In this chapter, we describe a generic method for profiling
hundreds of protein complexes in a single SEC-SWATH experiment. High-throughput LC–MS analysis allows quantification of
protein profiles across the size fractionation gradient within 1 day,
thereby enabling technical replicates and profiling of differential
conditions. The chapter ends with an application of an in-house
developed software tool for statistical scoring of protein complexes
from quantitative co-elution data.
2 Material
2.1 Cell Culture
and Tissue Samples
1. Jurkat T cells (ATCC, clone A3).
2. For adherent cell culture: A 70–80% confluent 15 cm dish
(145 cm
2
) or 20 Â 10
6 of adherently grown mammalian cells
(e.g., HEK293, HeLa). In principle any native protein samples
including tissues lysates can be used (see Note 1).
3. Phosphate buffer saline (PBS), without MgCl 2 or ClCl 2 ,
pH 7.4.
4. RPMI-1640.
5. FCS.
6. PenStrep.
7. Centrifuge.
2.2 Lysis Buffer
1. Stock solutions for native lysis buffer:
(a) 1 M HEPES pH 7.5 stored at 4
C.
(b) 5 M NaCl stored at room temperature (RT).
(c) 0.5 M NaF stored at RT.
(d) 20% NP-40 (IPEGAL
®
) stored at RT.
(e) 200 mM sodium vanadate stored at À20
C.
(f) 100 mM PMSF in EtOH (molecular grade) stored at
À20
C.
(g) Protease Inhibitor Cocktail (of 500Â mix, Sigma P8849).
2. HNN-buffer, pH 7.5: 50 mM HEPES pH 7.5, 150 mM NaCl,
50 mM NaF (filtered 0.22 μm). After sterile-filtration with a
top filter with 0.22 μm pore size, buffer can be stored at 4
C
for several weeks (see Note 2).
3. HNN-lysis buffer, pH 7.5, freshly prepared: HNN-buffer, supplemented with 0.5% NP-40 (IPEGAL
® ) detergent, 0.4 mM
Na 3 VO 4 , 1 mM PMSF, 1Â Protease Inhibitor Cocktail. If the
target protein complexes are DNA-bound, the HNN-lysis
buffer should be supplemented with nucleases (see Note 3).
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Andrea Fossati et al.
hundreds of protein complexes in a single SEC-SWATH experiment. High-throughput LC–MS analysis allows quantification of
protein profiles across the size fractionation gradient within 1 day,
thereby enabling technical replicates and profiling of differential
conditions. The chapter ends with an application of an in-house
developed software tool for statistical scoring of protein complexes
from quantitative co-elution data.
2 Material
2.1 Cell Culture
and Tissue Samples
1. Jurkat T cells (ATCC, clone A3).
2. For adherent cell culture: A 70–80% confluent 15 cm dish
(145 cm
2
) or 20 Â 10
6 of adherently grown mammalian cells
(e.g., HEK293, HeLa). In principle any native protein samples
including tissues lysates can be used (see Note 1).
3. Phosphate buffer saline (PBS), without MgCl 2 or ClCl 2 ,
pH 7.4.
4. RPMI-1640.
5. FCS.
6. PenStrep.
7. Centrifuge.
2.2 Lysis Buffer
1. Stock solutions for native lysis buffer:
(a) 1 M HEPES pH 7.5 stored at 4
C.
(b) 5 M NaCl stored at room temperature (RT).
(c) 0.5 M NaF stored at RT.
(d) 20% NP-40 (IPEGAL
®
) stored at RT.
(e) 200 mM sodium vanadate stored at À20
C.
(f) 100 mM PMSF in EtOH (molecular grade) stored at
À20
C.
(g) Protease Inhibitor Cocktail (of 500Â mix, Sigma P8849).
2. HNN-buffer, pH 7.5: 50 mM HEPES pH 7.5, 150 mM NaCl,
50 mM NaF (filtered 0.22 μm). After sterile-filtration with a
top filter with 0.22 μm pore size, buffer can be stored at 4
C
for several weeks (see Note 2).
3. HNN-lysis buffer, pH 7.5, freshly prepared: HNN-buffer, supplemented with 0.5% NP-40 (IPEGAL
® ) detergent, 0.4 mM
Na 3 VO 4 , 1 mM PMSF, 1Â Protease Inhibitor Cocktail. If the
target protein complexes are DNA-bound, the HNN-lysis
buffer should be supplemented with nucleases (see Note 3).
272
Andrea Fossati et al.