Microfluid Nanofluid (2013) 15:5764

DOI 10.1007/s10404-012-1134-0

RESEARCH PAPER

Activity of the integrated on-line trypsin microreactor

and nanoelectrospray emitter in acetonitrilewater
co-solvent mixtures
Ying Long Troy D. Wood

Received: 13 October 2012 / Accepted: 17 December 2012 / Published online: 28 December 2012
Springer-Verlag Berlin Heidelberg 2012

Abstract The on-line trypsin microreactor and nanoelectrospray emitter for peptide mass mapping was demonstrated
to be functional under aqueous conditions, but it is well
known that electrospray ionization works more efficiently
with organic co-solvents. Here, an activity assay was
developed to determine the activity of this integrated device
with acetonitrile as a co-solvent. Trypsin was immobilized
onto fused silica capillaries pulled to fine tips as integrated
microreactors coupled as nanoelectrospray ionization emitters. The model substrate Na-benzoyl-L-arginine ethyl ester
(2.520 lM) and an internal standard (Na-Z-L-arginine
(Z-Arg)) were dissolved in acetonitrile/water at various
ratios and infused through the immobilized trypsin microreactor. The trypsin digestion product Na-benzoyl-L-arginine (B-Arg) was detected by nanoelectrospray ionization
coupled to an ion trap mass spectrometer, and its abundance
compared to Z-Arg for quantification. The activity of
immobilized trypsin in the microreactor was determined by
measuring the ratio of the peak intensities of the hydrolysis
product B-Arg to Z-Arg internal standard (three replicates).
Kinetic parameters determined from LineweaverBurk
analysis indicate an enhancement of trypsin activity upon
immobilization and the addition of increasing ratios of acetonitrile up to 80 %, where Km is 0.14 mM and Vmax =
1.2 lM/s. Much lower immobilized trypsin activities were
noted at 100 % ammonium acetate or 100 % acetonitrile
than when the two solvents were mixed. The results clearly
indicate that immobilized trypsin retains high biocatalytic
activity in 2080 % acetonitrile and is highly compatible
with nanoelectrospray ionization mass spectrometry.
Y. Long
T. D. Wood (&)
Department of Chemistry, University at Buffalo,
State University of New York, 417 Natural Sciences Complex,
Buffalo, NY 14260-3000, USA
e-mail: twood@buffalo.edu

Keywords Nanofluidic integration
Trypsin

Microreactor
Electrospray ionization
Nanoelectrospray

1 Introduction
Proteomics is a field that has emerged in the last two
decades to tackle the immense task of characterizing the
complete composition of proteins within a biological
organism (Teng et al. 2010). This task is highly complex
because proteins are modified as a function of exposure to
biological stressors that an organism may encounter, and
thus certain proteins may be present in multiple related, but
not identical, forms. Furthermore, these post-translational
modifications are dynamic and can vary widely in their
relative abundance.
A key step in deducing proteomic information is the
digestion of proteins of interest into smaller peptides using
a proteolytic enzyme using the so-called bottom-up
approach followed by identification of the proteolytic
peptides using mass spectrometry (Lin et al. 2003). Enzymatic digestion has been accomplished conventionally in
solution, but there are a number of important drawbacks to
this procedure. In-solution enzymatic hydrolysis is of relatively low efficiency, takes lengthy periods of time
(624 h), and is complicated by the production of peptides
due to the autodigestion of the proteolytic enzyme.
As a consequence of these limitations of traditional
solution-phase proteolysis, techniques employing immobilized enzymes have become attractive. Immobilization is
known to improve the stability and performance of
enzymes (Weetall and Vann 1976; Wang et al. 2001;
Markvicheva et al. 1996; Sears and Clark 1993). First, an
immobilized enzyme reactor (IMER) can be fabricated by
immobilization of active enzyme onto a surface. IMER

