Our presented method improves the discussed SEC–MS workflow at several levels. It combines robust and reliable sample preparation with fast MS acquisition, irrespective of whether the data are
acquired in DDA or DIA. Further, we present a data analysis
protocol suitable for multiple SEC-SWATH data sets (for timing
see Note 26). As shown in Fig. 3, we are able to identify
111 CORUM annotated protein complexes with statistical significance from a single co-fractionation experiment. The enhanced
throughput of the protocol which reduces MS acquisition time to
1 day per sample, along with its operational flexibility opens the
possibility to apply SEC–MS as a screening technology for differential protein complex formation with high reproducibility across
multiple cellular states or patient cohorts.
5 Notes
1. For starting material, cells and tissues from various different
biological materials can be used. We normally process suspension or adherent mammalian cells, however, there are several
SEC–MS studies published already, for which different types of
biological material were used, including various different mammalian cells and plant tissues [34, 35]. Consider that for other
types of biological material it might be necessary to adapt the
lysis conditions and change to other detergents to isolate and
identify the complexes of interest. In practice, this includes
screenings of detergents (e.g., IGEPAL
®
, nonidet P40,
n-Dodecyl β-D-maltoside, Triton X-100) and testing of mild
lysis conditions (e.g., shear force, sonication, hypotonic
swelling) for its compatibility with the structural integrity of
protein complexes of interest as well as testing if an adverse
effect is present on the column. A good starting point for native
lysis conditions is to compare to published AP-MS
protocols [13].
2. SEC buffer supplemented with 0.025% NaN 3 (caution sodium
azide is toxic) can be stored at 4
C for a longer time. Before
usage, buffers must be filtered with sterile filters 0.22 μM.
3. The HNN-lysis buffer can be supplemented with Benzonase
(400 U/whole cell lysate of one 15 cm culture dish or
20 Â 10
6
) to digest DNA/RNA, thereby releasing DNA/RNA
bound protein complexes. It is recommended to incubate at
10
C for up to 45 min for efficient digestion of DNA/RNA.
The incubation temperature is a compromise between protein
complex stability and endonuclease activity.
4. Diluting the detergent by buffer exchange is a critical step.
Insufficient washing of the sample with detergent-free HNN
buffer through the Amicon filter will lead to residual detergent
288
Andrea Fossati et al.
acquired in DDA or DIA. Further, we present a data analysis
protocol suitable for multiple SEC-SWATH data sets (for timing
see Note 26). As shown in Fig. 3, we are able to identify
111 CORUM annotated protein complexes with statistical significance from a single co-fractionation experiment. The enhanced
throughput of the protocol which reduces MS acquisition time to
1 day per sample, along with its operational flexibility opens the
possibility to apply SEC–MS as a screening technology for differential protein complex formation with high reproducibility across
multiple cellular states or patient cohorts.
5 Notes
1. For starting material, cells and tissues from various different
biological materials can be used. We normally process suspension or adherent mammalian cells, however, there are several
SEC–MS studies published already, for which different types of
biological material were used, including various different mammalian cells and plant tissues [34, 35]. Consider that for other
types of biological material it might be necessary to adapt the
lysis conditions and change to other detergents to isolate and
identify the complexes of interest. In practice, this includes
screenings of detergents (e.g., IGEPAL
®
, nonidet P40,
n-Dodecyl β-D-maltoside, Triton X-100) and testing of mild
lysis conditions (e.g., shear force, sonication, hypotonic
swelling) for its compatibility with the structural integrity of
protein complexes of interest as well as testing if an adverse
effect is present on the column. A good starting point for native
lysis conditions is to compare to published AP-MS
protocols [13].
2. SEC buffer supplemented with 0.025% NaN 3 (caution sodium
azide is toxic) can be stored at 4
C for a longer time. Before
usage, buffers must be filtered with sterile filters 0.22 μM.
3. The HNN-lysis buffer can be supplemented with Benzonase
(400 U/whole cell lysate of one 15 cm culture dish or
20 Â 10
6
) to digest DNA/RNA, thereby releasing DNA/RNA
bound protein complexes. It is recommended to incubate at
10
C for up to 45 min for efficient digestion of DNA/RNA.
The incubation temperature is a compromise between protein
complex stability and endonuclease activity.
4. Diluting the detergent by buffer exchange is a critical step.
Insufficient washing of the sample with detergent-free HNN
buffer through the Amicon filter will lead to residual detergent
288
Andrea Fossati et al.