Methods for 20S Immunoproteasome and 20S Constitutive Proteasome Determination Based on SPRI Biosensors

Article abstract of DOI:10.1007/s12195-017-0478-7

The 20S proteasome, released into the circulation, is a novel cancer biomarker. It exists in two forms: the constitutive proteasome (20Sc) and the immunoproteasome (20Si), which both have separate diagnostic significance. The aim of this work was to develop new methods for 20Si and 20Sc determination. Five alternative specific biosensors usable with the surface plasmon resonance imaging (SPRI) technique for 20Si determination have been developed. Specific 20Si entrapment on the biosensor surface from an analyzed solution was achieved by means of an immobilized specific 20Si receptor. Four of the biosensors contain newly synthesized specific 20Si receptors, while the fifth contains the inhibitor ONX 0914. A method for 20Sc determination using an SPRI biosensor containing PSI inhibitor has been developed. By the introduction of an inhibitor blocking 20Si, 20Sc is selectively determined. All of the methods developed for 20Si and 20Sc determination exhibit good selectivity and satisfactory precision, recoveries and dynamic response ranges. 20Si and 20Sc were determined in blood plasma samples from healthy donors and patients with acute leukemia. In the case of these patients 20Si was the major component, and its level was more than one order of magnitude higher than in the healthy donors.

Full text of DOI:10.1007/s12195-017-0478-7

Cellular and Molecular Bioengineering, Vol. 10, No. 2, April 2017 (Ó 2017) pp. 174–185
DOI: 10.1007/s12195-017-0478-7
Methods for 20S Immunoproteasome and 20S Constitutive Proteasome
Determination Based on SPRI Biosensors
SANKIEWICZ ANNA,1 MARKOWSKA AGNIESZKA,2 LUKASZEWSKI ZENON,3 PUZAN BEATA,1
1
and GORODKIEWICZ EWA
1Department of Electrochemistry, Institute of Chemistry, University of Bialystok, Ciolkowskiego 1K, 15-245 Bialystok, Poland;
3
2Department of Organic Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland; and Faculty of
Chemical Technology, Poznan University of Technology, pl. Sklodowskiej-Curie 5, 60-965 Poznan, Poland
(Received 6 August 2016; accepted 17 January 2017; published online 24 January 2017)
Associate Editor James L. McGrath oversaw the review of this article.
Abstract—The 20S proteasome, released into the circulation,
is a novel cancer biomarker. It exists in two forms: the
constitutive proteasome (20Sc) and the immunoproteasome
(20Si), which both have separate diagnostic significance. The
aim of this work was to develop new methods for 20Si and
20Sc determination. Five alternative specific biosensors
usable with the surface plasmon resonance imaging (SPRI)
technique for 20Si determination have been developed.
Specific 20Si entrapment on the biosensor surface from an
analyzed solution was achieved by means of an immobilized
specific 20Si receptor. Four of the biosensors contain newly
synthesized specific 20Si receptors, while the fifth contains
the inhibitor ONX 0914. A method for 20Sc determination
using an SPRI biosensor containing PSI inhibitor has been
developed. By the introduction of an inhibitor blocking 20Si,
20Sc is selectively determined. All of the methods developed
for 20Si and 20Sc determination exhibit good selectivity and
satisfactory precision, recoveries and dynamic response
ranges. 20Si and 20Sc were determined in blood plasma
samples from healthy donors and patients with acute
leukemia. In the case of these patients 20Si was the major
component, and its level was more than one order of
magnitude higher than in the healthy donors.
mias (AML) exhibit 20S levels in blood serum elevated
by 1.5 orders of magnitude in comparison with the
control.10 However, 20S occurs in two forms, the
constitutive proteasome (20Sc) and the immunopro-
teasome (20Si), and the determination of both forms is
desirable. In the cases of ALL and AML, 20Si is the
dominant sub-form of 20S.18 Apart from acute leuke-
mia, 20Si plays a role in the pathogenesis of autoim-
mune diseases and neuropathologies.1,17,18 The
upregulation of immunoproteasome is a response to
challenges that induce stress and injury.6 In particular,
the immunoproteasome plays a role in the adaptive
immune response and is more efficient at eliciting
responses than the constitutive proteasome.4
Inhibition of 20Sc and 20Si is an efficient therapy in
the treatment of hematological malignancies.20 Selec-
tive inhibitors of 20Si or 20Sc, as well as inhibitors of
both forms, are used.
Both 20Sc and 20Si are barrel-shaped (MW
700 kDa) and composed of four rings. Each of the
rings contains seven distinct subunits. There are two
identical outer a rings and two inner b rings. The a-
rings control the entry of the substrate proteins into
the central catalytic chamber and bind the regulators.
The b rings contain three different catalytic sites. In the
case of 20Sc, the b1, b2, and b5 subunits are respon-
sible for caspase-like, trypsin-like and chymotrypsin-
like activities, for cleavage of proteins after acidic,
basic, and hydrophobic amino acids, respectively.7 In
the case of 20Si, the b2i and b5i subunits act similarly
to the b2 and b5 subunits in 20Sc. However, in the case
of the b1i subunit (also known as LMP2—low
molecular weight protein 2) the ability to cleave a
protein after an acidic residue almost vanishes, and
instead there appears an ability to act as a chy-
motrypsin-like subunit.5
Keywords—20S proteasome, Proteasome inhibitors, ONX
0914, PSI, Sensor, Surface plasmone resonance imaging.
