5. Non-denaturing acrylamide gel (12%)—for 10 ml mix the
following: 6 ml H 2 O, 1 ml 10Â concentrated TBE buffer,
80 μl 10% ammonium persulfate (APS), 8 μl TEMED (N,N,
N
0 ,N’-tetramethylethylenediamine), 3 ml 40% acrylamide:N,
N
0 -methylenebisacrylamide (1:19 mix).
6. DNA loading dye (Thermo Fisher).
7. Protein staining solution (45% methanol, 10% acetic acid,
0.25% Coomassie Brilliant Blue R250).
8. TBE buffer (90 mM Tris–Cl, 90 mM boric acid, 2 mM EDTA,
pH 8.3).
9. Fluorescent DNA stain GelRed (Thermo Fisher).
10. Deuterium oxide (D 2 O).
11. Quench buffer (500 mM glycine in LC-MS grade water,
pH 2.3 set with concentrated HCl).
12. Urea or Guanidine hydrochloride.
13. Liquid nitrogen.
14. Peltier cooled box or a polystyrene box filled with ice and water
mixture (see Note 4).
15. Two 6-port valves, one of them with injection needle port
(Rheodyne/IDEX).
16. PEEK (polyether ether ketone) tubing (OD 1/16
00 , ID—
250 μm and 750 μm) and stainless steel tubing (OD 1/16
00 ,
ID—125 μm).
17. UPLC (ultrahigh-performance liquid chromatography) fittings with ferrules (1/16
00 ).
18. Hamilton syringe with blunt end needle (100 μl).
19. Timer.
20. Columns with immobilized acidic protease—pepsin, rhizopuspepsin (protease type XVIII), nepenthesin-1, nepenthesin-2,
or aspergillopepsin (protease type XIII) immobilized on
POROS™ 20AL (Thermo Fisher Scientific) using previously
described [16, 17] protocols and packed into stainless steel
guard columns (2 mm  20 mm or 1 mm  20 mm, IDEX).
21. Desalting trap column—VanGuard Pre-column (ACQUITY
UPLC BEH C18, 130 A ˚ , 1.7 μm, 2.1 mm  5 mm, Waters).
22. Reversed-phase analytical column (ACQUITY UPLC BEH
C18, 130 A ˚ , 1.7 μm, 1 mm  100 mm, Waters).
23. Loading and desalting pump (1260 Infinity II Quaternary
pump (Agilent Technologies)).
24. Gradient pump (1290 series, Agilent Technologies).
25. LC loading solvent: 0.4% formic acid in water.
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Ruzena Filandrova et al.
following: 6 ml H 2 O, 1 ml 10Â concentrated TBE buffer,
80 μl 10% ammonium persulfate (APS), 8 μl TEMED (N,N,
N
0 ,N’-tetramethylethylenediamine), 3 ml 40% acrylamide:N,
N
0 -methylenebisacrylamide (1:19 mix).
6. DNA loading dye (Thermo Fisher).
7. Protein staining solution (45% methanol, 10% acetic acid,
0.25% Coomassie Brilliant Blue R250).
8. TBE buffer (90 mM Tris–Cl, 90 mM boric acid, 2 mM EDTA,
pH 8.3).
9. Fluorescent DNA stain GelRed (Thermo Fisher).
10. Deuterium oxide (D 2 O).
11. Quench buffer (500 mM glycine in LC-MS grade water,
pH 2.3 set with concentrated HCl).
12. Urea or Guanidine hydrochloride.
13. Liquid nitrogen.
14. Peltier cooled box or a polystyrene box filled with ice and water
mixture (see Note 4).
15. Two 6-port valves, one of them with injection needle port
(Rheodyne/IDEX).
16. PEEK (polyether ether ketone) tubing (OD 1/16
00 , ID—
250 μm and 750 μm) and stainless steel tubing (OD 1/16
00 ,
ID—125 μm).
17. UPLC (ultrahigh-performance liquid chromatography) fittings with ferrules (1/16
00 ).
18. Hamilton syringe with blunt end needle (100 μl).
19. Timer.
20. Columns with immobilized acidic protease—pepsin, rhizopuspepsin (protease type XVIII), nepenthesin-1, nepenthesin-2,
or aspergillopepsin (protease type XIII) immobilized on
POROS™ 20AL (Thermo Fisher Scientific) using previously
described [16, 17] protocols and packed into stainless steel
guard columns (2 mm  20 mm or 1 mm  20 mm, IDEX).
21. Desalting trap column—VanGuard Pre-column (ACQUITY
UPLC BEH C18, 130 A ˚ , 1.7 μm, 2.1 mm  5 mm, Waters).
22. Reversed-phase analytical column (ACQUITY UPLC BEH
C18, 130 A ˚ , 1.7 μm, 1 mm  100 mm, Waters).
23. Loading and desalting pump (1260 Infinity II Quaternary
pump (Agilent Technologies)).
24. Gradient pump (1290 series, Agilent Technologies).
25. LC loading solvent: 0.4% formic acid in water.
196
Ruzena Filandrova et al.