2 Materials
Unless otherwise stated, all solutions should be made using LC-MS
grade water.
2.1 Processing Intact
Proteins for Top Down
Proteomics Using
a MALDI-ToF Mass
Spectrometer
1. 0.1–5 μg of protein sample (see Note 1).
2. 0.1% v/v trifluoroacetic acid in 50% v/v acetonitrile.
3. MALDI matrix solution: Take an empty 0.65 mL microcentrifuge tube on the digital balance. Weigh out approximately
5 mg of sinapinic acid into the microcentrifuge tube. Add
50% acetonitrile, 0.1% TFA in water, to achieve a concentration
of 10 mg/mL. Bath sonicate for 5–10 min until the matrix is
fully dissolved. Matrix should be made fresh for each use (see
Note 2).
4. Calibration standard: Protein Standard II for Mass Spectrometry (see Note 3).
5. Brushed steel MALDI target plate.
2.2 Sample Dilution
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer
1. 1–10 μg of protein of interest (see Note 1).
2. Acetonitrile, LC-MS grade.
3. Water, LC-MS grade.
4. 10 mM ammonium acetate in LC-MS grade water (see Note 4).
5. Formic acid, LC-MS grade.
2.3 Protein
Precipitation
for Processing Intact
Proteins for Top-Down
Proteomics Using
an ESI Mass
Spectrometer
1. 10–50 μg of protein of interest.
2. Chloroform, LC-MS grade.
3. Methanol, LC-MS grade.
4. Acetonitrile, LC-MS grade.
5. Water, LC-MS grade.
6. 10 mM ammonium acetate in water, LC-MS grade (see
Note 4).
7. Formic acid, LC-MS grade (80% v/v in LC-MS grade water).
8. MS solvent: 5% acetonitrile and 0.1% formic acid in LC-MS
grade water.
2.4 Ultrafiltration
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer
1. 10–50 μg of protein of interest.
2. 3 kDa Molecular Weight Cutoff (MWCO) ultrafiltration device
(see Note 5).
3. Acetonitrile, LC-MS grade.
4. Water, LC-MS grade.
5. 25 mM and 10 mM ammonium acetate in water, LC-MS
grade.
536
Carolina Mehaffy et al.
Unless otherwise stated, all solutions should be made using LC-MS
grade water.
2.1 Processing Intact
Proteins for Top Down
Proteomics Using
a MALDI-ToF Mass
Spectrometer
1. 0.1–5 μg of protein sample (see Note 1).
2. 0.1% v/v trifluoroacetic acid in 50% v/v acetonitrile.
3. MALDI matrix solution: Take an empty 0.65 mL microcentrifuge tube on the digital balance. Weigh out approximately
5 mg of sinapinic acid into the microcentrifuge tube. Add
50% acetonitrile, 0.1% TFA in water, to achieve a concentration
of 10 mg/mL. Bath sonicate for 5–10 min until the matrix is
fully dissolved. Matrix should be made fresh for each use (see
Note 2).
4. Calibration standard: Protein Standard II for Mass Spectrometry (see Note 3).
5. Brushed steel MALDI target plate.
2.2 Sample Dilution
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer
1. 1–10 μg of protein of interest (see Note 1).
2. Acetonitrile, LC-MS grade.
3. Water, LC-MS grade.
4. 10 mM ammonium acetate in LC-MS grade water (see Note 4).
5. Formic acid, LC-MS grade.
2.3 Protein
Precipitation
for Processing Intact
Proteins for Top-Down
Proteomics Using
an ESI Mass
Spectrometer
1. 10–50 μg of protein of interest.
2. Chloroform, LC-MS grade.
3. Methanol, LC-MS grade.
4. Acetonitrile, LC-MS grade.
5. Water, LC-MS grade.
6. 10 mM ammonium acetate in water, LC-MS grade (see
Note 4).
7. Formic acid, LC-MS grade (80% v/v in LC-MS grade water).
8. MS solvent: 5% acetonitrile and 0.1% formic acid in LC-MS
grade water.
2.4 Ultrafiltration
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer
1. 10–50 μg of protein of interest.
2. 3 kDa Molecular Weight Cutoff (MWCO) ultrafiltration device
(see Note 5).
3. Acetonitrile, LC-MS grade.
4. Water, LC-MS grade.
5. 25 mM and 10 mM ammonium acetate in water, LC-MS
grade.
536
Carolina Mehaffy et al.