3.11 Differential
Analysis for Label-Free
1. Use Progenesis QI software (v 4.0). Do not filter the peptides’
features.
2. Obtain the mascot generic file (.mgf) generated from all
exported MS/MS spectra. Use SEQUEST (Proteome Discoverer v1.4) with the following parameters: peptide mass tolerance 20 ppm, fragment tolerance 0.6 Da, enzyme set as trypsin
and allowance up to one missed cleavages, dynamic modification of methionine oxidation (+16 Da), and fixed modification
of cysteine carbamidomethylation (+57 Da).
3. Filter the results with peptide rank 1 and peptide confidence
high (1% FDR).
4. After that, import this file into Progenesis QI software for
further analysis. The statistic criteria are ANOVA test with
p value less than 0.05 and fold changed !1.5.
3.12 System Biology
In order to obtain more information about the proteins identified,
several open-access software can be used. For example, Cytoscape
and Funrich software can be used to investigate possible interactions among all the identified proteins and their molecular functions. In particular, Cytoscape is an open-source software platform,
which is used to visualize complex networks and integrate these
with any type of attribute data [14]. Moreover, it is also a complementary tool to know the biological process, molecular functions,
and cellular components of the differential proteins. In addition,
FunRich software shows the cellular component, the biological
process, and the biological pathways among all proteins identified
[15]. Furthermore, there are different EV-related databases that
can be used, such as EVpedia and Vesiclepedia, to confirm that the
proteins identified were enriched in EVs. Finally, the EV biomarkers identified must be validated in an independent cohort of
patients bay target MS or other western blotting-based approaches
(e.g., enzyme-linked immunosorbent assay (ELISA) or
western blot).
4 Notes
1. Plasma is usually the preferred source of EVs because additional
EVs are released during clot formation when preparing serum.
2. The SSC on Vascular Biology of ISTH proposes that
EV-containing plasma must be isolated within 2 h after blood
collection.
3. Prostacyclin avoids unwanted platelet activation during centrifugation by raising the intracellular concentration of cAMP.
This prevents the release of EVs from platelets during
centrifugation.
Circulating EVs Clinical Proteomics
21
Analysis for Label-Free
1. Use Progenesis QI software (v 4.0). Do not filter the peptides’
features.
2. Obtain the mascot generic file (.mgf) generated from all
exported MS/MS spectra. Use SEQUEST (Proteome Discoverer v1.4) with the following parameters: peptide mass tolerance 20 ppm, fragment tolerance 0.6 Da, enzyme set as trypsin
and allowance up to one missed cleavages, dynamic modification of methionine oxidation (+16 Da), and fixed modification
of cysteine carbamidomethylation (+57 Da).
3. Filter the results with peptide rank 1 and peptide confidence
high (1% FDR).
4. After that, import this file into Progenesis QI software for
further analysis. The statistic criteria are ANOVA test with
p value less than 0.05 and fold changed !1.5.
3.12 System Biology
In order to obtain more information about the proteins identified,
several open-access software can be used. For example, Cytoscape
and Funrich software can be used to investigate possible interactions among all the identified proteins and their molecular functions. In particular, Cytoscape is an open-source software platform,
which is used to visualize complex networks and integrate these
with any type of attribute data [14]. Moreover, it is also a complementary tool to know the biological process, molecular functions,
and cellular components of the differential proteins. In addition,
FunRich software shows the cellular component, the biological
process, and the biological pathways among all proteins identified
[15]. Furthermore, there are different EV-related databases that
can be used, such as EVpedia and Vesiclepedia, to confirm that the
proteins identified were enriched in EVs. Finally, the EV biomarkers identified must be validated in an independent cohort of
patients bay target MS or other western blotting-based approaches
(e.g., enzyme-linked immunosorbent assay (ELISA) or
western blot).
4 Notes
1. Plasma is usually the preferred source of EVs because additional
EVs are released during clot formation when preparing serum.
2. The SSC on Vascular Biology of ISTH proposes that
EV-containing plasma must be isolated within 2 h after blood
collection.
3. Prostacyclin avoids unwanted platelet activation during centrifugation by raising the intracellular concentration of cAMP.
This prevents the release of EVs from platelets during
centrifugation.
Circulating EVs Clinical Proteomics
21