Neutron activation analysis and X-ray Rayleigh and Raman scattering of hair and nail clippings as noninvasive bioindicators for Cu liver status in Labrador Retrievers The heritability of chronic hepatitis in the Labrador Retriever is studied with the aim of identifying the related gene mutation . Identification of cases and controls is largely based on instrumental neutron activation analysis -LRB- INAA -RRB- Cu determination in liver biopsies . The burden for these companion animals may be reduced if nail clippings and hair -LRB- fur -RRB- could serve as a noninvasive indicator for the hepatic Cu concentrations . No correlation was found between hepatic Cu concentrations and Cu concentrations in hair and nail samples . However , hair and nail samples were also analyzed by X-ray tube excitation , taking advantage of the X-ray Compton , Rayleigh , and Raman scattering which reflects the organic components such as the type of melanin . Principal component analysis provided first indications that some differentiation between healthy and sick dogs could indeed be obtained from hair and nail analysis . Introduction Chronic hepatitis in dogs is a histological diagnosis , characterized by the presence of fibrosis , inflammation , and necrosis of the liver cells . Symptoms of chronic hepatitis are quite variable but the most common signs are lethargy , loss of appetite , and diarrhea . Pets may also drink and urinate more . As the disease progresses , many dogs develop yellowish gums , eyes , and skin -LRB- icterus/jaundice -RRB- , and a swollen abdomen which is filled with fluid -LRB- ascites -RRB- . In some cases toxins affect the nervous system and the dogs become blind and obtuse . This can progress to seizures , coma , and death . The disease is characterized , amongst others , by copper accumulation in the liver . Previous studies have shown elevated hepatic Cu concentrations in the order of 1,000 -- 2,000 mg kg − 1 compared with normal Cu levels of < 200 -- 500 mg kg − 1 . Such hepatic copper accumulation can result from increased uptake of copper , a primary metabolic defect in hepatic copper metabolism , or from altered biliary excretion of copper . Several studies have already been conducted at Utrecht University on inherited copper toxicosis and hepatic copper storage in companion dogs such as Bedlington Terriers -LSB- 1 -RSB- and Dobermanns -LSB- 2 -RSB- . Currently , one of the programs is investigating the genetic basis of the disease by examination of family members from Labrador Retrievers with copper-associated chronic hepatitis . The selected dogs undergo blood sampling , and retrieval of a liver biopsy -LRB- by use of the Menghini technique -RRB- . The liver biopsy is examined histologically and stained for copper granules . As this project deals with companion animals from the Labrador population in The Netherlands and not with test animals , cooperation of the dog owners is crucial . Following the Delft group 's involvement and over 20 years ' experience investigating nail clippings as a bioindicator for the trace element status in man -LSB- 3 , 4 -RSB- , a feasibility study was undertaken to assess the usefulness of nail clippings and hair -LRB- fur -RRB- as a noninvasive indicator for the Cu liver levels with the aim of replacing the transcuteaneous full-needle biopsy . Two approaches were tested . The most straightforward was to verify if Cu levels in nail clippings and hair would reflect the Cu liver levels . The other approach was initiated by the observation that a copper deficiency may result in hair/coat color changes . This is ascribed to the activity of cuproenzymes , which catalyze the biosynthesis of melanin from L-tyrosine . A copper deficiency causes , among others , pigmented hair on the head and face to lose its normal color and get a `` washed out '' appearance and become gray -LSB- 5 -RSB- . In 2005 , Bueno et al. demonstrated that X-ray Rayleigh peaks and especially the peakless part in the X-ray Raman scattering continuum in X-ray spectra obtained by tube excitation carry information on the organic constituents of biological materials -LSB- 6 , 7 -RSB- . A study of Poodles in which all spectral information was processed by dedicated principal component analysis -LRB- PCA -RRB- resulted in a clear differentiation of all dogs examined on the basis of their hair color and , moreover , the separation of healthy and sick dogs on the basis of the hair analysis . Hair pigmentation is due to two major types of melanin , viz. , eumelanin and phaeomelanin -LRB- Fig. 1 -RRB- . The type of melanin manifesting in the hair can be attributed to the hair color genotype -LSB- 8 , 9 -RSB- , whereas the intensity and distribution of the granules may also be affected by diseases in which sulfur metabolism is involved . Fig. 1Chemical structures of eumelanin and phaeomelanin Both approaches were tested here , i.e. , -LRB- i -RRB- Cu determination via instrumental neutron activation analysis -LRB- INAA -RRB- in liver biopsies , nail clippings , and hair samples ; and -LRB- ii -RRB- X-ray spectrum acquisition after X-ray tube excitation , followed by full spectrum data -LRB- i.e. all channel contents -RRB- processing by PCA , including the often neglected peakless part of the X-ray scattering region resulting from Compton , Rayleigh and Raman effects . Experimental Liver biopsy samples , nail clippings , and hair were collected from approximately 100 dogs with chronic hepatitis as well as from healthy animals . Nail clippings were taken from phalanx I -LRB- the thumb nail -RRB- , since this nail is not in contact with the ground , thus minimizing contamination problems . Nail clippings could not be collected from all animals as in some cases breeders had removed the nail permanently shortly after birth , since sometimes such a thumb nail hinders the dog in working trials . Hair was sampled from the median abdominal wall , just cranial to the belly button . The liver biopsy samples were freeze-dried at the veterinary clinic . The nail clippings and hair samples were analyzed without cleaning to prevent changes in the nail and hair morphology that might affect the scattering information from the X-ray fluorescence -LRB- XRF -RRB- analysis . It should be noted that a Cu contamination of the nail clipping and hair is highly unlikely ; the nail of the thumb nail is never in contact with , e.g. , soil , and external contamination of the hair was also unlikely . INAA The needle biopsies were transferred into polyethylene capsules and lyophilized , resulting in sample masses varying typically between 1.5 and 50 mg dry weight . Nail clippings had masses of 5 -- 50 mg , and the typical mass of the hair samples was approximately 100 -- 200 mg . The Cu levels were established by INAA via the determination of 66Cu . This is the radionuclide of choice although 64Cu is also produced ; however , the associated 511-keV peak is insufficient for reliable Cu determination , whereas the 1,345-keV peak provides insufficient sensitivity . Quantification of all element mass fractions was based on the single comparator method -LSB- 10 -RSB- . Samples were irradiated one-by-one for 3 min in the Hoger Onderwijs Reactor in a thermal neutron fluence rate of 1.7 × 1017 m − 2 s − 1 . A Zn flux monitor was included in each irradiation ; the neutron fluence rate was determined by measurement of the induced 69mZn activity . The activated biopsy , nail , and hair samples were measured 30 s after irradiation for 3 min at a 1-cm distance from the end-cap of a side-looking 12 % Ge detector . The samples were rotated during counting . The Zn monitors were measured in the same geometry for 60 s after a 4-min decay time . The samples were processed in batches of 14 samples , one internal quality control sample , and a blank . A sample of NBS 1577b Bovine Liver -LRB- certified Cu content 160 ± 8 mg kg − 1 -RRB- was used for internal quality control . Details of the irradiation facility , gamma-ray spectrometer , and associated quality assurance have been published elsewhere -LSB- 11 -RSB- . The spectrometer is equipped with ORTEC DSPEC-PLUS electronics , which allows for adequate dead-time correction -LSB- 12 -RSB- . It is obvious that , particularly in the nail and hair samples , several other elements such as Ca , Mg , and S were also quantified on the basis of their short halflife radionuclides , measured simultaneously with 66Cu . X-ray tube excitation The measuring procedure consisted of weighing approximately 100 mg of each hair sample and 10 mg of each nail sample directly into appropriate cells and submitting them to blank rhodium X-ray tube radiation , in triplicate . A common laboratory energy dispersive XRF instrument -LRB- EDX 700 , Shimadzu -RRB- was used under the same irradiation conditions : 50-kV applied voltage in the tube , 25 % dead time , 10-mm beam collimation , and 100-s irradiation time . It should be noted that the methodology of using the region in the X-ray spectrum resulting from scattering implies that only the spectrum 's shape is needed for the data processing by PCA . There is no need for quantification towards Cu mass fractions , and hence no reference materials were used for quality control as was the case in the hair , nail , and liver biopsy analysis by INAA . The chemometric method -LSB- 13 -RSB- used for data analysis was PCA , processed via the software The Unscrambler , version 9.2 , from Camo . Results and discussion Cu could not be determined in all available nail samples because of the small sample masses and the detection limit of the analytical protocol selected . Detection limits on the basis of 66Cu were typically in the order of 25 ng , corresponding to a minimum detectable Cu mass fraction of 5 mg kg − 1 in a 5-mg nail clipping sample . Cu levels in hair and nail are given in Fig. 2 , together with the associated Cu liver levels . Uncertainties are not depicted for clarity reasons ; typical uncertainties -LRB- combined standard uncertainty -RRB- were approximately 5 -- 20 % for the liver analyses , 15 -- 30 % for the nail , and 5 -- 10 % for the hair analyses . It is clear from Fig. 2 that the Cu levels in hair and nail do not reflect the Cu liver levels . This indicates that the gene -LRB- s -RRB- responsible for the Cu accumulation in the liver is -LRB- are -RRB- only active in the liver cells and not in the cells at the nail base or in the hair pocket . Fig. 2Cu levels in nail clippings and hair of Labrador Retrievers as a function of the Cu levels in liver biopsies taken from the same dogs : ○ hair , ● nail ; uncertainties are omitted for clarity -LRB- see text -RRB- Principal component analysis -LRB- PCA -RRB- results of the processing of the channel contents of the X-ray spectra of all hair samples are given in Fig. 3 , and the related factor loadings of PC1 and PC2 are shown in Fig. 4 -LRB- similar results were found for the processing of the channel contents of the X-ray spectra of the nail samples , although these are not shown here -RRB- . The PC1 loading graph indicates the dependent variables that explain most of the data variance , and the PC2 loading graph indicates variables that explain the second data variance . PC1 and PC2 are orthogonals and therefore independent of each other . Fig. 3PCA analysis of all hair samples , classified here by the hair color : □ yellow , * chocolate , ■ blackFig . 