3.4 Ultrafiltration
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer (See
Note 22)
1. Place protein sample in an ultrafiltration device containing a
3 kDa MWCO membrane (see Note 23).
2. Centrifuge the ultrafiltration device according to manufacturer’s instructions until only 1/10th of the initial volume
remains.
3. Add 10 mM ammonium acetate to the ultrafiltration device (see
Note 23).
4. Repeat step 2.
5. Repeat steps 3 and 4 two more times for a total of three buffer
exchanges.
6. Pipette out the retentate (concentrated protein sample).
7. Wash the membrane with a small amount of 10 mM ammonium acetate and pool with retentate from step 6 to ensure
maximum recovery.
8. Quantify the amount of protein using the bicinchoninic acid or
similar assay as per manufacturer’s instructions.
9. Prepare 0.1, 0.5, and 1 μg/μL dilutions in MS solvent (see
Note 21).
10. Inject protein samples to determine the best concentration to
achieve the optimum signal to noise (S/N) ratio for the protein
and proteoforms.
3.5 In-Gel Digestion
Processing for Bottom
up Proteomics (See
Note 24)
1. From a stained polyacrylamide gel, excise protein bands of
interest with a razor blade on a glass plate (see Notes 6 and 7).
2. Cut each band into small pieces (~1 mm by 1 mm) and place
the pieces in a deplasticized microcentrifuge tube.
3. To destain, cover the gel pieces with destain solution, vortex,
and incubate at 37
C for at least 30 min, up to overnight.
4. Centrifuge briefly to remove condensation from the upper tube
and lid.
5. Pipette the destain solution into a waste container, being sure
not to transfer any gel pieces with the destain solution (see
Note 25).
6. Repeat steps 3–5 up to three times until the gel is completely
destained (see Note 26).
7. Dry the gel pieces in the vacuum concentrator.
8. To the dried gel slices, add 0.5 μL of trypsin solution for every
1 μg of protein to achieve a 1:20 trypsin:protein ratio (see
Note 27).
9. Incubate at room temperature until the trypsin solution is
completely absorbed by the gel slices (see Note 28).
10. Add 10–15 μL of 0.2 M ammonium bicarbonate and gently
break apart the pelleted gel pieces using a pipet tip.
Proteomic Analyses
541
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer (See
Note 22)
1. Place protein sample in an ultrafiltration device containing a
3 kDa MWCO membrane (see Note 23).
2. Centrifuge the ultrafiltration device according to manufacturer’s instructions until only 1/10th of the initial volume
remains.
3. Add 10 mM ammonium acetate to the ultrafiltration device (see
Note 23).
4. Repeat step 2.
5. Repeat steps 3 and 4 two more times for a total of three buffer
exchanges.
6. Pipette out the retentate (concentrated protein sample).
7. Wash the membrane with a small amount of 10 mM ammonium acetate and pool with retentate from step 6 to ensure
maximum recovery.
8. Quantify the amount of protein using the bicinchoninic acid or
similar assay as per manufacturer’s instructions.
9. Prepare 0.1, 0.5, and 1 μg/μL dilutions in MS solvent (see
Note 21).
10. Inject protein samples to determine the best concentration to
achieve the optimum signal to noise (S/N) ratio for the protein
and proteoforms.
3.5 In-Gel Digestion
Processing for Bottom
up Proteomics (See
Note 24)
1. From a stained polyacrylamide gel, excise protein bands of
interest with a razor blade on a glass plate (see Notes 6 and 7).
2. Cut each band into small pieces (~1 mm by 1 mm) and place
the pieces in a deplasticized microcentrifuge tube.
3. To destain, cover the gel pieces with destain solution, vortex,
and incubate at 37
C for at least 30 min, up to overnight.
4. Centrifuge briefly to remove condensation from the upper tube
and lid.
5. Pipette the destain solution into a waste container, being sure
not to transfer any gel pieces with the destain solution (see
Note 25).
6. Repeat steps 3–5 up to three times until the gel is completely
destained (see Note 26).
7. Dry the gel pieces in the vacuum concentrator.
8. To the dried gel slices, add 0.5 μL of trypsin solution for every
1 μg of protein to achieve a 1:20 trypsin:protein ratio (see
Note 27).
9. Incubate at room temperature until the trypsin solution is
completely absorbed by the gel slices (see Note 28).
10. Add 10–15 μL of 0.2 M ammonium bicarbonate and gently
break apart the pelleted gel pieces using a pipet tip.
Proteomic Analyses
541