7. Run the second dimension with SDS-polyacrylamide gel electrophoresis (PAGE) using 11% polyacrylamide gels. The running conditions are 20 mA per gel for 1 h and then 40 mA per
gel for 4–6 h. All procedures are at 10
C using an Ettan Dalt
6 system.
8. Following the electrophoresis, scan directly the gels in a
Typhoon FLA 7000 scanner.
9. After scanning, fix the gels in fixing buffer for 1 h and stain
them with Sypro Ruby fluorescent dye for spot picking.
3.6 Differential
Image Analysis
1. Perform the differential image analysis with Progenesis SameSpots software (v 4.5).
2. Align the images manually and automatically using the
software.
3. Check the differentially regulated proteins present in the gels
considering significant when the fold change is at least 1.5 and
the p value is below 0.05.
4. Excise the differentially regulated spots manually from the gel
for MS analysis.
3.7 MS Analysis
In the present example, samples are analyzed using LC-MS/MS on
an EASY-nLC and a Bruker Amazon ETD ion trap.
1. Digest the spots in-gel with trypsin using a protocol defined by
Shevchenko et al. with minor modifications [13].
2. Separate the digested peptide mixtures dissolved in 0.1% formic
acid in EASY-nLC with a reverse-phase nanocolumn (Easy
column SC200).
3. Use a CID-ETD ion trap mass spectrometer equipped with a
Nanosprayer ionization source to analyze the ionized peptides.
The spectra were acquired in Enhanced Resolution mode.
4. Analyze the mass data with Data Analysis 4.0 and BioTools 3.2.
Search the results with the database Mascot v2.3.0 search tool
screening SwissProt (see Note 9).
5. Accept as positive only these identifications that obtain more
than 50% y-ions (CID fragmentation) or z-ions (ETD fragmentation) for peptide comprising at least eight amino acids
long and no missed tryptic cleavage site. Positive hits correspond to Mascot scores >40 plus the fulfillment of the above
criteria.
3.8 Gel-Free
Proteomics:
Label-Free
Another approach that can be used to compare different EVs from
different sources (e.g., health and disease) is label-free LC-MS/
MS. In this case, protein samples were digested with sequencing
grade modified trypsin using FASP (filter aided sample preparation
protocol). All steps are run at 13
C.
Circulating EVs Clinical Proteomics
19
gel for 4–6 h. All procedures are at 10
C using an Ettan Dalt
6 system.
8. Following the electrophoresis, scan directly the gels in a
Typhoon FLA 7000 scanner.
9. After scanning, fix the gels in fixing buffer for 1 h and stain
them with Sypro Ruby fluorescent dye for spot picking.
3.6 Differential
Image Analysis
1. Perform the differential image analysis with Progenesis SameSpots software (v 4.5).
2. Align the images manually and automatically using the
software.
3. Check the differentially regulated proteins present in the gels
considering significant when the fold change is at least 1.5 and
the p value is below 0.05.
4. Excise the differentially regulated spots manually from the gel
for MS analysis.
3.7 MS Analysis
In the present example, samples are analyzed using LC-MS/MS on
an EASY-nLC and a Bruker Amazon ETD ion trap.
1. Digest the spots in-gel with trypsin using a protocol defined by
Shevchenko et al. with minor modifications [13].
2. Separate the digested peptide mixtures dissolved in 0.1% formic
acid in EASY-nLC with a reverse-phase nanocolumn (Easy
column SC200).
3. Use a CID-ETD ion trap mass spectrometer equipped with a
Nanosprayer ionization source to analyze the ionized peptides.
The spectra were acquired in Enhanced Resolution mode.
4. Analyze the mass data with Data Analysis 4.0 and BioTools 3.2.
Search the results with the database Mascot v2.3.0 search tool
screening SwissProt (see Note 9).
5. Accept as positive only these identifications that obtain more
than 50% y-ions (CID fragmentation) or z-ions (ETD fragmentation) for peptide comprising at least eight amino acids
long and no missed tryptic cleavage site. Positive hits correspond to Mascot scores >40 plus the fulfillment of the above
criteria.
3.8 Gel-Free
Proteomics:
Label-Free
Another approach that can be used to compare different EVs from
different sources (e.g., health and disease) is label-free LC-MS/
MS. In this case, protein samples were digested with sequencing
grade modified trypsin using FASP (filter aided sample preparation
protocol). All steps are run at 13
C.
Circulating EVs Clinical Proteomics
19