11. Add 1 μL of 0.5 M DTT and incubate at 50
C for 20 min (see
Note 12).
12. Add 2.7 μL of 0.55 M IAA and incubate at room temperature
in the dark for 15 min (see Note 13).
13. Add 1 μL of 1% ProteaseMAX and 18 μL of 0.1 μg/μL trypsin.
Cover the tube with parafilm and incubate at 37
C for 3 h (see
Note 33).
14. Spin for 10 s in microcentrifuge. Add 27 μL of 2.5 %TFA (final
concentration 0.5%). Vortex and incubate at room temperature
for 5 min.
15. Dry down in vacuum concentrator (see Notes 30 and 34).
4 Notes
1. Protein samples should be dialyzed in 10 mM ammonium
bicarbonate or 1Â PBS for Maldi-ToF analysis and 10 mM
ammonium bicarbonate or 10 mM ammonium acetate for
ESI-MS analysis.
2. Many matrixes can be used for the analysis of intact proteins by
MALDI-ToF, and some are preferred when MALDI-ToF analysis is used for top down proteomics along with in-source decay
[28]. In this protocol, we describe the use of sinapinic acid as
MALDI matrix.
3. Protein Standard II is specifically for mass range of
~20,000–70,000 Da. When ordering standard, make sure the
mass range is appropriate for your protein of interest. Standard
can be resuspended per the manufacturer’s instructions and
then aliquoted for freezing. Thaw standard immediately
before use.
4. The final concentration of ammonium acetate is determined
empirically based on the solubility of the intact protein; however, the concentration should not be less than 2.5 mM. For
example, a soluble protein may be introduced into the mass
spectrometer in 50 mM ammonium acetate in 10% acetonitrile
and 0.1% formic acid. Conversely, a poorly soluble protein may
be introduced into the mass spectrometer in 5 mM ammonium
acetate in 50% acetonitrile and 0.1% formic acid. As a starting
point, protein sample should be dissolved in 10 mM ammonium acetate, 5% acetonitrile, and 0.1% formic acid and adjust
accordingly based on solubility in this solution.
5. The size of the membrane can be selected based on mass of
protein of interest. The molecular weight cutoff may be as
much as half of the molecular mass for the protein(s) of interest. A 3 kDa MWCO is recommended to recover all protein
sizes.
Proteomic Analyses
543
C for 20 min (see
Note 12).
12. Add 2.7 μL of 0.55 M IAA and incubate at room temperature
in the dark for 15 min (see Note 13).
13. Add 1 μL of 1% ProteaseMAX and 18 μL of 0.1 μg/μL trypsin.
Cover the tube with parafilm and incubate at 37
C for 3 h (see
Note 33).
14. Spin for 10 s in microcentrifuge. Add 27 μL of 2.5 %TFA (final
concentration 0.5%). Vortex and incubate at room temperature
for 5 min.
15. Dry down in vacuum concentrator (see Notes 30 and 34).
4 Notes
1. Protein samples should be dialyzed in 10 mM ammonium
bicarbonate or 1Â PBS for Maldi-ToF analysis and 10 mM
ammonium bicarbonate or 10 mM ammonium acetate for
ESI-MS analysis.
2. Many matrixes can be used for the analysis of intact proteins by
MALDI-ToF, and some are preferred when MALDI-ToF analysis is used for top down proteomics along with in-source decay
[28]. In this protocol, we describe the use of sinapinic acid as
MALDI matrix.
3. Protein Standard II is specifically for mass range of
~20,000–70,000 Da. When ordering standard, make sure the
mass range is appropriate for your protein of interest. Standard
can be resuspended per the manufacturer’s instructions and
then aliquoted for freezing. Thaw standard immediately
before use.
4. The final concentration of ammonium acetate is determined
empirically based on the solubility of the intact protein; however, the concentration should not be less than 2.5 mM. For
example, a soluble protein may be introduced into the mass
spectrometer in 50 mM ammonium acetate in 10% acetonitrile
and 0.1% formic acid. Conversely, a poorly soluble protein may
be introduced into the mass spectrometer in 5 mM ammonium
acetate in 50% acetonitrile and 0.1% formic acid. As a starting
point, protein sample should be dissolved in 10 mM ammonium acetate, 5% acetonitrile, and 0.1% formic acid and adjust
accordingly based on solubility in this solution.
5. The size of the membrane can be selected based on mass of
protein of interest. The molecular weight cutoff may be as
much as half of the molecular mass for the protein(s) of interest. A 3 kDa MWCO is recommended to recover all protein
sizes.
Proteomic Analyses
543