different species corresponding to the three monomers of the
CAK complex: namely, Cdk7 (MW: 40.1 kDa), Cyclin-H
(MW: 37.7 kDa), and MNAT1 (MW: 35.9 kDa) (Fig. 2b, c).
A species corresponding to the heterodimer Cdk7-Cyclin-H at
77.9 kDa is visible, confirming their direct interaction. Finally,
the physiologically active heterotrimeric CAK is identified at
113.9 kDa.
3.7 Protein/DNA
Complex Analysis on a
Q-TOF Synapt G2
The noncovalent interaction between the Estrogen-Related Receptor DNA binding domain (ERR-DBD) and its DNA response
element [24] can be monitored by direct infusion on a Synapt G2
platform. This transcription factor belongs to the steroid hormone
nuclear receptor family and shares strong similarity in its
DNA-binding domain (DBD) with that of the estrogen receptor
(ER). In vitro, ERR binds with high affinity inverted repeat REs
with a 3-bp spacing (IR3), but in vivo, it preferentially binds to
single half-site response elements extended at the 5
0 -end by 3 bp.
1. Desalt 50 μL of ERRα DBD-BE26PSIR3 complex [24] at
28 μM on a Zeba™ spin column equilibrated with 200 mM
ammonium acetate at pH 7.1 as explained in Subheading 3.1.
2. Use the TriVersa Nanomate™ (see Subheading 3.3) coupled to
the mass spectrometer and infuse the protein complex at 5 μM.
3. In the Tune page, start with the parameters used for the CsI
calibration and adapt the cone voltage at 140 V, the extraction
cone at 5 V and the backing pressure at 6 mbar (see Note 6).
4. Start a 2-min acquisition in the 1,000–6,000 m/z range.
5. The average full MS spectrum obtained is presented in Fig. 3a.
The deconvoluted spectrum of the protein/DNA complex
(see Subheading 3.5, step 5) highlights a 1:1 stoichiometry for
the protein/DNA complex (Fig. 3). The ERR-DBD binds two
zinc atoms and one DNA duplex (27,892.5 Da for full-length
DNA). Other species corresponding to truncated DNA associated with the protein with a 1:1 stoichiometry are also
detected (27,740.6 Da and 27,586.3 Da).
3.8 SEC Native MS
Coupling (ADH) on a QTOF Synapt G2Si
Size-exclusion chromatography (SEC) is commonly used both as a
purification and for analytical purposes to monitor sample heterogeneity. SEC is often coupled with UV Absorbance, Refractive
Index (RI), or Multi-Angle Laser Light Scattering (MALLS) to
estimate in particular the average molecular weight and the concentration of each component of the sample. SEC can also be
coupled with mass spectrometry (MS) to precisely measure the
molecular weight of each separated species. This approach is illustrated here by the characterization of yeast ADH oligomers on an
ACQUITY UPLC H-Class Bio coupled to a Q-TOF Synapt G2Si.
Native Mass Spectrometry
181
CAK complex: namely, Cdk7 (MW: 40.1 kDa), Cyclin-H
(MW: 37.7 kDa), and MNAT1 (MW: 35.9 kDa) (Fig. 2b, c).
A species corresponding to the heterodimer Cdk7-Cyclin-H at
77.9 kDa is visible, confirming their direct interaction. Finally,
the physiologically active heterotrimeric CAK is identified at
113.9 kDa.
3.7 Protein/DNA
Complex Analysis on a
Q-TOF Synapt G2
The noncovalent interaction between the Estrogen-Related Receptor DNA binding domain (ERR-DBD) and its DNA response
element [24] can be monitored by direct infusion on a Synapt G2
platform. This transcription factor belongs to the steroid hormone
nuclear receptor family and shares strong similarity in its
DNA-binding domain (DBD) with that of the estrogen receptor
(ER). In vitro, ERR binds with high affinity inverted repeat REs
with a 3-bp spacing (IR3), but in vivo, it preferentially binds to
single half-site response elements extended at the 5
0 -end by 3 bp.
1. Desalt 50 μL of ERRα DBD-BE26PSIR3 complex [24] at
28 μM on a Zeba™ spin column equilibrated with 200 mM
ammonium acetate at pH 7.1 as explained in Subheading 3.1.
2. Use the TriVersa Nanomate™ (see Subheading 3.3) coupled to
the mass spectrometer and infuse the protein complex at 5 μM.
3. In the Tune page, start with the parameters used for the CsI
calibration and adapt the cone voltage at 140 V, the extraction
cone at 5 V and the backing pressure at 6 mbar (see Note 6).
4. Start a 2-min acquisition in the 1,000–6,000 m/z range.
5. The average full MS spectrum obtained is presented in Fig. 3a.
The deconvoluted spectrum of the protein/DNA complex
(see Subheading 3.5, step 5) highlights a 1:1 stoichiometry for
the protein/DNA complex (Fig. 3). The ERR-DBD binds two
zinc atoms and one DNA duplex (27,892.5 Da for full-length
DNA). Other species corresponding to truncated DNA associated with the protein with a 1:1 stoichiometry are also
detected (27,740.6 Da and 27,586.3 Da).
3.8 SEC Native MS
Coupling (ADH) on a QTOF Synapt G2Si
Size-exclusion chromatography (SEC) is commonly used both as a
purification and for analytical purposes to monitor sample heterogeneity. SEC is often coupled with UV Absorbance, Refractive
Index (RI), or Multi-Angle Laser Light Scattering (MALLS) to
estimate in particular the average molecular weight and the concentration of each component of the sample. SEC can also be
coupled with mass spectrometry (MS) to precisely measure the
molecular weight of each separated species. This approach is illustrated here by the characterization of yeast ADH oligomers on an
ACQUITY UPLC H-Class Bio coupled to a Q-TOF Synapt G2Si.
Native Mass Spectrometry
181