INTRODUCTION
The 20S proteasome (20S), released into the circu-
lation, is a novel biomarker for the prognosis and
monitoring of patients suffering from various types of
cancers, as well as other pathologies.27 Patients with
acute lymphoblastic (ALL) and myeloblastic leuke-
Address correspondence to Gorodkiewicz Ewa, Department of
Electrochemistry, Institute of Chemistry, University of Bialystok,
Ciolkowskiego 1K, 15-245 Bialystok, Poland. Electronic mail:
ewka@uwb.edu.pl
174
1865-5025/17/0400-0174/0 Ó 2017 The Author(s). This article is published with open access at Springerlink.com

Methods for 20S Immunoproteasome and 20S Constitutive Proteasome
175
Two methods have been used for 20Si determina-
EXPERIMENTAL
tion: the enzyme-linked immunoabsorbent (ELISA)
test18,23,24 and semiquantitative western blot-
ting.3,18,21,29 These methods are broadly used in the
determination of biologically active species. However,
both methods are indirect ‘‘labeled methods’’. The use
of labels may be the reason for losing of the protein
functional properties.
An alternative method for immunoproteasome
detection may be a surface plasmon resonance imaging
(SPRI) biosensor. This is a label-free, surface-sensitive
spectroscopic technique used to examine the interac-
tion between biomolecules.13,28 SPRI detects changes
in the refractive index within a short distance from the
surface of a thin metal film caused by molecules bound
to the metal surface. The sensing surface usually con-
sist of glass coated with a thin metal layer (e.g. gold)
and a layer of active biomolecules as a receptor. The
antibody or inhibitor can be used for capturing of an
analyte from the solution.
Reagents
Fmoc-Phe-OH (Fmoc = 9-fluorenylmethyloxycar-
bonyl), chloranil, acetaldehyde, HOBt (1-hydroxyben-
zotriazole), Fmoc-nLeu-OH (Fmoc-2-APA-OH, APA 2
aminopentanoic acid), Fmoc-Ala-OH, Fmoc-Leu-OH,
Fmoc-hPhe-OH (hPhe homoPhe, 4-phenylbutyric acid)
HPLC solvent acetonitrile were purchased from Merck
(Novabiochem, Darmstadt, Germany, www.mer
ckmillipore.com/). TFA (trifluoroacetic acid), DIPEA
(diisopropylethylamine), DIC (diisopropylcarbodi-
imide), piperidine, TBTU (O-(benzotriazol-1-yl)-
N,N,N¢,N¢-tetramethyluronium
tetrafluoroborate,
NMP = 1-methyl-2-pyrrolidone and were obtained
from Iris Biotech GmbH (all Marktrewitz, Germany,
www.iris-biotech.de/). DCM (dichloromethane), DMF
(dimethylformamide) and methanol were the products
of Chempur (Piekary Slaskie, Poland, www.chempur.pl/).
DCM was used without further purification. DMF was
distillated over ninhydrin and stored under molecular
sieves 4A. Trypsin, chymotrypsin, Suc-Phe-pNA and
Bzl-L-Arg-pNA.HCl (Suc [succinyl], pNA [4-ni-
troanilide], Bzl [benzyl]) were purchased from Sigma
(Schnelldorf, Germany, www.sigmaaldrich.com/).
Phosphate buffered saline (PBS) were purchased from
Lublin Label Vaccines (Lublin, Poland, www.biom
ed.lublin.pl/).
SPRI biosensors have been successfully used for the
determination of biologically active substances such as
lysosomal proteases,8,9,11 20S proteasome,10 ubiquitin
carboxyl-terminal hydrolase L1 (UCHL1),18 aro-
matase,1
angiopoietin-226
and
transgelin-2
(TAGLN2).15 The determination of total 20S protea-
some concentration, i.e. without selective determina-
tion of sub-types of 20S, has been reported.10 Several
SPRI biosensor applications for clinical research have
been reported.9,12,16,25
20S proteasome (mammalian) (AFFINITI Research
Products Ltd, Mamhead, UK), commercial inhibitor
for immunoproteasome (ONX 0914) (Houston, USA,
www.selleckchem.com/), protein immunoproteasome
(Basepoint Business Centre, United Kingdom,
www.vivabioscience.com/), human albumin, cysteamine
The aim of this work was to develop methods for
the determination both 20Si and 20Sc i.e. both sub-
forms of 20S proteasome. This is entirely new
approach. The simplest method for 20Si determina-
tion was based on a specific biosensor, containing
20Si inhibitor as a receptor, with detection by the
surface plasmon resonance imaging (SPRI) technique.
