6. Pipette 1 μL of MALDI matrix solution on top of the standard
and mix well by pipetting up and down.
7. Samples and standards should be arranged on the plate such
that each sample is adjacent to a calibration standard as shown
in Table 1.
8. Submit target plate to a core facility for MALDI-ToF analysis;
be sure to include plate diagram with submission. The resolution and accuracy of the instrument should provide data indicating fraction purity and separation of proteins within 1 kDa
(see Fig. 2).
3.2 Sample Dilution
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer (See
Note 17)
1. Quantify protein sample using bicinchoninic acid or similar
assay as per manufacturer’s instructions.
2. Prepare dilutions of the protein of interest at 1:10, 1:20, and
1:50 (to achieve final concentration range of 0.1–2 μg/μL) in
ammonium acetate (2.5 mM to 250 mM) in 0–10% acetonitrile
and 0.1% formic acid (see Note 4).
3. Inject protein samples to determine the sample dilution that
achieves the optimum signal to noise (S/N) ratio for the protein and proteoforms.
3.3 Protein
Precipitation Method
for Processing Intact
Proteins for Top-Down
Proteomics Using
an ESI Mass
Spectrometer (See
Notes 18 and 19)
1. To the protein sample add chloroform:methanol:water to a
final ratio of 1:1:4.3 by volume (see Note 20).
2. Vortex briefly, followed by centrifugation at 9000 Â g for
1 min.
3. Discard the upper phase.
4. Wash with three volumes of methanol.
5. Vortex briefly and centrifuge at 9000 Â g for 2 min.
6. Discard the supernatant and air-dry the protein pellet until
fully dry.
7. Reconstitute the precipitated protein in a minimal volume of
80% (v/v) formic acid for 15 min at À20
C.
8. Fully resuspend protein to the same volume as starting material
with 10 mM ammonium acetate.
Table 1
Example of proper sample arrangement on a MALDI plate, with all unknown samples surrounding a
standard for calibration purposes
Sample 1
Sample 8
Sample 7
Sample 9
Sample 16
Sample 15
Sample 2
Standard
Sample 6
Sample 10
Standard
Sample 14
Sample 3
Sample 4
Sample 5
Sample 11
Sample 12
Sample 13
Proteomic Analyses
539
and mix well by pipetting up and down.
7. Samples and standards should be arranged on the plate such
that each sample is adjacent to a calibration standard as shown
in Table 1.
8. Submit target plate to a core facility for MALDI-ToF analysis;
be sure to include plate diagram with submission. The resolution and accuracy of the instrument should provide data indicating fraction purity and separation of proteins within 1 kDa
(see Fig. 2).
3.2 Sample Dilution
Method for Processing
Intact Proteins
for Top-Down
Proteomics Using
an ESI Mass
Spectrometer (See
Note 17)
1. Quantify protein sample using bicinchoninic acid or similar
assay as per manufacturer’s instructions.
2. Prepare dilutions of the protein of interest at 1:10, 1:20, and
1:50 (to achieve final concentration range of 0.1–2 μg/μL) in
ammonium acetate (2.5 mM to 250 mM) in 0–10% acetonitrile
and 0.1% formic acid (see Note 4).
3. Inject protein samples to determine the sample dilution that
achieves the optimum signal to noise (S/N) ratio for the protein and proteoforms.
3.3 Protein
Precipitation Method
for Processing Intact
Proteins for Top-Down
Proteomics Using
an ESI Mass
Spectrometer (See
Notes 18 and 19)
1. To the protein sample add chloroform:methanol:water to a
final ratio of 1:1:4.3 by volume (see Note 20).
2. Vortex briefly, followed by centrifugation at 9000 Â g for
1 min.
3. Discard the upper phase.
4. Wash with three volumes of methanol.
5. Vortex briefly and centrifuge at 9000 Â g for 2 min.
6. Discard the supernatant and air-dry the protein pellet until
fully dry.
7. Reconstitute the precipitated protein in a minimal volume of
80% (v/v) formic acid for 15 min at À20
C.
8. Fully resuspend protein to the same volume as starting material
with 10 mM ammonium acetate.
Table 1
Example of proper sample arrangement on a MALDI plate, with all unknown samples surrounding a
standard for calibration purposes
Sample 1
Sample 8
Sample 7
Sample 9
Sample 16
Sample 15
Sample 2
Standard
Sample 6
Sample 10
Standard
Sample 14
Sample 3
Sample 4
Sample 5
Sample 11
Sample 12
Sample 13
Proteomic Analyses
539