4PC1 loadings for hair samples -LRB- top -RRB- . PC2 loadings for hair samples -LRB- bottom -RRB- The variables included in the PCA analysis were from the channel contents between 0 and 40 keV , i.e. , covering the entire spectum recorded.The symbols in Fig. 3 reflect the hair color . The differentiation with respect to coat color results most likely from differences in type of melanin in Labradors with a black , liver -LRB- chocolate -RRB- , or yellow-colored coat . Table 1 shows the genotypes of the hair color inheritance of Labrador Retrievers -LSB- 8 -RSB- , in which each character represents a specific gene -LRB- denoted in pairs , of course -RRB- . An uppercase character represents a dominant gene , a lowercase character represents a recessive gene ; no character in a gene pair implies that it is not relevant for the genotype if the locus is occupied by a dominant or recessive gene . As produces black without any tan on the dog and there are other genes that can modify the black to liver -LRB- chocolate Labrador -RRB- . If As is present , in most cases the dog will be able to produce only eumelanin pigment . Some shades of liver -LRB- chocolate -RRB- , though a eumelanin pigment , overlap some shades of tan , a phaeomelanin pigment . The e , recessive red , overrides whatever gene is present at the A locus to produce a dog which shows only phaeomelanin pigment in the coat . The related melanin types are also included in Table 1 and the color differentiation by the principal component analysis is most probably based on the differences in melanin corresponding to the different genotypes . The wide spread of the groups might reflect homozygotes and heterozygotes for the genes involved . Table 1Labrador genotypes -LSB- 8 -RSB- for hair color inheritance and corresponding types of melaninColorGenotypeMelanin typeBlackAs -- B -- E -- EumelaninLiver -LRB- chocolate -RRB- As -- bb E -- EumelaninYellow , liver nose -- -- B -- eePhaeomelaninYellow , black nose -- -- bb eePhaeomelanin The PCA also revealed a differentiation to gender , as is further exemplified in Fig. 5 for nails . This is in agreement with earlier work for Poodles in which a similar gender separation was observed -LSB- 6 -RSB- . Fig. 5PCA analyses of all nail samples , classified by gender These results -LRB- Figs. 3 , 4 , 5 -RRB- indicate that PC1 allows for a separation of dogs by gender and PC2 for separation by color , particularly when using hair samples . The loading graphs of the PCs -LRB- Fig. 4 -RRB- show that sulfur and Rh scatter -LRB- Compton , Rayleigh , and Raman -RRB- explain the variance in PC1 . This is most likely attributed to variations in -LRB- sulfur bondings in -RRB- keratin structures . This effect contributes to almost 100 % of the data variance . The rest of it , PC2 , is not very large -LRB- approximately 1 -- 10 % -RRB- and allows for separations due to differences in sulfur , and to some extent with melanin . The physiological cause of the variation of the keratin structures in hair and nail by gender is not yet fully clear , but there are several indications that the structure of hair is different for males and females . It is well known that there is a relationship between testosterone and hair formation -LRB- andropogenic hair -RRB- . Andropogenic hair has a different growth rate and a higher weight than other hair . It has also been observed that the type of sulfur is different for females and males : rhombic -LRB- alpha -RRB- sulfur predominates in female hairs , monoclinical -LRB- beta -RRB- sulfur in male hairs -LSB- 14 -RSB- . Less information is available about nails . Genetic variation in the proteins of the human nail has been observed -LSB- 15 -RSB- , as well as a sex variation in lipid composition of human fingernail plates -LSB- 16 -RSB- . Another possible explanation for the separation of dogs in terms of genders in the hair analysis might be found in the results of Scott et al. -LSB- 17 -RSB- who concluded that the human melanocortin 1 receptor -LRB- MC1R -RRB- is regulated by , amongst others , specific endocrine sex hormones and by UV radiation . In confirmation of the latter study , Broekmans et al. -LSB- 18 -RSB- found that male subjects were more sensitive to UV irradiation than female subjects . It was already known that eumelanin is photoprotective , whereas phaeomelanin may contribute to UV-induced skin damage due to its potential to generate free radicals in response to UV radiation -LSB- 19 -RSB- . Skin melanin content , which was positively associated with hair color in men , was the main phenotypical determinant of sensitivity to UV irradiation . The PCA results could also be plotted as a function of the INAA Cu liver biopsy values . Results for hair and nail are given in Fig. 6 for a differentiation towards dogs with Cu liver levels of < 600 mg kg − 1 or > 600 mg kg − 1 . A Cu biopsy level of > 600 mg kg − 1 is considered to be positively indicative for liver Cu accumulation . In addition , plots were also made in which the hair and nail samples are identified by correlating with Cu biopsy values > and < 200 mg kg − 1 as well as at > and < 400 mg kg − 1 . At these lower Cu levels no clear differentiation is obtained , as could be expected , which confirms that the PCA is not producing an artifact , accidentally coinciding at the 600 mg kg − 1 Cu liver biopsy level . Fig. 6PCA results , nail X-ray spectral data vs. INAA Cu liver biopsy values -LRB- top -RRB- : ■ dogs with Cu liver levels 0 -- 600 mg kg − 1 , □ dogs with Cu liver levels > 600 mg kg − 1 , N = 60 . PCA results , hair X-ray spectral data vs. INAA Cu liver biopsy values -LRB- bottom -RRB- : ■ dogs with Cu liver levels 0 -- 600 mg kg − 1 , □ dogs with Cu liver levels > 600 mg kg − 1 , N = 43 In all PCA , it could be justified to remove some outliers on the basis of high leverage values and high energy variance residuals in PCs . High values of leverage and residuals are caused , for instance , by imperfect quality and positioning of the sample during the X-ray irradiation . In addition , outliers may sometimes result from the presence of dogs with strong parental relationships -LSB- 6 -RSB- . The strength of using the information of the scattered radiation is emphasized by the role of sulfur in the separation towards gender and Cu accumulation . Total sulfur , as determined by INAA via 37S , hardly shows any correlation with INAA Cu liver values -LRB- correlation coefficients 0.03 and 0.32 for nail and hair , respectively -RRB- . However , the INAA results provide only the total sulfur content , i.e. , sulfur present in keratin , melanin , and any other compound , whereas the PCs of the channel contents of the X-ray spectra , especially due to the peakless scattered region , apply to sulfur directly related to keratin and/or melanin . This explains the much better correlation of `` keratin/melanin '' sulfur with Cu liver values compared with total S with Cu . The results of our study show that a fairly sharp differentiation can be obtained between dogs with low and high Cu liver levels if PCA is performed on a dataset obtained from all channel contents of X-ray spectra obtained by X-ray tube excitation of nail or hair samples together with the spectral information of often neglected X-ray -LRB- Rayleigh , Raman -RRB- scattered radiation . A practical application of this technique would start with construction of a dataset using samples of known origin , encompassing all varieties expected . New and unknown samples can then be projected on this model , and subsequently classified . This approach will be further validated with new samples to be collected from Labrador Retrievers still being observed and treated along medicinal and dietary approaches . Conclusions Hair and nail clippings are widely used as bioindicators for the trace element status of man and , to a much lesser extent , also for animals . The results of this study emphasize that it is quite risky to assume a priori that the trace element levels in hair and nail reflect differences between classes of individuals , e.g. , `` healthy '' and `` sick '' people or animals . In this study related to chronic hepatitis , the Cu levels in liver biopsies are not reflected by Cu levels in hair and nail . The X-ray Rayleigh scattered and X-ray Raman continuum contain valuable information about the organic , low-Z elemental matrix composition of an object . In this study , first indications were obtained that the Cu accumulation possibly may have an effect on the nail and hair melanin , thus offering a potentially favorable outlook for noninvasive monitoring of the Cu liver status . An additional advantage of the approach is that a measurement by X-ray tube excitation takes only about 2 min per sample so that , once a database has been built up , the technique might be valuable for large-scale screening purposes .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