In this work, the commercial inhibitor ONX 0914 was
used for the construction of the biosensor. Addi-
tionally, new 20Si receptors were synthesized in order
to obtain a substance of simple chemical structure
suitable only for analytical purposes. A carboxyl
group was introduced into the compound structure
for immobilization of the receptor to a cysteamine
linker. Inhibiting effect of synthesized receptors on
the amidolytic activity of chymotrypsin and trypsin
has been determined. These enzymes are active parts
of 20Si and 20Sc. The analytical parameters of several
optional SPRI biosensors were optimized and the
biosensor’s ability to determine 20Si in biological
samples was tested. Apart from direct 20 Si determi-
nation, the newly synthesized receptors were used for
indirect determination of 20Sc i.e. the other form of
20S proteasome.
hydrochloride,
N-ethyl-N¢-(3-dimethylaminopropyl)
carbodiimide (EDC) were purchased from Sigma (all
Steinheim, Germany, www.sigmaaldrich.com). 1-oc-
tadecanothiol (ODM) and N-Hydroxysuccinimide
(NHS) was obtained from Aldrich (Munich, Germany,
www.sigmaaldrich.com). HBS-ES solution pH 7.4
(0.01 M HEPES, 0.15 M sodium chloride, 0.005%
Tween 20, 3 mM EDTA), acetic buffer pH 3.79–5.57,
phosphate buffer pH 7.17–8.04, carbonate buffer pH
8.50–9.86 (all Biomed, Lublin, Poland, www.biomed.pl/),
photopolimer ELPEMER SD 2054, hydrophobic
protective paint SD 2368 UV SG-DG (Peters, Kem-
pen, Germany, www.peters.de/) were used as received.
Aqueous solutions were prepared with MilliQ water
(SimplicityÒMILLIPORE).
Biological Samples
Plasma samples from healthy adult donors (n = 4)
and patients with acute lymphoblastic leukemia

176
ANNA et al.
(n = 5) were supplied by the Department of Hema-
tology, Medical University of Bialystok, Poland.
Plasma samples from patients with acute lymphoblas-
tic leukemia were diluted 10 times with PBS.
tion was performed by mass spectrometry using a
Bruker Daltonics Esquire 6000 (Bruker Daltonik
GmbH, Leipzig, Germany) with electrospray ioniza-
tion (ESI).
All of the samples were provided after obtaining the
consent of the Local Bioethical Commission for the
study of the biological material collected.
Enzymatic Investigations
Determination of amidolytic activity was performed
as described by Okada.22 The synthetic amidolytic
(chromogenic) substrates (Bzl-L-Arg-pNA.HCl for
trypsin and Suc-Phe-pNA for chymotrypsin) are se-
lected to give appropriate specificity for inhibition of
the serine proteases. A chromophore (para-nitroani-
line = pNA, yellow color) is cleaved from the C-ter-
minus amide bond by the protease, and the absorbance
can be detected.
PROCEDURES
Peptide Synthesis
The peptides shown in Table 1 were synthesized
manually using the standard Fmoc-based strategy.2
The first Fmoc amino acid was loaded onto the 2-
chlorotrityl chloride resin with a twofold molar excess
of DIPEA in DCM. Fmoc deprotection steps were
carried out with 20% (v/v) piperidine in DMF/NMP
(1:1, v/v) for 15 min. The coupling reactions of Fmoc
amino acids were performed in DMF/NMP/DCM
(1:1:1) using a molar ratio of amino acid/DIC/HOBt/
resin of 3:3:3:1. The reactions were monitored with the
Steward chloranil test.30 Cleavage from the resin was
carried out with TFA/water (95/5). After 2.5 h stirring,
the resin was filtered and washed with TFA. The
combined filtrates were concentrated under reduced
pressure. The crude peptide was precipitated and wa-
shed with cold diethyl ether, filtered, dissolved in water
and lyophilized.
The Waters system (Waters S.A.S., BP 608, 78056
Saint-Quentin, En Yvelines Cedex, France) was used
for analytical and semipreparatory HPLC (Phenom-
enex C18, Jupiter 90A, 4 micron, 250 9 10 mm; Phe-
nomenex C18, Jupiter 300A, 5 micron, 250 9 4 mm;
solvents: A, 0.1% aqueous TFA; B, 0.1% TFA in
acetonitrile, gradient 0–100% B in A in 30 min, ana-
lytical flow rate 1 mL minÀ1, preparatory flow rate
1.5 mL minÀ1 monitored at 220 nm). In the
semipreparatory HPLC the major peak fraction was
pooled and lyophilized. Molecular weight determina-
A detailed description of the method is given below.
The buffer and the enzyme solution contained:
a. borane buffer—0.5 mL (pH 7.5), enzyme: trypsin
(0.4 units mLÀ1), synthetic substrate: Bzl-L-Arg-
pNA.HCl (0.2 mL, 8 mM);
b. tris buffer—0.6 mL (pH 9.0), enzyme: chy-
motrypsin (0.4 units mLÀ1), synthetic substrate:
Suc-Phe-pNA (0.2 mL, 8 mM).
The buffer and 0.1 mL of enzyme solution were
added to 0.2 mL of the examined compound dissolved
in 0.15 M NaCl (1–11) (as control 0.15 M NaCl). The
mixture was incubated for 3 min at 37 °C, then the
synthetic substrate was added. After 20 min of incu-
bation, the reaction was stopped by adding 0.1 mL of
50% acetic acid, and the absorbance of the released p-
nitroaniline was measured at 405 nm (Spekol 1300,
Analytic Jena). Every value represents the average of
triplicate determination. The IC50 value was taken as
the concentration of the inhibitor which decreased the
absorbance at 405 nm by 50% compared with the
absorbance measured under the same conditions
without the inhibitor. The IC50 values obtained for the
inhibitors are given in Table 2.
