3.9 HomoOligomeric
Concanavalin A
Analysis on a HighResolution Q-TOF
Maxis II
Concanavalin A from Jack bean is a lectin commonly used as a
standard in Native MS experiments. Its physiological oligomeric
state is tetrameric although higher-order oligomers can often be
observed at high concentrations. We use it here to describe the
parameters allowing its quaternary structure assessment with a
Q-TOF Maxis II platform.
1. Resuspend 1.5 mg of Concanavalin A from Canavalia ensiformis (Jack bean, Sigma-Aldrich reference no. C5275) in 200 μL
of 10 mM ammonium acetate at pH 6.8 to reach a concentration around 50 μM for the tetrameric species.
2. Desalt 100 μL of the protein (see Subheading 3.2) on a Vivaspin™ 10 kDa cut-off membrane (6–8 cycles at 15,000 Â g and
+4
C during 8–10 min).
3. Connect the TriVersa Nanomate™ to the mass spectrometer.
Infuse the protein at 10 μM.
4. In the tune page, use the same parameters used for CsI calibration except for the isCID that should be set at 35 eV.
5. Start a 2-min acquisition in the 1,000–8,000 m/z range.
6. The average smoothed mass spectrum is represented in Fig. 5a.
The corresponding deconvoluted spectrum is obtained after
UniDec processing (Fig. 5b, see Subheading 3.5, step 5) and
allows the identification of Concanavalin A tetrameric species at
102,707 Da.
3.10 High Molecular
Weight Homooligomeric Protein on a
High-Resolution
Exactive™ Plus EMR
Orbitrap
The use of an Exactive Plus platform is illustrated by the characterization of the GroEL chaperone. This 57-kDa protein assembles as a
14-mer to form a double toroidal complex, which together with
the GroES co-chaperonin facilitates protein folding in an
ATP-dependent manner [25].
1. Use the freshly prepared and desalted GroEL protein (see Subheading 2.4).
2. Connect the TriVersa Nanomate™ (see Subheading 3.3) to the
mass spectrometer. Infuse the sample concentrated at 5 μM of
oligomer.
3. In the Tune page, set the following parameters: Positive mode
with a 1,000–20,000 m/z range. Activate the EMR mode and
set the trapping gas pressure at 7 u.a., the ion optics at 4 V
(inter, injection, and bent flatapoles), the S-lens RF voltage at
200 V, the temperature at 250
C, the fragmentation parameters at 25 eV and 150 eV for CID and CE values, respectively. Select a nominal resolution of 17,500.
4. Start a 2-min acquisition with a stable TIC and analyze the MS
spectrum.
The resulting averaged mass spectrum is presented in
Fig. 6a (full mass range) and Fig. 6b (zoom between 11,000
184
Ste ´ phane Erb et al.
Concanavalin A
Analysis on a HighResolution Q-TOF
Maxis II
Concanavalin A from Jack bean is a lectin commonly used as a
standard in Native MS experiments. Its physiological oligomeric
state is tetrameric although higher-order oligomers can often be
observed at high concentrations. We use it here to describe the
parameters allowing its quaternary structure assessment with a
Q-TOF Maxis II platform.
1. Resuspend 1.5 mg of Concanavalin A from Canavalia ensiformis (Jack bean, Sigma-Aldrich reference no. C5275) in 200 μL
of 10 mM ammonium acetate at pH 6.8 to reach a concentration around 50 μM for the tetrameric species.
2. Desalt 100 μL of the protein (see Subheading 3.2) on a Vivaspin™ 10 kDa cut-off membrane (6–8 cycles at 15,000 Â g and
+4
C during 8–10 min).
3. Connect the TriVersa Nanomate™ to the mass spectrometer.
Infuse the protein at 10 μM.
4. In the tune page, use the same parameters used for CsI calibration except for the isCID that should be set at 35 eV.
5. Start a 2-min acquisition in the 1,000–8,000 m/z range.
6. The average smoothed mass spectrum is represented in Fig. 5a.
The corresponding deconvoluted spectrum is obtained after
UniDec processing (Fig. 5b, see Subheading 3.5, step 5) and
allows the identification of Concanavalin A tetrameric species at
102,707 Da.
3.10 High Molecular
Weight Homooligomeric Protein on a
High-Resolution
Exactive™ Plus EMR
Orbitrap
The use of an Exactive Plus platform is illustrated by the characterization of the GroEL chaperone. This 57-kDa protein assembles as a
14-mer to form a double toroidal complex, which together with
the GroES co-chaperonin facilitates protein folding in an
ATP-dependent manner [25].
1. Use the freshly prepared and desalted GroEL protein (see Subheading 2.4).
2. Connect the TriVersa Nanomate™ (see Subheading 3.3) to the
mass spectrometer. Infuse the sample concentrated at 5 μM of
oligomer.
3. In the Tune page, set the following parameters: Positive mode
with a 1,000–20,000 m/z range. Activate the EMR mode and
set the trapping gas pressure at 7 u.a., the ion optics at 4 V
(inter, injection, and bent flatapoles), the S-lens RF voltage at
200 V, the temperature at 250
C, the fragmentation parameters at 25 eV and 150 eV for CID and CE values, respectively. Select a nominal resolution of 17,500.
4. Start a 2-min acquisition with a stable TIC and analyze the MS
spectrum.
The resulting averaged mass spectrum is presented in
Fig. 6a (full mass range) and Fig. 6b (zoom between 11,000
184
Ste ´ phane Erb et al.