4. Centrifuge.
5. Microplate shaker vortemp56 Labnet, or similar model.
6. Multichannel pipette (10–100 μL).
2.6 MS Setup
and Material
1. Sample loading and LC–MS buffers:
(a) MS Buffer A: 0.1% FA (formic acid) in water solution,
LC–MS grade (Thermo Fisher Scientific AG,
Switzerland).
(b) MS buffer B: 0.1% FA in ACN (acetonitrile), LC–MS
grade (Thermo Fisher Scientific AG, Switzerland).
(c) 2-propanol (Merck, Darmstadt, Germany).
(d) iRTs peptides kit (Biognosys AG, Switzerland).
(e) Beta-Galactosidase, Digested, 625 pmol/vial (Sciex,
MA, USA).
Table 1
Example of HPLC modules used for SEC separations for the presented SEC-SWATH workflow
LC Pump
1100 Series, Agilent
Degasser
1100 series, Agilent
LC injection system
1260 infinity series, Agilent
LC Thermostatted column compartments
1100 series, Agilent
LC detector
1100 series, Agilent
LC fraction collectors
1260 infinity series, Agilent
LC software
Agilent hybrid, OpenLab Agilent
Table 2
Proposed high-performance SEC column materials, separation ranges, and maximum loading
capacity (according to manufacturer) for whole cell lysates SEC–MS analysis
Column
Particle size/pore
size
Dimension
Separation
range
Max. loading
capacity
Yarra SEC4000
(Phenomenex)
3 μm, 500 A ˚
300x
7.8 mm
15 kDa to
1.5 MDa
1 mg
Biosep SEC-S4000
(Phenomenex)
5 μm, 500 A ˚
300Â
7.8 mm
15 kDa to
2.0 MDa
1 mg
SRT-C1000 (Sepax)
5 μm, 1000 A ˚
300Â
7.8 mm
50 kDa to
7.5 MDa
1 mg
SRT-C500 (Sepax)
5 μm, 500 A ˚
300Â
21.2 mm
15 kDa to
5 MDa
3–5 mg
274
Andrea Fossati et al.