Multidigestion in protease-immobilized microreactors / H. Yamaguchi et al. / Anal. Biochem. 407 (2010) 12–18
13
the microchannel, thereby resulting in a high enzyme-to-substrate
ratio and consequently allowing the protease-immobilized mic-
roreactor to perform rapid digestion. Furthermore, the immobi-
lized protease can be easily isolated and removed from the
protein digests prior to MS, thereby eliminating the influence of
fragments from the enzyme on MS results. Another advantage is
the possibility of multiple uses (reusability) due to low autodiges-
tion and high thermal or chemical stability [12–14]. Several meth-
ods for protease immobilization have been reported where the
enzyme was covalently bound, trapped, or physically adsorbed
onto different supports based on either silica and polymer particles
or monolithic materials [9–11,15]. We previously reported the pro-
cedure for immobilizing enzyme on the internal surface of the
poly(tetrafluoroethylene) (PTFE) tube by forming an enzyme poly-
meric membrane through a cross-linking reaction between Lys res-
idues on the protein surface [16]. Using this system, we reported
an efficient digestion method by the protease (trypsin [TY] or chy-
motrypsin [CT])-immobilized microreactor (TY–microreactor and
CT–microreactor, respectively) [14]. Although this microreactor
showed rapid digestion (5 min at 30 °C) compared with the con-
ventional in-solution digestion (18 h at 37 °C) [14], it needs to im-
prove the sequence coverage against the substrate protein that has
a resistance to proteolysis. The improved sequence coverage is
important to enhance the probability of identification and increase
the likelihood of detection of structural variants generated by
processes such as alternative splicing and posttranslational
modifications.
poly-Lys (MWs = 62,140 Da for CT and 4200 Da for AP, 10 mg/ml)
were mixed at a 1:1 volume ratio in 50 mM phosphate buffer
(PB, pH 8.0). The cross-linker in 50 mM PB (pH 8.0) (4% paraformal-
dehyde and 0.25% glutaraldehyde) and enzyme/poly-Lys solutions
were supplied to PTFE tube using a T-shaped connector that cre-
ates a laminar flow [13]. Solution introduction was performed at
different pumping rates (0.75
ll/min for the cross-linker and
0.5 l/min for the enzyme/poly-Lys) by an EconoFlow syringe
l
pump (Harvard Apparatus, Holliston, MA, USA). The polymeriza-
tion reaction was performed for 15 h at 4 °C. Other procedures
were the same as reported previously [13,16]. For the TY immobi-
lization microreactor, poly-Lys was omitted and polymerization
was performed as reported previously [14].
Digestion by microreactor
Proteolysis was carried out in buffer A (10 mM ammonium ace-
tate, pH 8.5) at 30 °C. Substrate proteins were dissolved to a con-
centration of 100
lg/ml in buffer A. The protein solution was
pumped through the microreactor at a flow rate of 1.2 to 15
ll/
min using a Pico Plus syringe pump. The peptide fragments were
collected in 1.5-ml test tubes and analyzed by MS and high-perfor-
mance liquid chromatography (HPLC) measurements.
To determine the kinetic parameters, Km and Vmax, the initial
velocities (v) were measured at various substrate concentrations
([S]) in buffer B (50 mM Tris–HCl, pH 8.0) at 30 °C. The substrate
solution was pumped through the microreactor at a flow rate of
The conventional approach using multidigestion by different
proteases for improved sequence coverage is based on parallel
digestions of the same samples and analyzes overlapping peptides.
However, this approach takes a long time and uses a multistep pro-
cedure. In this study, we prepared the tandem microreactor that
was connected by different protease-immobilized microreactors
using a Teflon connector. Connection was made easy because the
current microreactors were made of a PTFE tube. The multienzy-
matic reaction by the tandem microreactors can be used for rapid
analysis of protein sequence with high sequence coverage.
5.0 ll/min. Synthetic compounds (BAPA for TY–microreactor,
GPNA for CT–microreactor, and p-nitrophenylphosphate for AP–
microreactor) were used as substrates. The reaction was evaluated
as the amount of released p-nitroaniline or p-nitrophenol calcu-
lated from absorbance at 405 nm using a spectrophotometer (Mul-
tiskan JX, Thermo Fisher Scientific, Waltham, MA, USA). The data
were fitted to the Michaelis–Menten equation.
The stability of reusable microreactors was determined using
Cyt-C for TY and b-casein for CT in buffer A at 30 °C and was ana-
lyzed by MS. The concentration of each substrate was 100
lg/ml.
The flow rate of the substrate was 2.5 l/min. After each hydrolysis
l
reaction, the protease-immobilized microreactors were washed
with buffer B and stored at 4 °C.
Materials and methods
The chemical stability of the protease-immobilized microreac-
tors was tested using the synthetic compounds in buffer B with
or without 4 M urea at 30 °C. The flow rate of the substrate was
Materials
N-Tosyl-
L-phenylalanyl chloromethyl ketone-treated trypsin
5.0
ll/min (reaction time = 5.2 min). The concentrations of BAPA
(TY) was purchased from Worthington (Lakewood, NJ, USA).
Chymotrypsin (CT), cytochrome c (Cyt-C), glutaraldehyde, and
paraformaldehyde were obtained from Wako Pure Chemical (Osa-
a
-
for TY and GPNA for CT were 0.5 and 1 mM, respectively. For the
in-solution experiment, the concentration of protease was 40
ml and assays were performed for 5.2 min.
lg/
ka, Japan). b-Casein,
glutaryl- -phenylalanine p-nitroanilide (GPNA), pepsin A, and
poly- -Lys hydrobromides (MWs = 62,140 and 4200 Da) were pur-
a-cyano-4-hydroxycinnamic acid (CHCA), N-
L
L
In-solution digestion
chased from Sigma–Aldrich (St. Louis, MO, USA). Alkaline phospha-
tase (AP) was purchased from Biozyme Laboratories (South Wales,
UK). Benzoyl-L-arginine p-nitroanilide (BAPA) was obtained from
Peptide Institute (Osaka, Japan). p-Nitrophenylphosphate was ob-
tained from Nacalai Tesque (Kyoto, Japan). All other chemicals
were of analytical grade. PTFE tube was purchased from Flon
Chemical (Osaka, Japan).
The in-solution digestion was performed by adding proteases
into the substrate protein at a substrate-to-protease ratio of 50:1.
The reaction solution was incubated in buffer A at 37 °C for 18 h.
Acetic acid was added into the solution to stop the reaction.
MS analysis
Preparation of protease-immobilized microreactors
Electrospray ionization time-of-flight (ESI–TOF) MS measure-
ments were performed using a Mariner mass spectrometer (Ap-
plied Biosystems, Foster City, CA, USA) with a scan range of m/z
200 to 2500. The digested samples were dissolved in 50% aqueous
Protease-immobilized microreactors were prepared as reported
previously [13,16]. Briefly, enzymes were reacted with bifunctional
cross-linker agents, paraformaldehyde, and glutaraldehyde to facil-
acetonitrile and 1% formic acid at a concentration of 50 to 100
lg/
itate enzyme–enzyme covalent binding on PTFE tube (500
l
m i.d.
ml. Under 40 g/ml of the digested samples were difficult to detect
l
and 13 cm length). For the CT–microreactor and AP-immobilized
the signals using Mariner instrumentation. The acceleration volt-
microreactor (AP–microreactor), the enzyme (10 mg/ml) and
age was 4 kV. The electrospray signal was stabilized by a flow of