x-ray spectroscopy
chemometrics
trace elements
biological samples


Development

of

an

open-tubular

trypsin

reactor

for

on-line

digestion

of

proteins

A

study

was

initiated

to

construct

a

micro-reactor

for

protein

digestion

based

on

trypsin-coated

fused-silica

capillaries

Initially

surface

plasmon

resonance

was

used

both

for

optimization

of

the

surface

chemistry

applied

in

the

preparation

and

for

monitoring

the

amount

of

enzyme

that

was

immobilized

The

highest

amount

of

trypsin

was

immobilized

on

dextran-coated

SPR

surfaces

which

allowed

the

covalent

coupling

of

ng

mm

trypsin

Fused-silica

capillaries

were

modified

in

a

similar

manner

and

the

resulting

open-tubular

trypsin-reactors

having

a

pH

optimum

of

pH

display

a

high

activity

when

operated

at

°C

and

are

stable

for

at

least

two

weeks

when

used

continuously

Trypsin

auto-digestion

fragments

sample

carry-over

and

loss

of

signal

due

to

adsorption

of

the

protein

were

not

observed

On-line

digestion

without

prior

protein

denaturation

followed

by

micro-LC

separation

and

photodiode

array

detection

was

tested

with

horse-heart

cytochrome

C

and

horse

skeletal-muscle

myoglobin

The

complete

digestion

of

pmol

μL

horse

cytochrome

C

was

observed

when

the

average

residence

time

of

the

protein

sample

in

a

cm

μm

capillary

immobilized

enzyme

reactor

-LRB-

IMER

-RRB-

was

s.

Mass

spectrometric

identification

of

the

injected

protein

on

the

basis

of

the

tryptic

peptides

proved

possible

Protein

digestion

was

favorable

with

respect

to

reaction

time

and

fragments

formed

when

compared

with

other

on-line

and

off-line

procedures

These

results

and

the

easy

preparation

of

this

micro-reactor

provide

possibilities

for

miniaturized

enzyme-reactors

for

on-line

peptide

mapping

and

inhibitor

screening

Introduction

A

demand

for

smaller

enzyme

reactors

has

emerged

in

recent

years

as

a

consequence

of

ongoing

miniaturization

in

the

biochemical

and

analytical

sciences

These

micro-reactors

have

been

used

in

biocatalysis

and

biosensing

In

the

field

of

proteomics

the

reactors

are

a

tool

in

peptide

mapping

in

which

proteins

are

identified

via

peptide

fragment

identification

after

proteolysis

Currently

in

spite

of

its

limitations

most

of

these

analyses

are

conducted

by

means

of

gel

electrophoresis

followed

by

digestion

of

the

proteins

liquid

chromatographic

-LRB-

LC

-RRB-

separation

and

mass

spectrometric

-LRB-

MS

-RRB-

identification

of

the

peptides

-LSB-

-RSB-

The

most

time-consuming

step

in

this

procedure

is

digestion

of

the

protein

using

a

protease

In

general

every

protein

to

be

investigated

is

individually

incubated

with

the

protease

at

a

concentration

of

approximately

protein

weight

for

to

h

at

an

elevated

temperature

-LRB-

typically

°C

-RRB-

In

addition

to

the

long

incubation

time

needed

a

certain

level

of

auto-digestion

of

the

protease

can

be

expected

To

reduce

sample

handling

digestion

time

and

the

risk

of

sample

contamination

methods

for

the

on-line

digestion

of

proteins

have

been

developed

that

use

proteases

immobilized

on

a

solid

support

Immobilized

enzyme

reactors

have

been

developed

and

used

over

the

years

for

several

industrial

and

analytical

purposes

-LSB-

-RSB-

An

obvious

benefit

for

immobilizing

biocatalysts

is

the

fact

that

the

enzyme

can

be

used

in

several

catalytic

cycles

and

that

both

catalyst

and

reaction

mixture

can

easily

be

separated

Moreover

immobilized

enzymes

generally

show

an

improved

stability

even

at

more

extreme

reaction

conditions

Several

procedures

have

been

developed

for

immobilization

of

enzymes

e.g.

adsorption

or

encapsulation

in

a

matrix

or

membrane

Alternatively

and

more

often

used

is

the

covalent

attachment

of

biocatalysts

to

carrier

materials

which

allows

the

immobilization

of

a

large

amount

of

enzyme

for

a

high

activity

per

surface

area

Generally

particulate

large-pore

carrier

materials

are

used

such

as

controlled-pore

glass

-LSB-

-RSB-

silica

-LSB-

-RSB-

or

polymers

like

the

commercially

available

poroszyme

-LSB-

-RSB-

Current

research

in

the

production

of

immobilized

enzymes

is

focused

on

the

use

of

monolithic

materials

as

they

enable

efficient

fragmentation

of

proteins

-LSB-

-RSB-

Although

both

commercially

available

and

self-prepared

reversed-phase

capillary

monolithic

columns

have

successfully

passed

reproducibility

assessment

-LSB-

-RSB-

synthesis

of

monoliths

suitable

for

small-scale

enzyme

reactors

can

still

be

troublesome

Materials

suitable

for

the

fabrication

of

larger-scale

enzyme

reactors

are

commercially

available

from

BIA

Separations

-LRB-

Ljubljana

Slovenia

-RRB-

Although

it

is

possible

to

apply

an

immobilized

enzyme

reactor

-LRB-

IMER

-RRB-

positioned

after

the

separation

column

-LSB-

-RSB-

most

papers

dealing

with

on-line

digestion

of

protein

samples

position

the

IMER

upstream

of

the

separation

column

In

these

cases

the

sample

is

first

digested

and

the

resulting

peptide

fragments

are

separated

and

identified

by

LC

MS.