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Microfluid Nanofluid (2013) 15:5764

devices also perform digestion rapidly and show high

enzyme stability, reusability, and have relatively low
manufacturing costs. In addition, IMERs can be coupled
with separation techniques and/or with mass spectrometry
detection systems that are of high utility for protein characterization in the proteomics workflow.
The open-tubular immobilized trypsin reactor (IMTR)
coupled to low-flow ESI developed in our lab is characterized
with rapid digestion and high tryptic peptide coverage (Zhao
et al. 2006). In our initial report, the data were collected under
solution conditions favoring trypsin activity and for compatibility with low-flow ESI (pH *8.0, 80/20 10 mM ammonium acetate buffer/acetonitrile) (Zhao et al. 2006). Trypsin
immobilized onto a support does not exhibit identical biocatalytic activity as solution-phase trypsin; it has been noted
that the surface of the substrate support to which the enzyme is
immobilized plays a critical role in the retention of the
enzymes tertiary structure, which in turn has a considerable
influence on the thermal stability and the catalytic activity of
the immobilized enzyme in comparison to the enzyme under
native solution conditions (Girelli and Mattei 2005). Therefore, the immobilized enzyme has different kinetic properties
which can modify the MichaelisMenten constant (Km) and
maximum velocity (Vmax) (Girelli and Mattei 2005).
Biocatalysis in non-aqueous media has enlarged the
scope of application of enzymes beyond the aqueous media
(Gupta 1992). It is believed that the change of dielectric
constant of the medium affects the chemical reaction rates.
One aspect of the aqueous/organic cosolvents which raised
our attention is the enhancement of enzyme activity in the
presence of an organic co-solvent (Russell et al. 2001;
Strader et al. 2006; Tan and Lovrien 1972). While enzymes
may lose their activities gradually as the concentration of
organic solvent increases (Butler 1979; Batra and Gupta
1994), some recent reports show that trypsin is active even
with relatively high concentrations of acetonitrile (Russell
et al. 2001; Strader et al. 2006). We had never tested IMTR
Fig. 1 Hydrolysis of
Na-benzoyl-L-arginine ethyl
ester (BAEE) into Na-benzoylL-arginine (B-Arg) with Na-Z-Larginine (Z-Arg) as the internal
standard

activity except under relatively low concentrations of

organic solvent (B20 %) and were curious whether the
IMTR would function under such conditions which may be
very appropriate for coupling to nanoelectrospray.
Thus, an important test of our IMTRs applicability is the
activity of trypsin in the presence of an organic
co-solvent (acetonitrile). In order to characterize the relative
efficiency of IMTRs, it was necessary to develop a protocol to
measure the biocatalytic activity of immobilized trypsin. The
conditions used should be compatible with ESI if the IMTRs
are to be used for proteomics applications. Trypsin activity is
classically determined using spectrophotometric detection of
hydrolysis of Na-benzoyl-L-arginine ethyl ester (BAEE)
because of the chromophore in BAEE. The Michaelis constant (Km) and the maximum velocity (Vmax) are determined
in this manner (Schwert and Takenaka 1955).
Our trypsin activity assay for the integrated nanoelectrospray IMTR is based upon one developed using matrixassisted laser desorption ionization (MALDI) MS (Guo
et al. 2003; Xu et al. 2004) of off-line IMTR activity by the
Zou group, which itself was adapted from the spectrophotometric approach for measuring trypsin activity.
Trypsin activity is assayed by the hydrolysis of BAEE into
Na-benzoyl-L-arginine (B-Arg), which is depicted in Fig. 1.
Quantification of the product (B-Arg) is achieved using an
internal standard, Na-Z-L-arginine (Z-Arg), a species that is
not hydrolyzable by trypsin. BAEE produces an (M ? H)?
ion at 307 Da, while B-Arg produces an (M ? H)? at
279 Da and Z-Arg produces an (M ? H)? at 309 Da; their
different masses allow them to be distinguished from one
another in the nanoelectrospray mass spectrum. Relative
peak height ratios of Z-Arg/B-Arg in the nanoelectrospray
mass spectra enable an estimation of the solution concentration of B-Arg with exposure time. A plot of the reciprocal of the reaction rate (1/v) versus the reciprocal of the
substrate concentration (1/[S]) is constructed to obtain Km
and Vmax BAEE hydrolysis at different ratios of
NH2