TABLE 1. Synthesized compounds and their basic characteristics.
Yield (%) Retention time (min)
52 12.80
No.
Peptide
MW
[M+H]+
1
H-Phe-Leu-Phe-OH
H-Phe-Phe–Phe-OH
H-Phe-Ala-Phe-OH
H-Leu-Phe–Phe-OH
H-Phe-Leu–Leu-OH
H-Phe-Phe-Leu-OH
H-Phe-hPhe-Phe-OH
H-Leu-Leu-Phe-OH
H-Phe-Phe-OH
425.5
459.5
383.4
425.5
391.5
425.5
473.6
391.5
312.4
391.5
391.5
426.4
460.2
384.1
426.5
392.5
426.4
474.5
392.7
313.5
392.2
392.9
2
61
68
62
58
59
42
53
69
59
52
13.03
11.22
10.81
10.57
10.61
11.81
10.67
9.89
3
4
5
6
7
8
9
10
11
H-Leu-Phe-Leu-OH
H-Phe-Leu-nLeu-OH
10.34
9.98

Methods for 20S Immunoproteasome and 20S Constitutive Proteasome
177
All of the tripeptides obtained exhibit chymotrypsin
inhibition.
completed. A biosensor prepared in this way should be
used directly for analysis. A diagram of the chip ap-
pears in Fig. 1a.
Chip Preparation
Newly Synthesized Receptors Immobilization
Gold chip were manufactured as described in a
previous paper.11 The gold surface of the chip was
covered with photopolymer and hydrophobic paint.8
The chip has nine places each with 12 free gold sur-
faces. Using this chip, nine different examined solu-
tions can be simultaneously measured without mixing
the tested solutions. Twelve single SPRI measurements
can be performed from one solution.
The chip was immersed in a vessel with an ethanolic
solution of cysteamine for 2 h at room temperature.
After 2 h, the chip was rinsed with ethanol and water
and dried under a stream of argon. A chip prepared in
this manner can be stored and used for subsequent
measurements. Each of the synthesised inhibitor solu-
tions was activated with NHS (250 mM) and EDC
(250 mM) in the ratio 1: 1 and carbonate buffer (pH
8.5) as a suitable reaction medium. Next, 3 lL of this
solution was placed on each of nine active sites on the
amine-modified surface. The closed vessel with the
biosensor and under appropriate conditions of
humidity was incubated at 37 °C for 1 h. Next, the
biosensor was rinsed with water and dried under a
stream of argon. A biosensor prepared in this way
should be used directly for testing and analysis.
AFM measurements were performed to confirm the
creation of subsequent layers: gold, thiol (cysteamine
or ODM), inhibitor and immobilized immunoprotea-
some 20S. All measurements were done in ambient
conditions. The AFM pictures confirm that the de-
scribed stages of the creation of each layer on the
biosensor surface has really take place. This may be
concluded on the basis of the creation of different
structures after each stage.
AFM Measurements
Atomic Force Microscopy (AFM) enables obser-
vation of the surface of the biosensor during its
preparation. AFM measurements were performed to
confirm the creation of successive layers. All mea-
surements were performed using a commercial Ntegra
Prima scanning probe microscope (NT-MDT, Russia)
in tapping mode in ambient conditions. Ethalon
probes (NT-MDT, Russia), with resonance frequency
around 140 kHz and curvature radius less than 10 nm,
were used.
ONX 0914 Immobilization
Chips were immersed in an ethanolic solution 1-
octadecanothiol (ODM) (20 nM) for 24 h at room
temperature. Then the biosensors have been repeatedly
washed in ethanol and water, and dried under a stream
of argon. Chips prepared in this way can be stored and
used for subsequent measurements. The commercial
inhibitor ONX 0914 was then applied to the active sites
of the biosensor and incubated for 24 h at room tem-
perature. After that time, the process of immobiliza-
tion of the inhibitor by hydrophobic interaction was
SPRI Measurements
SPRI measurements were performed on a home-
made apparatus which has been successfully used and
described previously.8–11 The apparatus (Fig. 1b)
consists of a HeNe laser (JDS Uniphase, Edmund
Industrial Optics, USA), two glass lenses L1
TABLE 2. Inhibiting effect of synthesized peptides on the amidolytic activity of enzymes.
IC50 mM
No
Peptide
Trypsin
Chymotrypsin
1
PheLeuPhe
PhePhePhe
PheAlaPhe
LeuPhePhe
PheLeuLeu
PhePheLeu
PhehomoPhePhe
LeuLeuPhe
PhePhe
–
–
–
–
–
–
0.178 0.014
0.185 0.014
0.187 0.014
0.184 0.014
0.184 0.014
0.174 0.013
0.185 0.014
0.152 0.012
0.141 0.011
1.83 0.15
2
3
4
5
6
7
0.044 0.003
0.079 0.006
–
8
9
10
11
LeuPheLeu
PheLeu-norLeu
12 0.96
18 1.44
1.92 0.15

178
ANNA et al.
