane Interactions Using HDX-MS
469