resolution native MS [7]. Native MS is currently implemented in
most structural biology departments to complement conventional
structural biophysical techniques like crystallography, electron
microscopy, NMR, or SAXS. The methodology is versatile as any
type of multi-protein complex can be analyzed, ranging from
homo-oligomeric proteins [4] to hetero-oligomers [8, 9] or even
protein/nucleic assemblies [10, 11] and membrane complexes
[12–14]. Latest methodological breakthroughs in the field include
the direct analysis of complexes from cell lysates [15] or from native
membranes [13] and the release of a new instrument enabling the
complete analysis of very large complexes with unprecedented
resolution [16].
In this chapter, we illustrate the possibilities of native MS on
different types of multi-protein complexes and different MS platforms to assess stoichiometries. Data interpretation is usually performed by comparing the masses of the individual subunits of the
complexes obtained by classical MS analysis in denaturing conditions and the mass of the intact noncovalent multi-protein assembly
measured in native conditions.
2 Materials
Prepare all solutions using deionized water and analytical grade
reagents. Prepare and store all reagents at 4
C.
2.1 Desalting
Ammonium acetate: 200 mM solution in water (see Note 1). Weigh
1.54 g ammonium acetate (>98%) and transfer to a 100-mL cylinder. Add water to a volume of 95 mL. Mix and adjust pH to 7.4
with ammonium hydroxide (see Note 2). Make up to 100 mL with
water.
2.2 Cesium Iodide
Calibration Mix
Resuspend cesium iodide powder in 50/50 isopropanol/water
(v/v) to a final concentration of 2 mg/mL.
2.3 Ammonium
Hexafluorophosphate
Calibration Mix
Resuspend ammonium hexafluorophosphate powder in 50/50 isopropanol/water (v/v) to a final concentration of 1 mg/mL.
2.4 GroEL
Reconstitution
GroEL must be freshly prepared.
1. Prepare 9.445 mL of 1 M Tris-acetate by weighting 1.21 g of
Tris-base and top up to 10 mL with acetic acid.
2. Prepare 10 mL of reconstitution buffer, composed as follows:
200 μL Tris-acetate at 1 M previously prepared, 50 μL EDTA at
100 mM, 10.7 mg magnesium acetate tetrahydrate, 5.5 mg
ATP, 37.3 mg potassium chloride to reach final concentrations
of 20 mM, 0.5 mM, 5 mM, 1 mM and 50 mM, respectively.
174
Ste ´ phane Erb et al.
most structural biology departments to complement conventional
structural biophysical techniques like crystallography, electron
microscopy, NMR, or SAXS. The methodology is versatile as any
type of multi-protein complex can be analyzed, ranging from
homo-oligomeric proteins [4] to hetero-oligomers [8, 9] or even
protein/nucleic assemblies [10, 11] and membrane complexes
[12–14]. Latest methodological breakthroughs in the field include
the direct analysis of complexes from cell lysates [15] or from native
membranes [13] and the release of a new instrument enabling the
complete analysis of very large complexes with unprecedented
resolution [16].
In this chapter, we illustrate the possibilities of native MS on
different types of multi-protein complexes and different MS platforms to assess stoichiometries. Data interpretation is usually performed by comparing the masses of the individual subunits of the
complexes obtained by classical MS analysis in denaturing conditions and the mass of the intact noncovalent multi-protein assembly
measured in native conditions.
2 Materials
Prepare all solutions using deionized water and analytical grade
reagents. Prepare and store all reagents at 4
C.
2.1 Desalting
Ammonium acetate: 200 mM solution in water (see Note 1). Weigh
1.54 g ammonium acetate (>98%) and transfer to a 100-mL cylinder. Add water to a volume of 95 mL. Mix and adjust pH to 7.4
with ammonium hydroxide (see Note 2). Make up to 100 mL with
water.
2.2 Cesium Iodide
Calibration Mix
Resuspend cesium iodide powder in 50/50 isopropanol/water
(v/v) to a final concentration of 2 mg/mL.
2.3 Ammonium
Hexafluorophosphate
Calibration Mix
Resuspend ammonium hexafluorophosphate powder in 50/50 isopropanol/water (v/v) to a final concentration of 1 mg/mL.
2.4 GroEL
Reconstitution
GroEL must be freshly prepared.
1. Prepare 9.445 mL of 1 M Tris-acetate by weighting 1.21 g of
Tris-base and top up to 10 mL with acetic acid.
2. Prepare 10 mL of reconstitution buffer, composed as follows:
200 μL Tris-acetate at 1 M previously prepared, 50 μL EDTA at
100 mM, 10.7 mg magnesium acetate tetrahydrate, 5.5 mg
ATP, 37.3 mg potassium chloride to reach final concentrations
of 20 mM, 0.5 mM, 5 mM, 1 mM and 50 mM, respectively.
174
Ste ´ phane Erb et al.