com/msproteomicstools/msproteomicstools. For a detailed
instruction regarding installation check under http://
openswath.org/ page.
2.8 Protein
Co-Elution
Profiling Tools
1. R version > 3.3, freely available from https://www.r-project.
org
2. The CCprofiler R-package is available at https://github.com/
CCprofiler
3 Methods
An overview of the SEC-SWATH workflow is outlined in Fig. 1.
To avoid artifactual disruption of protein complexes, all steps
from the native lysis up to the SEC separation should be performed
either on ice or at 4
C and ideally conducted within 1 day. Depending on the number of SEC samples and replicates, this protocol may
result in a workload of up to 10 h for separation.
3.1 Tissue Culture
1. Grow Jurkat cells (ATCC, clone A3) in RPMI-1640 supplemented with 10% FCS and 1% PenStrep, in an incubator with
5% CO 2 . Maintain the cells at densities below 1 Â 10
6 cells/mL
and split them every 48 h. When splitting, check the cell
viability (e.g., Fluorescence-Based Viability Assays for Countess II FL Automated Cell Counter). The cell viability should be
95% or higher.
2. For harvesting, collect the volume of cell suspension containing 30 Â 10
6 cells (usually around 30 mL) into a fresh 50 mL
falcon and pellet the cells at 250 Â g at 4
C for 5 min on a
precooled centrifuge. Decant the supernatant culture medium
and gently resuspend the cell pellet with 10 mL of precooled
PBS. Avoid excessive pipetting to prevent mechanical cell lysis
during the wash stages. Pellet the cells again at 250 Â g at 4
C
for 5 min on a precooled centrifuge, decant the supernatant,
and gently resuspend the cell pellet with 5 mL of precooled
PBS. Transfer the cell suspension to a 15 mL falcon tube and
pellet cells at 250 Â g at 4
C for 5 min on a precooled
centrifuge.
3. Remove the supernatant and avoid leaving more than 50 μL of
PBS on the cell pellet.
4. Snap freeze the cells in liquid nitrogen and store the pellet at
À80
C. The frozen cell pellet can be stored for several months.
3.2 Native Lysis
1. Place frozen cells on ice and add fresh prepared HNN-lysis
buffer (HNN-buffer, pH 7.5: 50 mM HEPES pH 7.5,
150 mM NaCl, 50 mM NaF, supplemented with 0.5% NP-40
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Andrea Fossati et al.
instruction regarding installation check under http://
openswath.org/ page.
2.8 Protein
Co-Elution
Profiling Tools
1. R version > 3.3, freely available from https://www.r-project.
org
2. The CCprofiler R-package is available at https://github.com/
CCprofiler
3 Methods
An overview of the SEC-SWATH workflow is outlined in Fig. 1.
To avoid artifactual disruption of protein complexes, all steps
from the native lysis up to the SEC separation should be performed
either on ice or at 4
C and ideally conducted within 1 day. Depending on the number of SEC samples and replicates, this protocol may
result in a workload of up to 10 h for separation.
3.1 Tissue Culture
1. Grow Jurkat cells (ATCC, clone A3) in RPMI-1640 supplemented with 10% FCS and 1% PenStrep, in an incubator with
5% CO 2 . Maintain the cells at densities below 1 Â 10
6 cells/mL
and split them every 48 h. When splitting, check the cell
viability (e.g., Fluorescence-Based Viability Assays for Countess II FL Automated Cell Counter). The cell viability should be
95% or higher.
2. For harvesting, collect the volume of cell suspension containing 30 Â 10
6 cells (usually around 30 mL) into a fresh 50 mL
falcon and pellet the cells at 250 Â g at 4
C for 5 min on a
precooled centrifuge. Decant the supernatant culture medium
and gently resuspend the cell pellet with 10 mL of precooled
PBS. Avoid excessive pipetting to prevent mechanical cell lysis
during the wash stages. Pellet the cells again at 250 Â g at 4
C
for 5 min on a precooled centrifuge, decant the supernatant,
and gently resuspend the cell pellet with 5 mL of precooled
PBS. Transfer the cell suspension to a 15 mL falcon tube and
pellet cells at 250 Â g at 4
C for 5 min on a precooled
centrifuge.
3. Remove the supernatant and avoid leaving more than 50 μL of
PBS on the cell pellet.
4. Snap freeze the cells in liquid nitrogen and store the pellet at
À80
C. The frozen cell pellet can be stored for several months.
3.2 Native Lysis
1. Place frozen cells on ice and add fresh prepared HNN-lysis
buffer (HNN-buffer, pH 7.5: 50 mM HEPES pH 7.5,
150 mM NaCl, 50 mM NaF, supplemented with 0.5% NP-40
276
Andrea Fossati et al.