This

approach

is

often

employed

in

multi-dimensional

LC

methods

-LSB-

-RSB-

and

has

also

found

application

in

peptide

mapping

using

capillary

electrophoresis

-LSB-

-RSB-

Alternatively

as

recently

shown

by

Zhao

et

al.

-LSB-

-RSB-

and

Krenkova

et

al.

-LSB-

-RSB-

who

covalently

coupled

trypsin

to

the

wall

of

fused-silica

nanoelectrospray

emitters

a

protein

sample

can

be

analyzed

by

direct

infusion

into

a

mass

spectrometer

This

paper

describes

the

development

of

trypsin-modified

open-tubular

micro-reactors

The

chemistry

was

controlled

and

optimized

using

surface

plasmon

resonance

-LRB-

SPR

-RRB-

a

technique

allowing

sensitive

and

real-time

monitoring

of

surface

reactions

such

as

protein

binding

-LSB-

-RSB-

The

surface

modification

resulting

in

the

highest

enzyme

immobilization

yield

was

used

to

covalently

immobilize

the

trypsin

on

the

inside

wall

of

a

fused-silica

capillary

The

constructed

trypsin

micro-reactor

which

is

compatible

with

micro

and

nano-LC

was

further

characterized

The

influence

of

reaction

time

pH

temperature

and

reactor

stability

were

investigated

with

the

model

substrate

insulin

B-chain

The

reactor

was

also

applied

to

digestion

of

the

proteins

cytochrome

C

and

myoglobin

The

produced

peptides

were

analyzed

by

liquid

chromatography

mass

spectrometry

Experimental

Materials

The

SPR

equipment

used

was

from

IBIS

Technologies

-LRB-

currently

available

from

Eco

Chemie

Utrecht

The

Netherlands

-RRB-

equipped

with

a

polycarbonate

cuvet

The

gold-sensor

disks

purchased

from

SSENS

-LRB-

Hengelo

The

Netherlands

-RRB-

were

positioned

on

the

IBIS-prism

using

index-matching

oil

from

R.P.

Cargille

Laboratories

-LRB-

Cedar

Grove

USA

-RRB-

PEEK

nuts

unions

tubing

and

loops

were

from

Upchurch

-LRB-

Santa

Monica

USA

-RRB-

Manual

injections

during

the

preparation

of

the

reactors

were

performed

using

a

Rheodyne

injector

-LRB-

Inacom

Veenendaal

The

Netherlands

-RRB-

equipped

with

a

PEEK

loop

Model

HPLC

pumps

from

Shimadzu

-LRB-

Kyoto

Japan

-RRB-

were

used

for

reactor

preparation

and

activity

determinations

The

water

used

for

washing

and

to

prepare

buffers

was

produced

by

a

Sartorius

Arium

ultrapure

water

system

-LRB-

Nieuwegein

The

Netherlands

conductivity

MΩ

cm

-RRB-

The

model

UV

absorbance

detector

was

equipped

with

a

capillary

flow-cell

-LRB-

i.d.

-RRB-

and

was

obtained

from

Applied

Biosystems

-LRB-

Nieuwerkerk

a/d

IJssel

The

Netherlands

-RRB-

On-line

digestion

experiments

with

micro-HPLC

separations

were

conducted

using

LC-Packings

instruments

and

columns

-LRB-

Amsterdam

The

Netherlands

-RRB-

The

equipment

consisted

of

an

injector

-LRB-

Famos

-RRB-

nanovalve

column

switcher

-LRB-

Switchos

-RRB-

nanopump

-LRB-

Ultimate

-RRB-

and

a

photodiode-array

detector

-LRB-

PDA

-RRB-

equipped

with

a

micro

flow

cell

-LRB-

nL

-RRB-

The

reversed-phase

pre-columns

were

mm

with

Å

particles

The

mm

reversed-phase

micro-column

contained

Å

PepMap

particles

The

mass

spectrometer

was

an

Agilent

LC/MSD

XCT

ion

trap

-LRB-

Amstelveen

Netherlands

-RRB-

Acetic

acid

boric

acid

calcium

chloride

-LRB-

-RRB-

ethanol

ethanolamine

-LRB-

EA

-RRB-

hydrochloric

acid

-LRB-

HCl

-RRB-

hydrogen

peroxide

sodium

dihydrogen

phosphate

sodium

chloride

sodium

hydroxide

-LRB-

NaOH

-RRB-

and

sulfuric

acid

were

purchased

from

Merck

-LRB-

Darmstadt

Germany

-RRB-

HPLC

grade

acetonitrile

and

ethanol

were

from

Biosolve

-LRB-

Valkenswaard

The

Netherlands

-RRB-

-LRB-

-RRB-

acid

-LRB-

HEPES

-RRB-

tris-hydroxymethylaminoethane

-LRB-

TRIS

-RRB-

urea

and

acetone

were

purchased

from

Acros

Organics

-LRB-

Geel

Belgium

-RRB-

Benzamidin

N

-LRB-

-RRB-

N-ethylcarbodiimide

hydrochloride

-LRB-

EDC

-RRB-

benzoyl-arginine

ethyl

ester

-LRB-

BAEE

-RRB-

iodoacetamide

N-hydroxysuccinimide

-LRB-

NHS

-RRB-

and

carboxyl-modified

dextran

-LRB-

CMD

-RRB-

were

purchased

from

Fluka

-LRB-

Buchs

Switzerland

-RRB-

Horse-heart

cytochrome

C

insulin

B

-LRB-

oxidized

-RRB-

polyoxyethylenesorbitan

monolaurate

-LRB-

Tween

-RRB-

and

porcine

pancreas

trypsin

were

purchased

from

Sigma

-LRB-

St

Louis

USA

-RRB-

The

specific

activity

of

trypsin

was

determined

according

to

the

method

of

Schwert

and

Takenaka

-LSB-

-RSB-

using

BAEE

as

substrate

and

turned

out

to

be

U

mg

Aminopropyltriethoxysilane

-LRB-

APTES

-RRB-

carbonyl

diimidazole

-LRB-

CDI

-RRB-

glycidoxypropyltrimethoxysilane

-LRB-

GOPS

-RRB-

and

mercaptoethanol

-LRB-

ME

-RRB-

were

from

Aldrich

Chemical

Company

-LRB-

Milwaukee

USA

-RRB-

Amino-modified

dextran

-LRB-

AMD

-RRB-

was

from

Unavera

ChemLab

-LRB-

Mittenwald

Germany

-RRB-

Fused-silica

capillaries

were

purchased

from

Bester

-LRB-

Mijdrecht

The

Netherlands

-RRB-

Piranha

solution

was

prepared

by

mixing

part

hydrogen

peroxide

and

parts

concentrated

sulfuric

acid

-LRB-

caution

aggressive

solution

-RRB-

TRIS

digestion

buffer

consisted

of

mmol

L

TRIS

and

mmol

L

adjusted

to

pH

by

use

of

mol

L

HCl

solution

Methods

Preparation

of

dextran-coated

SPR

sensor

disks

-LRB-

Fig.

