Waters Enzymate BEH Pepsin Columns - Manual (Page 4)

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[ CARE AND USE MANUAL ]
Enzymate BEH Pepsin Column
c. Digesting/Trapping Flow Rate
The recommended starting digestion flow rate is at 100 μL/min
using 0.1% formic acid (pH 2.5) in water. Decreasing the digestion
flow rate increases the contact time between the proteins and
the immobilized pepsin enzyme. This can increase digestion
efficiency but will result in longer digestion times. Increasing
the flow rate decreases the contact time between the proteins
and the immobilized pepsin enzyme and can decrease digestion
efficiency.
d. Quenching
A recommended quenching solution is 100 mM potassium
phosphate, pH 2.50 dissolved in water (50 mM K
2
HPO
4
and 50 mM
in KH
2
PO
4
adjusted to exactly pH 2.50 with concentrated HCl).
e. Protein Injection
Typical protein buffers include Tris (tris(hydroxymethyl)
aminomethane), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid), or PBS (phosphate buffered saline). Between 10 and 50 pmol
of protein can be injected on column.
f. Trapping Time
The digested peptides can be trapped and desalted on the trap
column for several minutes (and for as little as thirty seconds)
depending on the buffer concentration before eluting to the
ACQUITY UPLC Column. Increasing the trapping time by decreasing
the flow rate can increase digestion efficiency but may cause
back-exchange (i.e., deuterium for hydrogen).
g. pH and Solvent Compatibility
The Enzymate BEH Pepsin Column is NOT
compatible with organic solvents, detergents, or
high pH. Therefore, DO NOT expose the Enzymate
BEH Pepsin Column to high concentrations of
organic solvents, detergents, or high pH solvents (pH > 4.5).
The Enzymate BEH Pepsin Column can tolerate a very low
concentration of organic solvent (≤ 5%) which can be injected
during a column cleaning procedure as described in section i.
h. Temperature
The Enzymate BEH Pepsin Column is maintained at a digestion
temperature of between 10–20 °C. Increasing digestion
temperature may increase digestion efficiency but may also
promote back-exchange (i.e., deuterium for hydrogen).
i. Column Washing/Cleaning
Under normal operating conditions, the digestion buffer (0.1%
formic acid in water, pH 2.5) should be sufficient to remove
residual proteins and/or peptides from the Enzymate BEH Pepsin
Column and the trap column, thereby reducing injection-to-
injection carryover. However, if carryover is observed, prepare
a column cleaning solution of 1.5 M guanidine hydrochloride/
acetonitrile/formic acid (95.2/4/0.8). Inject 100 μL of this
cleaning solution and allow it to flow through the Enzymate
BEH Pepsin Column and the trap column. Inject a blank solution
to ensure no injection-to-injection carryover is observed. If
carryover is observed, repeat the cleaning solution injection
protocol until no injection-to-injection carryover is observed,
j. Column Storage
Store the Enzymate BEH Pepsin Column at 4 °C in 0.1% formic
acid in water when not in use. Make certain that the column end
caps are secured tightly in place to ensure that the Enzymate BEH
Pepsin Column does not dry out.
IV. CHECKING FOR ENZYMATIC ACTIVIT Y
The Enzymate BEH Pepsin Column should be checked periodically
to ensure acceptable enzymatic activity. Waters recommends
checking the enzymatic activity by injecting a standard
protein (e.g., Waters HDX Phosphorylase B Check Standard, PN
186006930 ) onto the Enzymate BEH Pepsin Column, followed
by LC-MS analysis to detect the peptides from the on-column
proteolysis. Acceptable performance criteria for the Enzymate
BEH Pepsin Column activity can be subjective. In general, if the
Phosphorylase B sequence coverage from the LC-MS analysis
is ≥ 75%, the Enzymate BEH Pepsin Column has maintained its
optimum enzymatic activity.