concentration. Alternatively, pipette tips containing reversephase chromatography media (such as ZipTip C18 for proteins
<20 kDa and C4 for proteins >20 kDa) may be used to
concentrate and desalt the protein of interest.
2. Freshly prepare the matrix solution. The chosen matrix will
depend on protein size and glycosylation state. HCCA
(α-cyano-4-hydroxycinnamic acid, a.k.a. α-CHCA) or DHB
(2,5-dihydroxybenzoic acid) are typically used for peptides
and proteins below 20 kDa. SuperDHB (DHB with 10%
(w/w) 2-hydroxy-5-methoxybenzoic acid) or sinapinic acid
for larger proteins. Follow the instrument manufactures’
instructions for matrix preparation. However, 25 mg/mL in
50% (v/v) acetonitrile and 0.1% (v/v) TFA in HPLC water is
typically effective.
3. The protein sample can be mixed with the matrix directly on
the MALDI plate, or the mixture can be prepared beforehand
in a tube and a droplet is then deposited on the target plate.
Typically, 1μL of protein is applied to the target plate and 1μL
of matrix is immediately added. Alternatively, the sample and
the matrix can be mixed by repeated pipetting before the
deposit, which is still in the form of a droplet, is allowed to dry.
4. Acquire data on the MALDI time-of-flight (TOF) mass spectrometer. It is recommended to use the minimum laser power
required to get sufficient signal to noise in the spectrum to
detect the protein as a high laser power leads to a loss of
resolution and precision on the m/z.
5. Calibrate the experiment with a standard protein mixture as
recommended by the manufacturer.
2.3 Protein
Structural Integrity
and Stability
2.3.1 CD Spectroscopy
The signal/noise (S/N) ratio in a CD spectrum is strongly dependent on the UV absorbance of the sample—a low absorbance gives
little measurable differential absorption (i.e., a low CD signal), and
high absorbance means that little light reaches the detector. Optimal signal to noise is obtained a little below 1 AU, and an absorbance of the sample between 0.5 and 1.5 AU will give good results
(above 2 AU insufficient light will reach the detector to obtain a
reliable measurement). Protein sequence (for near-UV) and buffer
composition, their concentrations, and cuvette path length will
alter the total absorbance. It is necessary to select or adjust those
quantities that can be changed to obtain a satisfactory S/N for each
sample.
Choice of Cuvette, Buffer,
and Sample Preparation
There are a variety of cuvettes available with path lengths ranging
from 0.01 to 10 mm. The required concentration of the protein
sample depends on the spectral range and type of cuvette. For
far-UV CD, typically, a 1-mm cuvette is used, and for this, a good
30
Bertrand Raynal et al.
<20 kDa and C4 for proteins >20 kDa) may be used to
concentrate and desalt the protein of interest.
2. Freshly prepare the matrix solution. The chosen matrix will
depend on protein size and glycosylation state. HCCA
(α-cyano-4-hydroxycinnamic acid, a.k.a. α-CHCA) or DHB
(2,5-dihydroxybenzoic acid) are typically used for peptides
and proteins below 20 kDa. SuperDHB (DHB with 10%
(w/w) 2-hydroxy-5-methoxybenzoic acid) or sinapinic acid
for larger proteins. Follow the instrument manufactures’
instructions for matrix preparation. However, 25 mg/mL in
50% (v/v) acetonitrile and 0.1% (v/v) TFA in HPLC water is
typically effective.
3. The protein sample can be mixed with the matrix directly on
the MALDI plate, or the mixture can be prepared beforehand
in a tube and a droplet is then deposited on the target plate.
Typically, 1μL of protein is applied to the target plate and 1μL
of matrix is immediately added. Alternatively, the sample and
the matrix can be mixed by repeated pipetting before the
deposit, which is still in the form of a droplet, is allowed to dry.
4. Acquire data on the MALDI time-of-flight (TOF) mass spectrometer. It is recommended to use the minimum laser power
required to get sufficient signal to noise in the spectrum to
detect the protein as a high laser power leads to a loss of
resolution and precision on the m/z.
5. Calibrate the experiment with a standard protein mixture as
recommended by the manufacturer.
2.3 Protein
Structural Integrity
and Stability
2.3.1 CD Spectroscopy
The signal/noise (S/N) ratio in a CD spectrum is strongly dependent on the UV absorbance of the sample—a low absorbance gives
little measurable differential absorption (i.e., a low CD signal), and
high absorbance means that little light reaches the detector. Optimal signal to noise is obtained a little below 1 AU, and an absorbance of the sample between 0.5 and 1.5 AU will give good results
(above 2 AU insufficient light will reach the detector to obtain a
reliable measurement). Protein sequence (for near-UV) and buffer
composition, their concentrations, and cuvette path length will
alter the total absorbance. It is necessary to select or adjust those
quantities that can be changed to obtain a satisfactory S/N for each
sample.
Choice of Cuvette, Buffer,
and Sample Preparation
There are a variety of cuvettes available with path lengths ranging
from 0.01 to 10 mm. The required concentration of the protein
sample depends on the spectral range and type of cuvette. For
far-UV CD, typically, a 1-mm cuvette is used, and for this, a good
30
Bertrand Raynal et al.