-RRB-

Fig.

Surface

modifications

used

to

immobilize

the

enzyme

in

fused-silica

capillaries

a

GOPS-modified

b

GOPS/AMD-modified

c

APTES/CMD/AMD-modified

surface

In

the

last

case

the

CDI

activation

and

enzyme

-LRB-

enzyme

-RRB-

coupling

are

as

in

b

The

SPR

sensor

disks

were

extensively

cleaned

in

freshly

prepared

piranha

solution

After

h

the

disks

were

thoroughly

rinsed

with

water

dried

in

a

stream

of

nitrogen

gas

and

immediately

incubated

for

h

in

a

mmol

L

solution

of

ME

in

ethanol

in

order

to

produce

a

self-assembled

monolayer

-LRB-

SAM

-RRB-

containing

hydroxyl

functionalities

After

SAM

formation

the

disks

were

washed

with

ethanol

and

water

and

dried

with

nitrogen

gas

The

resulting

ME

disks

were

incubated

for

h

with

a

GOPS

solution

in

ethanol

after

which

the

disks

were

washed

with

ethanol

dried

with

nitrogen

gas

and

stored

overnight

at

°C

Dextran-modified

sensors

were

prepared

by

incubating

GOPS-modified

sensor

disks

for

h

at

room

temperature

with

a

AMD

solution

in

a

mmol

L

borate

buffer

pH

After

removing

the

solution

the

disks

were

washed

with

water

ethanol

and

water

dried

in

a

gentle

flow

of

nitrogen

gas

and

stored

at

room

temperature

in

a

closed

box

until

use

Alternatively

ME

disks

were

incubated

for

h

with

a

APTES

solution

in

acetone

After

washing

drying

and

storing

overnight

in

the

oven

similar

to

the

GOPS

disks

the

surfaces

were

incubated

for

h

with

a

solution

containing

CMD

in

water

containing

mmol

L

EDC

and

mmol

L

NHS

After

removing

the

solution

the

disks

were

washed

with

water

and

ethanol

and

incubated

for

min

in

a

solution

containing

mmol

L

CDI

in

acetone

These

activated

surfaces

were

washed

with

ethanol

blown

to

dryness

with

nitrogen

and

incubated

overnight

with

a

solution

of

AMD

in

water

After

removing

the

solution

the

disks

were

washed

with

water

ethanol

and

water

dried

in

a

gentle

flow

of

nitrogen

gas

and

stored

at

room

temperature

in

a

closed

box

until

use

SPR

experiments

The

GOPS-modified

SPR

sensor

disks

were

incubated

overnight

with

μL

of

a

solution

containing

mg

mL

trypsin

and

μg

mL

benzamidin

in

mmol

L

borate

buffer

pH

-LRB-

Fig.

-RRB-

Both

types

of

dextran-modified

sensor

disk

were

activated

for

min

with

μL

mmol

L

CDI

in

dry

acetone

After

washing

the

surface

with

water

to

remove

the

last

traces

of

acetone

the

sensors

were

incubated

overnight

with

μL

mg

mL

trypsin

and

μg

mL

benzamidin

in

mmol

L

borate

buffer

pH

-LRB-

Fig.

and

c

respectively

-RRB-

The

remaining

esters

were

inactivated

by

incubating

the

disks

for

min

with

mol

L

EA

in

mmol

L

borate

buffer

pH

The

successive

steps

in

the

immobilization

were

monitored

with

SPR

and

the

amount

of

covalently

coupled

enzyme

was

calculated

from

the

recorded

angle

shift

During

the

experiments

the

SPR

system

was

thermostatted

at

°C

Preparation

of

dextran-coated

fused-silica

capillaries

In

order

to

generate

a

proper

surface

for

silanization

fused-silica

capillaries

were

cleaned

for

min

with

mol

L

NaOH

solution

at

a

flow-rate

of

μL

min

The

capillary

was

then

washed

for

min

with

mol

L

HCl

for

min

with

water

and

finally

for

min

with

ethanol

In

order

to

prepare

dextran-coated

capillaries

the

capillaries

were

flushed

for

min

with

a

GOPS

solution

in

ethanol

After

this

step

the

capillaries

were

closed

with

silicon

plugs

and

dried

overnight

at

°C

After

silanization

the

capillaries

were

flushed

with

methanol

at

a

flow-rate

of

μL

min

after

which

the

capillaries

were

chemically

modified

in

flow

using

injections

with

a

Rheodyne

manual

injector

equipped

with

a

PEEK

loop

in

a

way

similar

to

that

described

above

in

the

SPR

section

Washing

steps

were

conducted

at

flow

rates

of

μL

min

overnight

incubations

at

a

flow

rate

of

μL

min

All

chemistries

are

outlined

in

Fig.

Enzyme

activity

determination

Insulin

B

chain

was

used

as

a

substrate

to

determine

the

activity

of

trypsin

Injections

of

μL

of

a

concentration

of

μmol

L

in

digestion

buffer

were

introduced

into

the

reactor

that

was

kept

at

the

indicated

temperature

using

a

water

bath

To

determine

the

enzyme

activity

in

solution

trypsin

was

incubated

in

these

solutions

also

-LRB-

final

trypsin

concentration

μg

mL

-RRB-

At

time

intervals

from

to

min

samples

were

taken

and

the

activity

was

stopped

by

the

addition

of

μL

TFA

in

water

Buffers

that

were

used

to

determine

the

effect

of

pH

on

activity

were

MES

-LRB-

pH

to

-RRB-

MOPS

-LRB-

pH

to

-RRB-

TRIS

-LRB-

pH

to

-RRB-

and

CHES

-LRB-

pH

to

-RRB-

The

buffers

were

adjusted

to

pH

by

use

of

mol

L

NaOH

and

were

prepared

at

a

mmol

L

concentration

also

containing

mmol

L

To

determine

the

effect

of

temperature

the

temperature

during

incubation

was

varied

between

°C

and

°C

Both

the

on-line

and

off-line

samples

were

analyzed

using

micro-HPLC

with

PDA

detection

as

outlined

below

The

insulin

B

conversion

of

both

immobilized

trypsin

and

the

enzyme

in

solution

was

calculated

from

the

peak

areas

of

substrate

and

products

On-line

protein

digestion

in

micro-HPLC

For

the

on-line

peptide

and

protein

digestion

experiments

the

trypsin-modified

capillaries

were

positioned

between

the

LC

injector

and

a

valve

as

shown

in

Fig.

and

were

immersed

in

a

thermostatted

water

bath

set

at

°C

unless

mentioned

otherwise

Protein

samples

were

prepared

in

digestion

buffer

and

were

transported

through

the

capillary

towards

a

valve

using

a

acetonitrile

solution

containing

TFA

The

peptide

fragments

formed

during

digestion

were

concentrated

on

an

RP

trapping

column

-LRB-

TC

-RRB-

and

salts

and

other

buffer

components

present

in

the

sample

were

removed

By

switching

the

valve

the

trapping

column

is

in

series

with

the

RP

micro

column

and

an

acetonitrile

gradient

started

The

gradient

was

composed

of

two

solutions

-LRB-

A

-RRB-

acetonitrile

in

water

containing

TFA

and

-LRB-

B

-RRB-

acetonitrile

in

water

containing

TFA

In

min

the

gradient

changed

linearly

from

to

B

followed

by

min

at

B

and

min

at

B

The

eluent

was

monitored

with

the

PDA

detector

in

the

range

from

to

nm

Fig.

