6. Take care to observe and adjust the volume to equalize volumes
in the concentrator tubes, if it is needed.
7. A method to quantify proteins is required to proceed with this
protocol. After the desalted sample is concentrated, before
removing the sample from the concentrator, its concentration
can be monitored to surpass 1 μg/μL, allowing for dilution to
1 μg/μL with ammonium bicarbonate (50 mM). Avoid heating the sample over 4
C to prevent protein degradation.
8. To prevent ionic suppression, steps 4–7 can be repeated.
9. This is a 1:100 wt:wt ratio of enzyme:protein.
10. Incubate the samples for at least 12 h and up to 18 h maximum.
11. Distribute pooled or individual samples or quality controls
(QCs) according to your experimental design. We prefer to
use individual and randomized samples alternating with QC
injections.
12. When opening the chromatogram, open a time point near the
beginning of the elution and move forward in time until background noise is washed out by the peptide signal. Do the same
for the end of the elution period, moving backward. These
should be used as the start and end points for the integration
range. The total area under the curve can then be compared
between samples to create ratios of peptide signal to calculate
any fine-tuning adjustments necessary due to undigested proteins and other contaminants.
13. Remember to maximize the software window, and be careful to
not cross the lines under multiply charged state and over
multiply charged state.
14. It is recommended that the file be checked using an appropriate
text editor to verify that the file was saved properly. On rare
occasions, rule files are saved empty.
15. Save as UDMS
E method before changing. This ensures that
the original method is not overwritten.
16. An FDR of 1% can be selected here.
17. Ungrouping proteins may provide more information; however,
the data are more complex and need more processing to get the
desired answers. Deselect this if the collaborating bioinformatician or biostatistician requires.
18. The processing must be done for each of the fraction groups
before combining the fractions (using a built-in tool with
Progenesis) and proceeding with the experiment.
19. This procedure must be done for each of the five fractions.
Human Blood Plasma Investigation Employing 2D UPLC-UDMS
E Data. . .
163
in the concentrator tubes, if it is needed.
7. A method to quantify proteins is required to proceed with this
protocol. After the desalted sample is concentrated, before
removing the sample from the concentrator, its concentration
can be monitored to surpass 1 μg/μL, allowing for dilution to
1 μg/μL with ammonium bicarbonate (50 mM). Avoid heating the sample over 4
C to prevent protein degradation.
8. To prevent ionic suppression, steps 4–7 can be repeated.
9. This is a 1:100 wt:wt ratio of enzyme:protein.
10. Incubate the samples for at least 12 h and up to 18 h maximum.
11. Distribute pooled or individual samples or quality controls
(QCs) according to your experimental design. We prefer to
use individual and randomized samples alternating with QC
injections.
12. When opening the chromatogram, open a time point near the
beginning of the elution and move forward in time until background noise is washed out by the peptide signal. Do the same
for the end of the elution period, moving backward. These
should be used as the start and end points for the integration
range. The total area under the curve can then be compared
between samples to create ratios of peptide signal to calculate
any fine-tuning adjustments necessary due to undigested proteins and other contaminants.
13. Remember to maximize the software window, and be careful to
not cross the lines under multiply charged state and over
multiply charged state.
14. It is recommended that the file be checked using an appropriate
text editor to verify that the file was saved properly. On rare
occasions, rule files are saved empty.
15. Save as UDMS
E method before changing. This ensures that
the original method is not overwritten.
16. An FDR of 1% can be selected here.
17. Ungrouping proteins may provide more information; however,
the data are more complex and need more processing to get the
desired answers. Deselect this if the collaborating bioinformatician or biostatistician requires.
18. The processing must be done for each of the fraction groups
before combining the fractions (using a built-in tool with
Progenesis) and proceeding with the experiment.
19. This procedure must be done for each of the five fractions.
Human Blood Plasma Investigation Employing 2D UPLC-UDMS
E Data. . .
163