colored models for four distinct time points). If only a single
model is used, it is highly recommended to include the deuterium incorporation timecourses (Fig. 3f), as this provides
essential information on the dynamics of the deuterium incorporation difference.
4 Notes
1. Peptide digestion is achieved using an acid-functional protease
such as pepsin, which is active at a pH range of 1–4. In the
methods described here, pepsin is immobilized on a column so
that it can be incorporated within the fluidics flowpath,
enabling online digestion. To maximize the digestion efficiency, the fluidics system was optimized to include two immobilized pepsin columns stored at different temperatures, the
first at 10
C and the second at 2
C. Storing the columns at
different temperatures facilitates a balance between favoring
proteolytic cleavage and minimizing back exchange. At
10
C, proteolytic cleavage is more efficient; however this
temperature is less effective at minimizing back exchange.
Moreover, amide hydrogen back exchange is decreased at
lower temperatures [42], a temperature less favorable for cleavage. There is a second function to having the two columns in
series as it increases the back pressure, which has been shown to
improve digestion efficiency [43]. In our system, the back
pressure of the two pepsin columns should never exceed
2500 psi. Typically the pressure reaches its maximum during
the digestion/desalting phase, when the pepsin columns are in
line with the C18 trap column (Schematic 1). Sequence coverage can be maximized by optimizing the quench composition
(see Note 4), increasing digestion time by decreasing the flow
rate over pepsin, or increasing protein amount.
2. A key factor to consider for obtaining best results is optimizing
the protein–membrane interaction conditions. Protein and
lipid concentrations, lipid presentation, vesicle size, and membrane compositions are all factors that require careful optimization. Lipids can be presented in membrane mimics, bicelles,
nanodiscs, and nanotubes. Membrane compositions and vesicle
size must be optimized so as to mimic the cell membrane of
interest (plasma membrane, golgi membrane, mitochondrial
membrane, etc). These parameters can all be optimized using
a variety of biophysical assays such as protein-lipid fluorescence
resonance energy transfer (FRET) assays, lipid sedimentation,
lipid flotation, surface plasmon resonance (SPR). Final concentrations of both proteins and vesicles should be carefully optimized in order to maximize interaction kinetics while
minimizing problematic scenarios associated with high protein
480
Jordan T. B. Stariha et al.
model is used, it is highly recommended to include the deuterium incorporation timecourses (Fig. 3f), as this provides
essential information on the dynamics of the deuterium incorporation difference.
4 Notes
1. Peptide digestion is achieved using an acid-functional protease
such as pepsin, which is active at a pH range of 1–4. In the
methods described here, pepsin is immobilized on a column so
that it can be incorporated within the fluidics flowpath,
enabling online digestion. To maximize the digestion efficiency, the fluidics system was optimized to include two immobilized pepsin columns stored at different temperatures, the
first at 10
C and the second at 2
C. Storing the columns at
different temperatures facilitates a balance between favoring
proteolytic cleavage and minimizing back exchange. At
10
C, proteolytic cleavage is more efficient; however this
temperature is less effective at minimizing back exchange.
Moreover, amide hydrogen back exchange is decreased at
lower temperatures [42], a temperature less favorable for cleavage. There is a second function to having the two columns in
series as it increases the back pressure, which has been shown to
improve digestion efficiency [43]. In our system, the back
pressure of the two pepsin columns should never exceed
2500 psi. Typically the pressure reaches its maximum during
the digestion/desalting phase, when the pepsin columns are in
line with the C18 trap column (Schematic 1). Sequence coverage can be maximized by optimizing the quench composition
(see Note 4), increasing digestion time by decreasing the flow
rate over pepsin, or increasing protein amount.
2. A key factor to consider for obtaining best results is optimizing
the protein–membrane interaction conditions. Protein and
lipid concentrations, lipid presentation, vesicle size, and membrane compositions are all factors that require careful optimization. Lipids can be presented in membrane mimics, bicelles,
nanodiscs, and nanotubes. Membrane compositions and vesicle
size must be optimized so as to mimic the cell membrane of
interest (plasma membrane, golgi membrane, mitochondrial
membrane, etc). These parameters can all be optimized using
a variety of biophysical assays such as protein-lipid fluorescence
resonance energy transfer (FRET) assays, lipid sedimentation,
lipid flotation, surface plasmon resonance (SPR). Final concentrations of both proteins and vesicles should be carefully optimized in order to maximize interaction kinetics while
minimizing problematic scenarios associated with high protein
480
Jordan T. B. Stariha et al.