FIGURE 1. Schematic illustration of the sensor’s active part (a) and the apparatus (b).
(f = 3 mm) and L2 (f = 300 mm), two polarizers (P1
and P2), a mirror, a prism and a CCD camera (QI-
Cam, QImaging, Canada). NIH Image J version 1.42
software was used for evaluation of the SPRI images in
2D form.
duced into the analyzed solution (blood plasma or
blood plasma diluted with PBS buffer) in the volume
necessary to achieve a concentration of 15 lg mLÀ1
.
SPRI measurements for the protein biosensor array
were performed as described elsewhere.8,11 Briefly, the
measurements were performed at a fixed angle of
incident light and the reflectivity was simultaneously
measured cross an entire chip surface. The contrast
values obtained for all pixels across a particular sample
single spot were integrated. The signal was measured
twice on the basis of registered images, after immobi-
lization of the inhibitor and after immunoproteasome
interaction. The SPRI signal, which is proportional to
the coupled protein, was obtained by subtraction
between the signals before and after interaction with
protein for each spot separately.
RESULTS AND DISCUSSION
Biosensor Based on ONX 0914 Inhibitor
The biosensor was made by immobilization of the
inhibitor via ODM linker. The ODM was anchored
onto the gold surface by a linker thiol group. ONX
0914 was immobilized due to hydrophobic interaction
between the octadecyl group of ODM and the in-
hibitor, as is described above (ONX 0914 immobiliza-
tion).
Optimization of ONX 0914 Concentration
A background correction was applied i.e. some of
the sites on the biosensor covered with PBS buffer were
used as a control. Non-specific binding was monitored
by measuring the SPRI signal at a site on the chip
without the receptor (ligand). Minimization of non-
specific binding was achieved by preparing samples in
PBS buffer (NaCl an KCl concentration ~200 mM) at
pH 7.4 near the isoelectric point of the protein.
In the case of indirect 20Sc determination, the in-
hibitor (ONX 0914 or tripeptide) solution was intro-
Concentration of receptor is a significant parameter
at SPRI measurement, and ought therefore to be
optimized. At constant concentration of the analyte to-
be-determined, the dependence of the analytical signal
as a function of receptor concentration exhibits a
Langmuirian shape with formation of a plateau. Cor-
rect SPRI measurement should be performed with the
receptor concentration corresponding to this plateau.
The dependence of the analytical signal of 20Si on
ONX 0914 concentration was investigated as its con-

Methods for 20S Immunoproteasome and 20S Constitutive Proteasome
179
centration ranged between 0.5 and 25 lg mLÀ1, at a
constant 20Si concentration of 2.8 lg mLÀ1 and pH
7.4. Time of contact of the 20Si solution with the
biosensor was 10 min. The dependence is shown in
Fig. 2a. The gradual formation of a plateau is evidence
of saturation of the biosensor surface with ONX 0914.
A further increase in ONX 0914 concentration does
not cause an increase in surface ONX 0914 concen-
tration. On the basis of curve A in Fig. 2, a concen-
tration 15 lg mLÀ1 was selected as the optimal ONX
0914 concentration for further investigations.
Calibration Curve for 20Si
The calibration curve for 20Si determination with a
biosensor consisting of ODM linker and the inhibitor
ONX 0914 as the receptor was performed in optimized
conditions. SPRI measurements were performed in a range
between 1 and 15 lg mLÀ1. The results are shown in
Fig. 3a. The curve is of typical of Langmuirian type. The
plateau of the curve corresponds to saturation of active
sites of the biosensor. The initial section of the curve
(Fig. 3) is linear and is useful for analytical purposes.
Specificity of the Developed Biosensor
The specificity of the developed biosensor, especially
against 20Sc, is crucial factor for the device’s applica-
tion. Specificity was investigated at different ratios of
20Si and 20Sc and various 20Si concentrations. Addi-
tionally, tolerance of human albumin was checked.
The results are shown in Table 3.
It is clear that a 5-time excess of 20Sc has a small,
analytically acceptable effect on the 20Si determina-
tion. ONX 0914 is an irreversible inhibitor of
immunoproteasome. It is an a¢b¢-epoxyketone which
blocks the b5i-subunit 20–40 times more selectively
than other active sites of the immunoproteasome.19
20Sc at a lower ratio against 20Si has a negligible effect
on the 20Si determination, as does a 1000-fold excess
of human albumin. The recoveries and precision of
determination, the latter represented by confidence
ranges, are well acceptable for analytical purposes.
Examples of 20Si determination in real samples are
given in a further part of the paper.
FIGURE 2. Dependence of SPRI signal on receptor concen-
tration: (a) ONX 0914 inhibitor, (b) inhibitor 5. 20Si concen-
tration: 2.8 lg mL21, pH 7.4.
Biosensors Based on Newly Synthesised Receptors
Because the ONX 0914 inhibitor was synthesized
for therapeutic purposes, an attempt to synthesized a
receptor of simpler chemical structure was undertaken.
Apart from the active part necessary for specific 20Sc
entrapment, the new receptor was expected to have a
FIGURE 3. Calibration curve for 20Si with biosensor con-
sisting of ODM linker and ONX 0914 inhibitor as the receptor.
