14. For “Ion matching requirements,” select 2 fragments per peptide, 5 fragments per protein, and 1 peptide per protein. Click
to save parameters and then click next.
15. Under the “Protein Quantitation” tab, select Relative Quantitation Using Hi-N option and choose “3” as the number of
peptides to measure per protein. Check the box “Use protein
grouping” (see Note 17).
16. Click “Finish” to start automatic processing (see Note 18).
17. Under the “Experiment Design Setup” tab, the samples must
be grouped into one condition, corresponding to each analyzed fraction (see Note 19).
18. Under the“Refine Identifications” tab, filter for and remove
peptides which have an absolute mass error greater than
20 ppm.
19. After processing each of the five fractions, load Progenesis
® QI
for Proteomics software, and under the tab “Combine Analysed Fractions,” click “Recombine analyzed fractions.”
20. Select the five fractions to recombine, import the selected
experiments, and put them in ascending order. Click on
“Section Complete.”
21. Rename each sample if needed, select whether the experiment
design is between-subject or within-subject design and group
samples according to their experimental groups.
3.7 In Silico
Analysis:
Available Tools
1. Cytoscape, Reactome, DAVID, and Panther are useful and free
applications and tools that help analyze and visualize pathway
enrichment, gene ontology, and molecular investigation.
2. Ingenuity Pathway Analysis, which is paid software, is able to
perform pathway and functional correlation analyses.
4 Notes
1. We employed the A280 method of quantification using a
DS-11 Spectrophotometer (Denovix Corporation), but the
BSA method or fluorometric methods can be used.
2. Information about column variance may be required. Depletion or addition of known amounts of a nonhuman protein
standard may allow evaluating the efficiency of the column.
3. Empower Pro 2 Software is used to process data and recording.
4. Wash the sample loop with buffer A with at least three times the
sample volume between the runs.
5. Take care to avoid air entering into the system through the
syringe, if manual injection is employed.
162
Licia C. Silva-Costa et al.
to save parameters and then click next.
15. Under the “Protein Quantitation” tab, select Relative Quantitation Using Hi-N option and choose “3” as the number of
peptides to measure per protein. Check the box “Use protein
grouping” (see Note 17).
16. Click “Finish” to start automatic processing (see Note 18).
17. Under the “Experiment Design Setup” tab, the samples must
be grouped into one condition, corresponding to each analyzed fraction (see Note 19).
18. Under the“Refine Identifications” tab, filter for and remove
peptides which have an absolute mass error greater than
20 ppm.
19. After processing each of the five fractions, load Progenesis
® QI
for Proteomics software, and under the tab “Combine Analysed Fractions,” click “Recombine analyzed fractions.”
20. Select the five fractions to recombine, import the selected
experiments, and put them in ascending order. Click on
“Section Complete.”
21. Rename each sample if needed, select whether the experiment
design is between-subject or within-subject design and group
samples according to their experimental groups.
3.7 In Silico
Analysis:
Available Tools
1. Cytoscape, Reactome, DAVID, and Panther are useful and free
applications and tools that help analyze and visualize pathway
enrichment, gene ontology, and molecular investigation.
2. Ingenuity Pathway Analysis, which is paid software, is able to
perform pathway and functional correlation analyses.
4 Notes
1. We employed the A280 method of quantification using a
DS-11 Spectrophotometer (Denovix Corporation), but the
BSA method or fluorometric methods can be used.
2. Information about column variance may be required. Depletion or addition of known amounts of a nonhuman protein
standard may allow evaluating the efficiency of the column.
3. Empower Pro 2 Software is used to process data and recording.
4. Wash the sample loop with buffer A with at least three times the
sample volume between the runs.
5. Take care to avoid air entering into the system through the
syringe, if manual injection is employed.
162
Licia C. Silva-Costa et al.