NH2

Trypsin

NH

NH

NH

O
O

OEt

N -benzoyl-L-arginine (B-Arg)

N-benzoyl-L-arginine ethyl ester (BAEE)

NH2
O

OH

product

substrate

NH
NH
O
O

OH

N-Z-L-arginine (Z-Arg)

123

NH

NH

NH

NH

Microfluid Nanofluid (2013) 15:5764

acetonitrile/buffered aqueous solvents is measured using

the integrated nanoelectrospray emitter/IMTR.

2 Experimental
2.1 Materials
Fused silica capillaries (i.d. = 75 lm, o.d. = 360 lm)
were purchased from Polymicro Technologies, Inc. (Phoenix,
AZ). Glutaraldehyde, 3-aminopropyltriethoxy-silane (3-APTES), ammonium bicarbonate, (TPCK)-trypsin from bovine
pancreas, Na-benzoyl-L-arginine ethyl ester hydrochloride
(BAEE), and sodium azide were from Sigma-Aldrich Co. (St.
Louis, MO). Na-Z-L-arginine was purchased from Bachem
(Torrance, CA). Tris was purchased from Bio-Rad Laboratories (Hercules, CA). Sodium cyanoborohydride was purchased
from Acros Organics (Geel, Belgium). Sodium hydroxide and
calcium chloride were purchased from J.T. Baker (Phillipsburg, NJ). Hydrochloric acid was purchased from Fisher Scientific (Fair Lawn, NJ). HPLC grade acetonitrile and methanol
were purchased from Aldrich (Milwaukee, WI). The water was
purified by a Milli-Q system (Nippon Millipore, Tokyo, Japan).

59

Instrument Co. (Novato, CA) P-2000 laser-based capillary

puller (Valaskovic et al. 1995; White and Wood 2003).
Parameters were optimized to produce emitters with short
tapers and relatively thick-walled orifices. The emitters were
dipped into the solution of polyaniline (Monsanto, St. Louis,
MO) to provide conductivity needed for nanoelectrospray
(Maziarz et al. 2000). To avoid emitter clogging during the
coating process, a stream of air was blown through the
emitters during dipping. Evaporation of the PANI solution in
air creates a coating layer of about 2030 nm in thickness (as
determined by SEM). After coating, the emitters were
removed and stored until used.
2.4 Sample preparation
TPCK-treated trypsin was prepared at 2 mg/ml in 50 mM
Tris, 10 mM CaCl2 (pH = 8.16). BAEE was prepared as
solutions of 5, 10, and 15 lM in 80/20 10 mM NH4HCO3/
ACN (pH = 8.10). Z-Arg was prepared as 10 lM in 80/20
10 mM NH4HCO3/ACN (pH = 8.10) solution. Solutions
of BAEE and Z-Arg were mixed before injection into the
IMTR capillary.

2.2 Preparation of IMTRs

2.5 Kinetic assay of immobilized trypsin

Segments of bare fused silica capillaries (30 cm, i.d.

75 lm, o.d. 360 lm) were pretreated by 1 M NaOH at
room temperature for 1 h, followed by H2O for 1 h and
methanol for 1 h. The capillaries were dried overnight in
an oven at 110 C. The immobilization of trypsin onto the
inner wall surface of a capillary was performed according
to the following procedure. First, silanization in 10 %
APTES solution was performed at 95 C for 3 h after
activating the capillary with concentrated HCl for 1 h. This
was followed by activation of 2 % glutaraldehyde at room
temperature for another 4 h. Finally, TPCK-treated trypsin
(2 mg/ml in 50 mM Tris, 10 mM CaCl2, pH = 8.16) was
circulated through the capillary at room temperature for
24 h. To suppress the reversibility of the formed Schiffs
base (and to stabilize the bound enzyme), trypsin was
coupled to the inner wall surface in the presence of a weak
reducing agent, NaCNBH3. Any trypsin not immobilized
onto the inner wall surface of the capillary was rinsed out
by flushing the capillary with 80/20 10 mM NH4HCO3/
ACN (pH = 8.10) solution. The capillary with immobilized trypsin was stored with 10 mM Tris, 10 mM CaCl2,
0.02 % NaN3 (pH = 8.18) in a refrigerator until use.