Concentration of ONX 0914: 15 lg mL21, pH 7.4. Interaction
time: 10 min.
TABLE 3. Influence of proteasome 20Sc or human albumin on the determination of 20Si concentration by SPRI biosensor with
ONX 0914 as a receptor.
Potential interferent
Ratio 20Si vs. interferent
Added 20Si (lg mLÀ1
)
Found 20Si (lg mLÀ1
)
Recovery (%)
20Sc
20Sc
1:1
1:5
2.8
2.8
7.0
2.8
2.82 0.44
3.08 0.22
7.31 0.67
2.94 0.12
101
110
104
105
20Sc
Albumin
5:1
1:1000
Confidence limits were calculated for 12 independent measurements for each concentration at a 95% confidence level.
20Si, immunoproteasome; 20Sc, constitutive proteasome.

180
ANNA et al.
carboxylic group for covalent bonding to a linker. The
synthesis of 11 receptor candidates, as well as their
basic properties and enzymatic activity, were described
above under Procedures. Examination of the selectivity
of these tripeptides is described below.
Selectivity of Synthesised Receptors
A crucial factor for the application of newly synthe-
sised potential receptors is their selectivity. Selectivity
for 20Si or 20Sc was checked using SPRI measurements.
For this purpose, SPRI measurements were performed
for 11 newly synthesised potential receptors and for
ONX 0914, known to be a selective 20Si inhibitor.14
Measurements were performed as described above at
three different concentrations of potential receptors (1,
10, 100 lg mLÀ1) at a fixed concentration of 20Si
(2.8 lg mLÀ1) or 20Sc (2.8 lg mLÀ1). An increase in the
SPRI signal of less than 20% was considered to imply
lack of ability to bind 20Si or 20Sc. The obtained results
are shown in Table 4.
Tripeptides 1,2,3 and 7 are able to bind both 20Si
and 20Sc, like the PSI inhibitor, while tripeptide 4 does
not capture any form of 20S. Tripeptides 6 and 9 ex-
hibit selectivity to 20Sc. However, the results of pre-
liminary attempts to use them as 20Sc-specific
receptors were discouraging. The expected selective
inhibition of 20Si is exhibited by tripeptides 5, 8, 10
and 11, as well as by ONX 0914, which is known as
selective 20Si inhibitor.
TABLE 4. The ability of newly synthesised potential recep-
tors and ONX 0914 to bind 20Si or 20Sc.
Determined substance
Potential receptors
20Si
20Sc
1
+
+
+
–
+
–
+
+
–
+
+
+
+
+
+
–
–
+
+
–
+
–
–
–
2
3
4
5
6
7
8
9
10
11
ONX 0914
Commercially available proteasome was considered as 20Sc.
+, bound; À, lack of bonding.
FIGURE 4. Calibration curves for biosensors with receptors: 5 (a), 8 (b), 10 (c) and 11 (d), pH 7.4, interaction time: 10 min.

Methods for 20S Immunoproteasome and 20S Constitutive Proteasome
181
TABLE 5. Influence of proteasome 20Sc (2.8 lg mL21) on the determination of 20Si concentration by SPRI biosensors with
receptors 5, 8, 10 and 11.
Receptor
Ratio 20Si vs. 20Sc
Added 20Si (lg mLÀ1
)
Found 20Si (lg mLÀ1
)
Recovery (%)
5
1:1
1:1
1:1
1:1
2.80
2.80
2.80
2.80
2.97 0.40
2.88 0.44
2.89 0.60
2.99 0.47
106
103
103
107
8
10
11
Confidence limits were calculated for 12 independent measurements for each concentration at a 95% confidence level.
shown in Figs. 4a–4d for receptors 5, 8, 10 and 11
respectively. Generally, all four calibration curves have
the same shape, with straight initial sections which are
useful for analytical purposes. All straight sections
have an R2 value close to 1. The linear sections of the
curves for receptors 5 and 8 begin from 0, while the
remaining two have intercepts similar to the calibra-
tion curve for the ONX 0914 inhibitor.
Specificity of the Developed Biosensors with Receptors
5, 8, 10 and 11
This investigation is crucial for the determination of
20Si in the presence of 20Sc. Series of model solutions
FIGURE 5. Calibration curve of total 20Sc and 20Si with
biosensor based on PSI receptor (25 lg mL21), pH 7.4.
containing both 20Si (2.8 lg mLÀ1
)
and 20Sc
(2.8 lg mLÀ1) were determined using four developed
biosensors specific for 20Si. The results are shown in
Table 5. The confidence limits of the results are a
measure of measurement precision.
Optimization of Receptor Concentration
The dependence of the analytical signal of 20Si on
receptor concentration was investigated with
a
The results show the very small influence of 20Sc on
the results of 20Si determination. Thus, the developed
biosensors can be used for selective 20Si determina-
tion. The precision of the measurements is acceptable.
The results are very similar to those obtained with
biosensors based on the ONX 0914 inhibitor.
biosensor having receptor 5 within a receptor concen-
tration range between 0.5 and 25 lg mLÀ1, at a con-
stant 20Si concentration of 2.8 lg mLÀ1 and pH 7.4.