used

for

on-line

protein

digestion

using

a

trypsin-modified

fused-silica

capillary

For

detection

a

PDA

or

an

ion-trap

MS

was

used

ESI-MS

was

conducted

in

the

positive-ion

mode

with

the

capillary

voltage

set

at

V

The

flow

rate

and

temperature

of

the

nitrogen

drying

gas

were

L

min

and

°C

respectively

The

sequence

of

the

peptide

fragments

was

determined

by

using

the

mass

spectrometer

in

auto-MS

MS

mode

fragmenting

the

two

peptides

that

were

most

abundantly

present

when

the

signal

reached

threshold

The

MS

MS

result

was

analyzed

by

a

Mascot

database

search

-LRB-

-RRB-

Results

and

discussion

Surface

chemistry

and

enzyme

immobilization

As

shown

before

-LSB-

-RSB-

modification

of

an

SPR

sensor

surface

with

a

dextran

hydrogel

leads

to

less

non-specific

adsorption

of

proteins

compared

with

unmodified

surfaces

The

presence

of

such

a

layer

also

enhances

the

immobilization

capacity

of

biomolecules

compared

with

monolayer-based

coatings

Due

to

the

flexible

nature

of

the

dextran

chains

the

accessibility

also

is

often

improved

compared

with

molecules

immobilized

on

a

flat

surface

As

the

amount

of

protein

present

in

a

capillary

after

immobilization

can

not

be

determined

easily

SPR

sensors

were

used

as

a

model

to

investigate

the

effect

of

the

different

surface

modifications

on

the

amount

of

trypsin

that

could

be

attached

covalently

Hydroxyl

functionalities

necessary

to

enable

silanization

are

introduced

using

mercaptoethanol

-LRB-

ME

-RRB-

but

all

other

surface

modifications

are

carried

out

in

exactly

the

same

way

both

on

the

SPR

sensor

surfaces

and

in

the

fused-silica

capillaries

Trypsin

immobilization

after

silanization

with

GOPS

by

reaction

of

the

trypsin

primary

amines

and

the

glycidyl

function

of

GOPS

results

in

an

SPR

angle

shift

of

m

-LRB-

n

-RRB-

which

equals

an

amount

of

trypsin

of

ng

mm

covalently

attached

to

the

surface

which

is

close

to

monolayer

coverage

of

trypsin

When

the

GOPS-silanized

surface

is

modified

with

AMD

resulting

in

a

dextran

hydrogel

the

amount

of

trypsin

that

can

be

immobilized

after

CDI

activation

increases

to

ng

mm

-LRB-

n

-RRB-

To

further

increase

the

amount

of

trypsin

an

intermediate

dextran

layer

was

added

Therefore

fused-silica

capillaries

were

APTES

silanized

and

modified

with

CMD

and

AMD

subsequently

The

amount

of

trypsin

that

could

covalently

be

attached

to

these

layers

was

determined

with

SPR

and

proved

to

be

ng

mm

-LRB-

n

-RRB-

As

this

amount

is

significantly

more

than

obtained

with

the

GOPS/AMD

surface

all

further

experiments

have

been

conducted

with

fused-silica

capillaries

the

surface

of

which

is

modified

with

dextran

in

this

way

Assuming

that

the

investigated

surface

modification

allows

a

similar

amount

of

trypsin

to

be

immobilized

per

surface

area

in

fused-silica

capillaries

the

quantity

of

enzyme

immobilized

on

the

dextran-modified

capillaries

is

more

than

five

times

larger

per

surface

area

compared

with

other

open-tubular

trypsin

micro-reactors

described

in

the

literature

-LSB-

-RSB-

These

reactors

first

described

by

Amankwa

and

Kuhr

-LSB-

-RSB-

are

based

on

immobilized

avidin

and

at

saturation

pmol

biotinylated

trypsin

was

immobilized

in

a

long

capillary

of

i.d.

This

amount

equals

ng

trypsin

mm

capillary

surface

which

is

less

than

monolayer

coverage

Characterization

of

the

enzyme

reactor

Fused-silica

capillaries

were

modified

with

dextran

as

described

above

and

the

activity

and

characteristics

of

the

trypsin

micro-reactor

were

assessed

with

the

oligopeptide

insulin

B

chain

a

amino

acids

long

insulin

fragment

When

insulin

B

is

exposed

to

the

enzyme

present

in

the

reactor

hydrolysis

at

the

arginine

present

at

position

and

to

a

lesser

extent

the

lysine

at

position

is

observed

Using

the

aforementioned

substrate

insulin

B

the

pH

optimum

was

determined

for

the

enzyme

in

solution

and

the

immobilized

enzyme

As

can

be

observed

in

Fig.

the

optimum

pH

value

for

both

free

and

immobilized

trypsin

is

pH

Therefore

all

further

experiments

are

conducted

at

this

pH

value

Fig.

of

-LRB-

a

-RRB-

pH

and

-LRB-

b

-RRB-

temperature

on

the

relative

activities

of

trypsin

in

solution

-LRB-

circles

-RRB-

and

trypsin

immobilized

on

an

APTES/CMD/AMD-modified

fused-silica

surface

-LRB-

triangles

-RRB-

In

b

the

maximum

insulin

B

conversion

rate

determined

under

experimental

conditions

is

pmol

min

μg

for

the

enzyme

in

solution

and

pmol

min

μg

for

a

i.d.

microreactor

operated

at

a

flow

rate

of

μL

min

Also

the

effect

of

temperature

on

the

activity

of

both

the

immobilized

trypsin

and

the

enzyme

in

solution

was

determined

in

the

range

°C

to

°C

As

can

be

observed

in

Fig.

the

activity

of

trypsin

increases

with

temperature

to

reach

a

maximum

around

°C

and

decreases

at

higher

temperatures

The

immobilized

enzyme

shows

a

higher

activity

compared

with

the

enzyme

in

solution

which

may

be

due

to

an

often

observed

higher

stability

of

immobilized

enzymes

also

under

more

extreme

conditions

From

the

data

the

activation

energy

can

be

determined

For

the

immobilized

enzyme

a

value

of

kJ

mol

can

be

calculated

and

for

the

enzyme

in

solution

a

value

of

kJ

mol

This

means

that

for

the

immobilized

enzyme

the

temperature

has

less

effect

on

the

activity

than

for

the

enzyme

in

solution

As

the

highest

activity

is

observed

at

°C

all

further

experiments

are

conducted

at

that

temperature

For

the

enzyme

in

solution

a

specific

activity

of

pmol

min

μg

is

determined

at

a

concentration

of

pmol

μL

insulin

B

and

pH

and

°C

Under

similar

conditions

in

a

microreactor

of

μL

with

a

μm

i.d.

and

operated

at

a

flow

rate

of

μL

min

an

amount

of

pmol

insulin

B

is

converted

In

such

a

reactor

a

total

of

μg

trypsin

might

be

present

taking

the

earlier

SPR

observations

into

account

which

means

that

the

immobilized

enzyme

is

both

accessible

and

still

active

after

immobilization

The

specific

activity

after

immobilization

is

higher

than

observed

in

recent

papers

for

the

protease

pepsin

immobilized

on

beaded

chitosan

-LSB-

-RSB-

and

trypsin

photoimmobilized

in

a

fused-silica

capillary

-LSB-

-RSB-

The

stability

during

operation

of

the

open-tubular

reactor

was

tested

at

°C

and

pH

Activity

tests

using

the

model

substrate

show

that

the

activity

of

a

reactor

that

is

continuously

in

operation

is

constant

for

at

least

two

weeks

The

enzyme

trypsin

dissolved

in

digestion

buffer

and

incubated

for

h

at

°C

loses

of

its

activity

and

after

three

days

no

activity

is

measured

On-line

digestion

of

proteins

The

on-line

digestion

of

horse

cytochrome

C

is

accomplished

as

described

in

the

Methods

section

Samples

are

submitted

to

on-line

digestion

in

a

long

trypsin-modified

APTES/CMD/AMD-coated

capillary

with

an

i.d.