The activity of immobilized trypsin was determined at

room temperature by monitoring the catalytic hydrolysis of
substrate BAEE into B-Arg with Z-Arg as the internal
standard. A range of substrate concentrations (2.520 lM)
was mixed with an equal volume of 10 lM internal standard Z-Arg. The mixture was delivered through the nanoESI emitter with immobilized trypsin and introduced into
MS by using nanoESI. Relative peak height ratios of
Z-Arg/B-Arg in the mass spectra were analyzed.

2.3 Nanoelectrospray emitter fabrication

The fused silica nanoESI emitters with immobilized trypsin
were prepared by pulling heated glass capillaries with a Sutter

2.6 Effect of acetonitrile on kinetics of immobilized

trypsin
Acetonitrile was chosen to be the organic solvent to demonstrate the effect of organic solvent on enzyme assay in
part because it has been shown to be highly compatible with
high trypsin activity in previous studies (Russell et al. 2001;
Strader et al. 2006). This study was performed with
increasing concentrations (0100 %) of acetonitrile in
10 mM NH4HCO3/ACN (pH = 8.10). The activity of
immobilized trypsin in the acetonitrile/aqueous mixture was
determined at room temperature by monitoring catalytic
hydrolysis of a range of BAEE substrate concentrations
(540 lM before mixing) at different concentrations of
acetonitrile mixed with an equal volume of 10 lM Z-Arg as
internal standard. The relative peak height ratios of Z-Arg/
B-Arg were analyzed from the resulting mass spectra.

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Microfluid Nanofluid (2013) 15:5764

2.7 Nanoelectrospray conditions

All experiments were performed on a modified Thermo
Electron LCQ Advantage quadrupole ion trap mass spectrometer (San Jose, CA). The ESI source was replaced by a
home-built nanoESI source. Samples were introduced into
the nanoESI source using polyaniline-coated emitters with
immobilized trypsin developed in our laboratory. The
emitters were positioned *2 cm from the inlet of the mass
spectrometer and supplied with ?4.7 kV spray voltage to
form positive ions. The capillary temperature was set at
210 C. The sheath gas and auxiliary gas were all at 0.

3 Results and discussion

3.1 Quantitative evaluation of the immobilized
versus free trypsin activity
As shown in Fig. 2, the hydrolyzing reaction is observed
through the appearance of B-Arg signal and a reduction of
BAEE signal intensity. Activity of the immobilized
enzyme was determined by determining the ratio of peak
intensity of B-Arg to Z-Arg. Z-Arg was used not only as an
internal standard for quantitative analysis but also as a
standard to evaluate spectrum quality.
To investigate the activity of the immobilized enzyme,
the conditions for digestion were compared. To achieve
compatibility between the biochemical assay and MS
detection conditions, 80/20 10 mM NH4HCO3/ACN was
selected initially. Five enzymatic hydrolyses with different
concentrations of BAEE substrate in the mixed solution
2.5, 5, 7.5, 15, and 20 lMwere compared in terms of

Fig. 3 ESI mass spectra for the conversion of BAEE to B-Arg

catalyzed by immobilized trypsin. The BAEE concentrations used are
a 2.5; b 5; c 7.5; d 15 and e 20 lM, respectively
Fig. 2 ESI mass spectrum that monitors the enzymatic production of
the product B-Arg (m/z 279) from the substrate BAEE (m/z 307) in the
immobilized trypsin microreactor. The internal standard Z-Arg is
detected at m/z 309