The dependence is shown in Fig. 2b. Due to the same
mass of the other inhibitors, it was presumed that the
curves for inhibitors 8,10 and 11 have similar shape
and plateau. The gradual formation of a plateau is
evidence of saturation of the biosensor surface with the
receptor. A further increase in the concentration of
receptor 5 does not cause an increase in the receptor
surface concentration. It should be noted that the
analytical signal obtained with the use of receptor 5 is
lower than when the inhibitor ONX 0914 is used as the
receptor. 15 lg mLÀ1 was selected as the optimal
concentration for further investigation on the basis of
curves in Fig. 2.
Determination of 20Sc in the Presence of 20Si
Apart from direct application of the newly synthe-
sized tripeptides and ONX 0914 inhibitor for 20Si
determination, these compounds can be used indirectly
for the determination of 20Sc. A biosensor containing
inhibitor PSI can be used for this purpose. This
biosensor is selective for the total 20S concentration
i.e. the sum 20Si and 20Sc.17 Selective blocking of 20Si
should result in specific 20Sc determination. For this
purpose, a biosensor for selective 20S determination
based on PSI inhibitor was made, and a calibration
graph was constructed as shown in Fig. 5.
Calibration Curves of 20Si with Biosensors Having
Receptors 5, 8, 10 and 11
A series of measurements was performed to examine
the possibility of 20Sc determination in the presence of
20Si blocked by inhibitor ONX 0914 or tripeptides 5,
8, 10 and 11 used as 20Si inhibitors. The experiments
were performed at a 1:1 ratio of 20Sc and 20Si. The
The calibration curves for 20Si determination with
four biosensors consisting of cysteamine linker and
receptors 5, 8, 10 and 11 were obtained in optimized
conditions. SPRI measurements were performed
within a range between 1 and 15 lg mLÀ1. Results are

182
ANNA et al.
TABLE 6. Influence of proteasome 20Si (2.8 lg mL21) on the determination of 20Sc concentration by SPRI biosensors using 5, 8,
10, 11 and ONX 0914 inhibitors as blocking solutions.
Receptor
Ratio 20Sc vs. 20Si
Added 20Sc (lg mLÀ1
)
Found 20Sc (lg mLÀ1
)
Recovery (%)
5
1:1
1:1
1:1
1:1
1:1
1:1
2.80
2.80
2.80
2.80
2.80
2.80
2.93 0.36
2.60 0.35
2.69 0.33
2.60 0.53
2.55 0.54
5.31 0.45*
105
93
96
93
91
95
8
10
11
ONX 0914
PSI
Confidence limits were calculated for 12 independent measurements for each concentration at a 95% confidence level.
*Expected total 20Sc and 20Si concentration is equal to 5.60.
TABLE 7. A comparison of average concentrations: (a) immunoproteasome determined on the synthesized inhibitors and ONX
0914, (b) proteasome determined on the receptor PSI with blocking immunoproteasome, (c) proteasome determined on the
receptor PSI without blocking immunoproteasome.
Inhibitor no as receptor
5
8
10
11
ONX 0914
(a) Immunoproteasome (20Si), C = (lg mLÀ1
)
1.19 0.32
41.05 10.18
Healthy donors, n = 4
Patients with acute leukemia, n = 5
1.35 0.47
46.78 12.17
1.19 0.32
44.11 10.28
1.32 0.49
44.23 10.45
1.42 0.45
39.36 5.46
(b) Proteasome (20Sc) (with blocking immunoproteasome), C = (lg mLÀ1
)
Inhibitor no as blocker
Healthy donors, n = 4
Patients with acute leukemia, n = 5
5
8
1.17 0.61
10
2.08 0.51
8.21 0.42
11
1.76 0.59
9.54 0.53
ONX 0914
1.67 0.43
8.90 4.89
1.64 0.84
10.55 4.73
11.43 8.32
)
(c) Proteasome (20S) (without blocking immunoproteasome), C = (lg mLÀ1
Healthy donors, n = 4
Patients with acute leukemia, n = 5
2.80 0.70
43.00 10.40
Confidence limits were calculated for 12 independent measurements for each concentration at a 95% confidence level.
results in Table 6 show that the concept can be realized
successfully and that 20Sc can be selectively deter-
mined. Generally, the recoveries of 20Sc in this
experiment can be acceptable. It should be noted that
independent determination of 20Si and 20Sc and
comparison of the results with independent determi-
nation of total 20S is the most credible option.
The second series was performed with five blood
plasma samples from acute leukemia patients. The
samples were diluted with PBS buffer so as to fit the
linear section of the calibration graph. The measure-
ments were performed as described above. The results
are shown in the lower part of Table 7. Each of the
blood plasma samples was subjected to determination
12 times. The confidence limits of the results reflect the
variation of concentration in particular samples as well
as the precision error.
Determination of 20Si and 20Sc Concentration in
Biological Samples
No significant difference between the measurements
of 20Si concentration with each of the five developed
biosensors is observed, or in 20Sc determination using
the newly synthesized tripeptides or ONX 0914 as 20Si
inhibitors. Each of these tripeptides can be used
equivalently in place of ONX 0914 for analytical pur-
poses. The total 20Si and 20Sc concentrations in blood
plasma are generally in agreement with independent
measurement of the sum of 20Si and 20Sc.