of

μm

-LRB-

total

volume

μL

-RRB-

or

with

an

i.d.

of

μm

and

a

length

of

cm

-LRB-

μL

-RRB-

or

cm

-LRB-

μL

-RRB-

When

the

protein

is

not

reduced

the

heme-moiety

will

remain

covalently

attached

to

the

peptides

containing

the

protein

sulfhydryls

By

monitoring

the

heme-containing

peptides

at

nm

the

progress

of

the

digestion

and

the

amount

of

undigested

protein

can

be

determined

The

results

are

summarized

in

Table

Table

of

the

on-line

digestion

experiments

for

cytochrome

C

The

experiments

were

conducted

at

pH

and

°C

-LRB-

n

-RRB-

capillary

ID

-LRB-

μm

-RRB-

reactor

volume

-LRB-

μL

-RRB-

amount

injecteddigestion

time

-LRB-

s

-RRB-

undigested

protein

-LRB-

SD

-RRB-

-LRB-

μg

mL

-RRB-

-LRB-

pmol

-RRB-

-LRB-

-RRB-

-LRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

502.75248201650502.75124101650502.7512.4101650

The

experiments

were

conducted

at

pH

and

°C

-LRB-

n

-RRB-

As

expected

when

a

limited

amount

of

enzyme

activity

is

present

in

a

reactor

-LRB-

cm

μm

-RRB-

for

increasing

concentrations

cytochrome

C

a

larger

amount

of

protein

is

undigested

Nevertheless

many

tryptic

peptides

are

still

generated

As

can

be

expected

an

increase

in

exposure

time

of

the

substrate

with

the

immobilized

enzyme

will

result

in

an

improved

digestion

yield

A

longer

contact

time

is

achieved

by

increasing

the

reactor

volume

by

using

a

longer

enzyme-modified

capillary

Additionally

the

enzyme-to-substrate

ratio

is

increased

which

is

accomplished

by

changing

the

surface-to-volume

ratio

by

using

a

capillary

with

a

smaller

internal

diameter

As

can

be

observed

in

Table

decreasing

the

i.d.

of

the

capillary

leads

to

improved

digestion

for

a

reactor

of

equal

volume

due

to

a

higher

surface-to-volume

ratio

and

hence

a

higher

amount

of

enzyme

The

use

of

a

cm

μm

capillary

allows

the

complete

digestion

of

up

to

pmol

-LRB-

μg

mL

-RRB-

of

cytochrome

C

in

less

than

min

including

the

sample

concentration

and

removal

of

salts

by

the

trapping

column

Chromatograms

for

the

on-line

digestions

obtained

with

capillaries

with

an

i.d.

of

μm

are

shown

in

Fig.

Figure

shows

a

blank

run

in

a

capillary

containing

no

enzyme

and

Fig.

and

c

present

on-line

digestions

for

a

micro-reactor

of

μL

operated

at

μL

min

and

μL

min

respectively

As

discussed

above

incomplete

digestion

of

the

injected

cytochrome

C

will

lead

to

the

presence

of

multiple

peptide

fragments

containing

the

heme

group

as

is

visible

in

Fig.

The

digestion

is

complete

when

the

sample

exposure

time

is

s

-LRB-

flow

rate

μL

min

-RRB-

as

both

intermediate

products

and

the

undigested

protein

-LRB-

retention

time

min

in

Fig.

-RRB-

which

are

visible

at

a

wavelength

of

nm

as

outlined

above

are

no

longer

observed

An

injection

of

off-line-digested

cytochrome

C

showed

a

similar

chromatogram

as

is

shown

in

Fig.

Fig.

obtained

from

injection

of

pmol

horse

cytochrome

C

in

capillary

digestion

systems

monitored

at

nm

and

nm

The

experiments

were

conducted

with

an

APTES-CMD-AMD

derivatised

fused-silica

capillary

of

-LRB-

a

-RRB-

mm

not

containing

trypsin

operated

at

μL

min

-LRB-

blank

-RRB-

-LRB-

b

-RRB-

mm

trypsin-modified

operated

at

μL

min

-LRB-

average

sample

residence

time

s

-RRB-

-LRB-

c

-RRB-

as

-LRB-

b

-RRB-

but

operated

at

μL

min

-LRB-

average

sample

residence

time

s

-RRB-

For

clarity

the

beginning

of

the

chromatogram

displaying

the

nm

signal

is

offset

as

indicated

The

on-line

digestion

of

horse

cytochrome

C

is

also

monitored

with

mass

spectrometry

The

effect

of

flow

rate

and

hence

incubation

time

on

the

digestion

of

the

protein

and

the

number

of

peptides

identified

with

a

Mascot

database

search

is

determined

and

summarized

in

Table

In

this

table

the

undigested

amounts

of

protein

which

have

been

determined

using

the

PDA

detector

are

also

shown

At

a

flow

rate

of

μL

min

protein

digestion

is

complete

and

many

peptides

are

matched

resulting

in

high

sequence

coverage

and

Mascot

score

With

higher

flow

and

hence

decreasing

incubation

time

the

amount

of

protein

that

remains

undigested

increases

and

consequently

fewer

peptides

are

produced

and

observed

However

even

at

relatively

high

flow

rates

an

adequate

amount

of

peptides

is

still

formed

and

the

protein

can

be

identified

on

the

basis

of

the

fragments

present

Nevertheless

the

ion

intensity

is

low

and

some

peptides

are

not

retrieved

as

they

are

below

the

threshold

for

auto-MS

MS.

Table

summarizes

the

peptides

observed

and

matched

using

the

MS

MS

data

and

a

Mascot.database

search

of

proteins

digested

at

a

flow

rate

of

μL

min

A

base-peak

chromatogram

-LRB-

BPC

-RRB-

of

the

digestion

of

cytochrome

C

under

these

conditions

is

shown

in

Fig.

The

MS

MS

fragmentation

of

one

of

the

peptides

is

presented

in

Fig.

Table

overview

of

the

on-line

digestion

experiments

for

cytochrome

C

-LRB-

n

-RRB-

flow

rate

-LRB-

μL

-RRB-

digestion

time

-LRB-

s

-RRB-

sequence

coverage

-LRB-

-RRB-

total

no

peptides

matchedMascot

scoreundigested

protein

-LRB-

-RRB-

1017661134760.3533662150131.8283662364918.5116587278330

Table

fragments

observed

by

MS

in

the

on-line

digestion

of

cytochrome

C

and

myoglobin

The

experiments

were

conducted

at

pH

and

°C

at

a

flow

rate

of

μL

min

using

a

trypsin

reactorPeptide

sequenceTheoretical

m/zExperimental

m/zPositionMissed

cleavageCytochrome

C

-LRB-

sequence

coverage

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

sequence

coverage

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

digestion

of

pmol

cytochrome

C

at

a

flow

rate

of

μL

min

-LRB-

a

-RRB-

base

peak

chromatogram

the

numbers

correspond

to

the

matched

peptides

in

Table

and

-LRB-

b

-RRB-

MS

MS

fragmentation

of

the

peptide

TGPNLHGLFGR

with

m/z

showing

several

of

the

matched

fragment

ions

In

further

experiments

horse

myoglobin

was

digested

on-line

This

protein

is

generally

regarded

as

difficult

to

digest

-LSB-

-RSB-

When

μL

of

a

μmol

L

solution

in

buffer

is

injected

at

a

flow

rate

of

μL

min

-LRB-

s

exposure

time

-RRB-

the

injected

protein

is

completely

digested

as

was

observed

with

UV

detection

-LRB-

data

not

shown

-RRB-

Using

mass

spectrometric

analysis

different

peptides

are

observed

and

matched

using

the

MS

MS

data

and

a

Mascot

database

search

resulting

in

a

sequence

coverage

of

The

matched

peptides

are

summarized

in

Table

and

a

BPC

of

the

on-line

digestion

of

myoglobin

is

shown

in

Fig.