123

enzyme activity, which is reflected by the ratio of ion

intensity of the product B-Arg to internal standard Z-Arg
(5 lM) in the ESI mass spectra. After hydrolysis (flow

Microfluid Nanofluid (2013) 15:5764

61

immobilized enzymes is the consequence of diffusion limitations, which reflects the change in trypsins intrinsic
kinetics (Sears and Clark 1993). Another possibility is that
the partial blockage of active site by the support may hinder
the binding of the substrate to the active sites of enzyme,
thereby increasing Km so that a higher concentration of
substrate is needed to achieve half of the maximum value
(Sears and Clark 1993).
3.2 Quantitative investigation of the effect
of acetonitrile on the immobilized trypsin activity

Fig. 4 Plots of the ion intensity ratio of B-Arg/Z-Arg for the

conversion of BAEE to B-Arg in the immobilized trypsin reactor as a
function of substrate BAEE concentration at different ratios of
acetonitrile/10 mM NH4HCO3 (pH = 8.10)

rate = 30 ll/h in 15 cm immobilized trypsin fused silica,

calculated as 1.3 min residence time), the velocity of
enzyme activity increases proportionally with an increase
in BAEE substrate concentration. Figure 3ae shows the
ESI mass spectra. As is evident, even with the short residence times in the microreactor used here, throughout the
range of BAEE concentrations employed, conversion into
the product B-Arg (m/z 279) is complete; no residual
BAEE (m/z 307) remains. Figure 4 plots a comparison of
B-Arg/Z-Arg to evaluate the conversion of BAEE into
B-Arg by trypsin as a measure of enzymatic efficiency.
Kinetic parameters are determined by constructing a
LineweaverBurk plot (Fig. 5).
The enzymatic activity of immobilized trypsin in the
microreactor behaves differently from free trypsin. Kinetic
parameters for the immobilized trypsin were determined as
Vmax = 0.79 lM/s and Km = 0.092 mM from Fig. 5 based
on the MichaelisMenten equation. For free trypsin,
the measured enzymatic reaction constants are Vmax =
1.50 lM/s and Km = 0.070 mM, respectively (Guo et al.
2003). The Vmax value of the immobilized trypsin is lower
than that of the free trypsin. This may have resulted from a
loss of some of the available active sites of immobilized
trypsin due to denaturation of a portion of the enzyme
during the immobilization process, and/or due to partial
blockage of the active sites of the immobilized trypsin with
different orientations on the capillary wall. The Km value
represents the substrate concentration at which the reaction
rate is half of Vmax. The Km value of immobilized trypsin is
higher than that of free trypsin. This is consistent with literature findings that suggest that a higher Km for

The catalytic properties of immobilized trypsin in acetonitrile/water co-solvent mixtures were examined. According to the thermodynamic model of enzyme (protein)
denaturation in organic co-solvents, protein molecules in
aqueous solution have a hydration shell formed from the
hydrogen bonds between water and the protein surface.
This water shell is believed to be an integral part of the
protein for its structure and function (Kuntz and Kauzmann
1974; Rupley et al. 1983). Replacement of bound water
molecules with organic solvents (Gekko and Timasheff
1981; Gekko and Morikawa 1981) will lead to the dramatic
change of the protein structure, i.e., denaturation. Therefore, it is assumed based on this model that dehydration is
the primary driving force of protein denaturation.
A quantitative relationship between physicochemical
properties of organic solvents and their denaturing strength
is established from the molecular mechanism of the denaturation process. The term denaturation capacity (DC) was
introduced to assess the relative ability of different organic
solvents to denature proteins. The higher DC of the solvent
means stronger denaturing ability. The scale of DC is
useful in practice to predict quantitatively the threshold
concentrations of organic solvents at which proteins can
still retain their native properties. The experimental
observations of most studied organic solvents are in good
agreement with the theory except for some bad solvents
like formamide which do not comply with the denaturation
model and DC scale (Khmelnitsky et al. 1991).
Here, acetonitrile (ACN) is used as a model because it is a
polar organic solvent miscible with water and is a widely
utilized solvent compatible with ESI. Thus, mixtures of
ACNwater are of moderate polarity and low viscosity. ACN
also possesses a relatively high denaturation capacity (64.3
for a-chymotrypsin) (Khmelnitsky et al. 1991). Moreover, it
has been found that addition of ACN results in an increase in
free trypsin activity (Russell et al. 2001; Strader et al. 2006;
Batra and Gupta 1994). In one report, the maximum activity
of trypsin was reached in 10 % (v/v) of ACN, and the activity
was gradually lost as the percentage of ACN increased up to
60 % (Batra and Gupta 1994).

