Covalent immobilization of trypsin on glutaraldehyde-activated silica for protein fragmentation

Figures & data

Table I. Immobilization of trypsin on silica support

		Specific activity^a (nmol/min/mg)
Immobilization method	Immobilization yield (%)	A	B	C	Optimum temperature ˚C	Optimum pH
Silica-GA	63	92	106	90	45	9.5
Silica-GA-Dah	60	103	115	97	45	9.5
Silica-GA- PEG2000	50	114	118	111	45	10
Silica-GA- PEG5000	63	79	102	95	45	10

^a Specific activity of immobilized trypsin in A: 0.1 M phosphate buffer (pH 8); B: 30% ACN + 0.1% TFA; C: 50% ACN + 0.1% TFA.

Figure 1. Thermal stability of free (♦) and immobilized trypsin [(▪) Silica-GA-Trypsin ; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin] at 45°C (A) and 55°C (B).

Figure 2. pH stability of free (♦) and immobilized trypsin [(▪) Silica-GA-Trypsin; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin].

Figure 3. Storage stability of free (♦) and immobilized trypsin [(▪) Silica-GA-Trypsin; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin].

Figure 4. Operational stability of immobilized trypsin [(▪) Silica-GA-Trypsin; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin].

Figure 5. BSA fragmentation with: A: free trypsin (overnight degradation in buffered solution); B: immobilized trypsin-solid phase module (15 min degradation in 30% ACN); and C: online trypsin reactor coupled with HPLC (30 min online degradation in column with 0.01 mL/min flow rate) [HPLC conditions for peptide separation: Flow rate: 0.4mL/min; Column temperature: 50°C; Mobile phases A: 0.1% TFA, B: ACN; Elution: 1%-50% ACN gradient; Wavelength: 220nm].