It is clear that in the case of patients with acute
leukemia, 20Si is the major form of proteasome and its
concentration is highly elevated compared with the
level of 20Si in the blood plasma of healthy subjects. In
the case of healthy donors, the two proteasome forms
occurs at approximately equal concentrations.
In order to verify the developed methods for 20Si
and 20Sc determination, two series of measurements of
the concentration of these analytes in blood plasma
were performed. The first series consisted of four
samples of human blood plasma of healthy donors.
20Si was determined using the five developed biosen-
sors separately. 20Sc was determined with the use of a
biosensor based on PSI inhibitor. 20Si in the samples
was inactivated by the addition of inhibitors at a
concentration of 15 lg mLÀ1. ONX 0914, 5, 8, 10 and
11 were used separately in each measurement series.
The total concentration of 20 Si and 20Sc was also
determined i.e. without inhibitor. The results are
shown in Table 7.

Methods for 20S Immunoproteasome and 20S Constitutive Proteasome
Advantages and Limitations of the Developed Methods CONCLUSIONS
183
The five developed direct methods for circulating
20Si determination offer an alternative to the ELISA
method, which is the only available quantitative
method to date. In contrast to ELISA, these methods
are direct and label-free. All five methods are equiva-
lent in terms of analytical characteristics, i.e. selectiv-
ity, linear response range, precision and recovery. A
certain advantage over the other methods is demon-
strated by those using receptors 5 and 8, because the
linear sections of their calibration graphs have practi-
cally no intercepts (see Fig. 4). The similarity of the
analytic characteristics of the four methods based on
newly synthesized receptors is due to the fact that these
receptors are isomers (see Table 1). Surprisingly, the
method using ONX 0914 as the receptor exhibits very
similar analytical characteristics, despite the difference
in chemical structure and in the manner of immobi-
lization on the biosensor surface (hydrophobic inter-
action vs. covalent bonding for the other receptors). A
certain limitation of all of the developed direct meth-
ods is the relatively narrow range of linear response,
comprising approximately a half of an order of mag-
nitude. This limitation can be overcome by the dilution
of samples with buffer.
The five developed indirect methods for constitu-
tive 20S determination exhibit poorer analytical
characteristics than the developed methods for 20Si
determination. Recoveries are mediocre even under
model investigation; the worst is for the method using
ONX 0914. However, these methods are unique be-
cause they are the first available methods for 20Sc
determination. (It should be noted that 20S concen-
tration, i.e. 20Si and 20Sc, is frequently called ‘con-
stitutive 20S’.) The lack of alternatives makes these
methods useful.
We have developed five biosensors usable with the
SPRI technique, suitable for specific 20Si determina-
tion in the presence of 20Sc i.e. the other form of
proteasome. One of these biosensors contains com-
mercial ONX 0914 inhibitor bound to the biosensor
via an ODM linker by hydrophobic forces. Four other
biosensors contain newly synthesized receptors cova-
lently bound to the biosensors via a cysteamine linker.
These four receptors were selected from 11 newly
synthesized potential receptors candidates. All five
developed biosensors have similar characteristics in
terms of selectivity, recoveries, precision and dynamic
response ranges.
The same substances used as receptors were applied
for indirect determination of 20Sc. Here a biosensor
with inhibitor PSI was used, such a biosensor cannot
distinguish between 20Si and 20Sc. However, 20Si was
inactivated by the inhibitors previously used as recep-
tors. Satisfactory analytical characteristics were
obtained for all five versions of 20Sc determination. All
five new constructed biosensors were used for 20Si
determination in blood serum samples, as well as for
20Sc determination after blocking of 20Si. Both 20S
proteasome types were determined in the blood plasma
of patients with acute leukemia and of healthy donors.
20Si was the major type of 20S proteasome, and its
level was more than one order of magnitude higher
than in the healthy donors.
CONFLICT OF INTEREST
Sankiewicz Anna, Markowska Agnieszka, Lu-
kaszewski Zenon, Puzan Beata and Gorodkiewicz Ewa
declare that they have no conflict of interest.
Experiments with blood plasma confirm the
applicability of all five direct and five indirect meth-
ods for simultaneous determination of 20Si and 20Sc.
Cross-examination of the results (Table 7) by inde-
pendent determination of 20S concentration (i.e.
20Si + 20Sc) show that the sum of 20Si and 20Sc
determined separately exceeds the results of indepen-
dent measurements by approximately 10% in the case
of healthy donors and approximately 20% in the case
of patients with leukemia. The reason for this dif-
ference may be the unequal response of 20Si and 20Sc
in independent measurement (the determination is
calibrated with 20Sc). Certainly, these few experi-
ments with blood plasma are insufficient for conclu-
sions to be drawn concerning leukemia; they
demonstrate only the applicability of the newly
developed analytical tools.
ETHICAL STATEMENTS
All human subject research was carried out in
accordance with guidelines approved by the Local
Bioethical Commission. Informed consent was
obtained from all patients for being included in the
study. No animal studies were carried out by the au-
thors for this article.
OPEN ACCESS
This article is distributed under the terms of the
Creative Commons Attribution 4.0 International Li-
cense (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution, and re-

184
ANNA et al.
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