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Immobilization is known to improve the efficiency and

stability of enzymes due to greater retention of enzyme
activity compared to the free enzyme. Of particular
importance here is trypsins stability and performance in
ACN. The activities of the immobilized trypsin at different
concentrations of ACN (v/v) in 10 mM NH4HCO3
(pH = 8.10) are investigated here. Enzymatic hydrolyses
with different concentrations of BAEE substrate (2.5, 5,
7.5, 15, and 20 lM) in 0, 20, 40, 60, 80, 100 % acetonitrile
(three replicates for each) were conducted. The enzyme
activity was evaluated by the ratio of ion intensity of the
product B-Arg to the internal standard Z-Arg (5 lM). After
hydrolysis, at a flow rate 30 lL/h with 15 cm immobilized
trypsin fused silica (all their exposure times are 1.3 min),
Fig. 4 shows the ratio of B-Arg/Z-Arg for the conversion
of BAEE to B-Arg in the immobilized trypsin at different
percentages of acetonitrile. Kinetic parameters of Vmax and
Km were determined from LineweaverBurk plots in Fig. 5
of the reciprocal initial reaction rates, 1/v, versus the
reciprocal of the substrate concentration, 1/[S], for different
percentages of ACN. These are summarized in Table 1.
The resultant Vmax values show a sudden jump from 0 to
20 % ACN, then a small but gradual increase from 20 to
80 % ACN, and finally a significant decrease upon reaching 100 % ACN. In comparison, the values of Km only
Fig. 5 LineweaverBurk plots
of the reciprocal initial reaction
rates, 1/v, versus the reciprocal
of the substrate concentration,
1/[S], for different ratios of
acetonitrile/10 mM NH4HCO3
(pH = 8.10)

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Microfluid Nanofluid (2013) 15:5764

exhibit slight changes in the range 2080 % ACN, and

shows decreases at 0 and 100 % ACN. These results prove
that there is an enhancement of immobilized trypsin
activity upon addition of an increasing amount of ACN to
an aqueous solvent, although in pure ACN activity
decreases markedly. We hypothesize that the kinetic
activity of immobilized trypsin may remain stable in these
acetonitrile/aqueous co-solvents because immobilization
may enable the retention of trypsins native structure, and
the support may provide a similar stabilization effect to
minimize the denaturation by ACN, although it loses
substantial enzymatic activity in 100 % ACN. It is an
interesting finding that the kinetic activity of immobilized
trypsin is not particularly sensitive to the content of ACN
in aqueous solution, which may be explained that a small
amount of water in this co-solvent system can maintain the
biocatalytic activity of trypsin and decrease the sensitivity
of immobilized trypsin to the content of organic solvent
(Goradia et al. 2006). These results which show that
enzymatic activity increases upon addition of ACN are
consistent with earlier results for free trypsin (Russell et al.
2001; Strader et al. 2006; Batra and Gupta 1994), which
may be attributed to the increased solubility of the substrate
upon addition of ACN (Griebenow and Klibanov 1996)
without significant unfolding of trypsin (Simon et al. 1998).