with

an

MS

MS

spectrum

of

one

of

the

tryptic

peptides

Both

the

degree

of

digestion

and

the

sequence

coverage

are

adequate

compared

with

other

systems

that

often

use

a

high

percentage

of

modifier

to

enhance

digestion

as

the

absence

of

denaturing

agents

during

digestion

leads

to

little

or

no

digestion

of

myoglobin

-LSB-

-RSB-

Therefore

these

reactors

are

generally

used

for

direct

infusion

into

MS

or

off-line

protein

digestion

as

the

presence

of

high

concentrations

of

methanol

or

acetonitrile

in

the

digestion

buffer

will

seriously

impede

on-line

protein

digestion

in

combination

with

RP-LC

Fig.

from

on-line

digestion

of

pmol

myoglobin

at

a

flow

rate

of

μL

min

-LRB-

a

-RRB-

base

peak

chromatogram

the

numbers

correspond

with

the

matched

peptides

in

Table

and

-LRB-

b

-RRB-

the

MS

MS

spectrum

of

the

peptide

HGTVVLTALGGILK

with

m/z

Amankwa

and

Kuhr

-LSB-

-RSB-

reported

that

proteins

and

peptides

adsorb

to

the

capillary

surface

of

their

trypsin

reactor

Reactor

fouling

and

as

a

result

sample

carry-over

is

also

observed

when

trypsin-modified

particulate

polymers

or

monoliths

are

used

-LSB-

-RSB-

Adsorption

of

proteins

and

peptides

to

these

relatively

hydrophobic

columns

is

prevented

by

the

addition

of

up

to

methanol

to

the

digestion

buffer

For

dextran-coated

surfaces

it

is

known

that

the

non-specific

adsorption

of

protein

is

minimal

and

depends

on

the

character

of

the

oligosaccharide

layer

-LSB-

-RSB-

During

protein-digestion

experiments

on

SPR

sensor

surfaces

modified

identically

with

the

capillary

reactors

no

adsorption

during

incubation

is

observed

-LRB-

data

not

shown

-RRB-

Similarly

during

on-line

digestion

using

the

dextran

coated

capillaries

sample

adsorption

and

carry-over

are

not

observed

as

repeated

sample

injections

result

in

identical

chromatograms

and

blank

buffer

injections

do

not

show

peptide

fragments

On

basis

of

the

latter

result

and

the

long

lifetime

of

the

reactor

trypsin

auto-digestion

is

probably

absent

The

developed

IMER

has

been

applied

for

the

digestion

of

horse

cytochrome

C

and

myoglobin

in

buffer

without

prior

pretreatment

followed

by

separation

and

MS

MS

sequencing

The

time

needed

to

obtain

complete

protein

digestion

and

sample

trapping

is

min

with

an

average

sample

residence

time

of

s

The

total

analysis

time

digestion

and

LC

separation

is

min

The

developed

micro-reactor

is

competitive

when

compared

with

other

systems

used

for

on-line

protein

digestion

described

in

literature

-LSB-

-RSB-

In

the

capillary

IMERs

described

by

Amankwa

and

Kuhr

-LSB-

-RSB-

and

Bossi

et

al.

-LSB-

-RSB-

the

complete

digestion

of

native

proteins

was

only

obtained

when

the

protein

resided

in

the

trypsin-coated

capillary

for

min

The

peptide

separation

was

carried

out

with

CE

in

these

latter

cases

Many

reactors

use

a

protein

denaturation

step

to

make

the

protein

more

susceptible

to

proteolytic

action

The

digestion

efficiency

can

be

enhanced

by

partial

denaturation

by

addition

of

acetonitrile

to

the

buffer

-LSB-

-RSB-

or

by

sonication

-LSB-

-RSB-

However

the

latter

has

not

yet

been

used

in

an

automated

set-up

and

the

presence

of

high

amounts

of

modifier

will

make

reversed-phase

chromatography

in

a

subsequent

step

for

the

separation

of

the

fragments

produced

during

protein

digestion

very

difficult

The

described

reactors

were

efficient

in

terms

of

time

needed

for

digestion

and

the

digestion

result

but

were

as

a

rule

used

for

direct

infusion

into

the

MS

or

used

for

off-line

digestion

Alternatively

protein

denaturation

is

accomplished

using

chemicals

such

as

SDS

guanidine

HCl

or

urea

A

recent

example

is

the

miniaturized

on-line

proteolysis

capillary

LC

system

as

described

by

Samskog

et

al.

-LSB-

-RSB-

who

employed

a

column

packed

with

poroszyme

The

sample

contained

guanidine.HCl

for

protein

denaturation

and

was

diluted

with

buffer

also

containing

methanol

prior

to

injection

The

total

time

needed

for

digestion

trapping

and

removal

of

the

high

concentration

of

salt

was

min

The

system

needed

periodic

regeneration

to

counteract

the

effects

of

residual

salts

in

the

analyte

Conclusions

To

study

the

immobilization

of

the

protein

trypsin

in

a

fused-silica

capillary

a

number

of

surface

modifications

was

tested

In

order

to

investigate

the

amount

of

protein

that

can

be

covalently

attached

to

these

surfaces

SPR

sensor

disks

are

modified

with

these

coatings

to

mimic

the

capillary

surface

The

SPR

measurements

show

that

the

best

results

were

obtained

using

a

dextran

coating

with

an

intermediate

layer

The

resulting

open-tubular

trypsin

reactors

having

a

pH

optimum

of

pH

display

a

high

activity

when

operated

at

°C

and

are

stable

for

at

least

two

weeks

when

used

continuously

The

capillary

reactors

show

flow-dependent

catalysis

For

a

capillary

IMER

the

conversion

of

the

insulin

B

chain

increases

with

decreasing

flow

and

hence

a

longer

residence

time

The

same

is

observed

for

the

digestion

of

horse

cytochrome

C

The

complete

digestion

of

pmol

μL

horse

cytochrome

C

is

observed

without

the

need

of

protein

denaturation

reduction

or

alkylation

when

the

average

residence

time

of

the

protein

sample

in

a

cm

μm

capillary

IMER

is

s.

For

the

proteins

used

in

this

study

trypsin-reactors

were

described

that

were

capable

of

faster

digestion

using

denaturing

agents

like

acetonitrile

to

enhance

the

digestion

process

However

the

presence

of

such

agents

would

seriously

hamper

direct

analysis

of

the

peptides

formed

using

RPLC

and

MS.

Identification

of

the

proteins

cytochrome

C

and

myoglobin

is

possible

by

the

tryptic

peptides

that

are

produced

on-line

separated

by

micro-RPLC

and

analyzed

using

mass

spectrometry

with

auto

MS

MS.

The

open-tubular

reactor

can

be

produced

easily

reproducibly

and

inexpensively

and

can

be

used

for

other

applications

such

as

enzyme-inhibitor

studies

Future

research

will

focus

on

the

development

of

miniaturized

multi-dimensional

analysis

systems

employing

on-line

digestion

using

these

IMERs

The

capillary

enzyme

reactors

show

no

backpressure

and

seem

promising

for

coupling

to

other

analytical

techniques

such

as

capillary

electrophoresis

and

surface

plasmon

resonance

O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O

trypsin reactor
dextran hydrogel
surface plasmon resonance
liquid chromatography