Microfluid Nanofluid (2013) 15:5764

63

Table 1 Kinetic parameters for different concentrations of acetonitrile in 10 mM NH4HCO3 buffer (pH = 8.10)
Percentage of
acetonitrile (v/v) (%)

Km (mM)

Vmax (lM/s)

0.037

0.12

20

0.092

0.79

40
60

0.098
0.10

0.83
0.86

80

0.14

1.2

100

0.043

0.13

The subsequent reduction of trypsin activity in 100 % ACN

is consistent with the hypothesis that at very high acetonitrile concentrations some proteins may begin to precipitate (Santos et al. 2008), which could be the case for
BAEE, although we did not note such precipitation at a
macroscopic level.
3.3 Broader implications of the technology
Rapid and complete trypsin digestion is a major goal
in proteomics platforms because it enables increased
throughput. Further, accurate quantitation of peptides that
may serve as biomarkers is a major application of digestion
platforms. As such, it is important to consider the dual
IMTR nanoelectrospray emitter in this context.
Our dual IMTR nanoelectrospray emitter enables direct
mass spectrometric detection of trypsin hydrolysates with
greater than 90 % digestion efficiency in little over a
minute (Zhao et al. 2006). This is several minutes earlier
than the microwave-induced method (Sun et al. 2006), and
our platform enables direct on-line detection of the trypsin
hydrolysates by a mass spectrometer. Another method for
rapid trypsin digestion has been developed that incorporates a high-pressure sample loop within a liquid chromatography system, called the fast online digestion system
(FOLDS) (Lopez-Ferrer et al. 2008). Both our dual IMTR
nanoelectrospray emitter and FOLDS compare similarly
with respect to both digestion time and efficiency. The
advantages of the dual IMTR nanoelectrospray emitter
technology are that it does not require mixing a solution of
trypsin directly with the sample to be analyzed, and replicate injections can be acquired under identical exposure
conditions. Unlike FOLDS, however, the IMTR nanoelectrospray emitter has not been coupled to an autosampler, which is a subject worthy of future research. A more
recently employed method to obtain rapid trypsin digestion
is to utilize high intensity focused ultrasound (HIFU) to
proteins in gel slabs, which also enables very efficient
digestions in only a minute (Lopez-Ferrer et al. 2005).
Detection of tryptic peptides produced by HIFU is done

off-line followed by injection into a liquid chromatography-mass spectrometer, so the total analysis time is slower
than that described here.
It is also important to recognize that ultimately enzymatic digestion procedures need to be able not only to map
and identify proteins, but also to quantify them in the
process of biomarker determination. It has been pointed out
that it is possible to do reproducible trypsin digestion under
a controlled platform of reagents across laboratories using
the approach of multiple reaction monitoring coupled to
stable isotope dilution mass spectrometry (Addonna et al.
2007). Thus, if the dual IMTR nanoelectrospray emitter
device discussed here can be shown to have high reproducibility, it may have important potential for quantitative
proteomics as well.

4 Conclusions
In this work, the kinetic characterization of immobilized
trypsin on the surface of fused silica has been examined
with the dual IMTR nanoelectrospray emitter developed in
our lab (Zhao et al. 2006). It is found that immobilization
changes trypsins intrinsic kinetics compared to the free
trypsin using an assay where BAEE served as a substrate
for immobilized trypsin. Immobilized trypsin in an acetonitrile/aqueous co-solvent shows good stability and high
biocatalytic activity of immobilized trypsin from 20 to
80 % acetonitrile. These results demonstrate that rapid
digestion and immediate nanoelectrospray mass spectrometry detection using the dual-purpose immobilized
trypsin microreactor/nanoelectrospray emitters is efficient,
even in the presence of relatively high proportions of
acetonitrile. This apparatus should thus be beneficial to
peptide mapping applications in proteomics.
Acknowledgments The authors would like to thank The Mark
Diamond Research Fund and the Department of Chemistry at the
University at Buffalo for partial financial support of this work.

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