Ultrasensitive Ambient Mass Spectrometry Immunoassays: Multiplexed Detection of Proteins in Serum and on Cell Surfaces

Article abstract of DOI:10.1021/jacs.8b10853

The sensitive and accurate determination of multiple protein biomarkers for clinical diagnosis is in high demand. Here, an ambient mass spectrometry immunoassay platform that possesses advantages of high sensitivity, multiplexed quantitation, low sample c

Full text of DOI:10.1021/jacs.8b10853

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Ultrasensitive Ambient Mass Spectrometry Immunoassays:
Multiplexed Detection of Proteins in Serum and on Cell Surfaces
Shuting Xu, Wen Ma, Yu Bai, and Huwei Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10853 • Publication Date (Web): 19 Dec 2018
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Ultrasensitive Ambient Mass Spectrometry Immunoassays: Multiplexed
Detection of Proteins in Serum and on Cell Surfaces
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Shuting Xu, Wen Ma, Yu Bai , Huwei Liu
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular
Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular
Engineering, Peking University, Beijing, 100871, P. R. China
in MS immunoassays has become a topic of interest in
ABSTRACT: The sensitive and accurate determination of
multiple protein biomarkers for clinical diagnosis is in high
recent years. Multiple proteins tagged with elements have
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been detected by inductively coupled plasma MS.
demand. Here, an ambient mass spectrometry
immunoassay platform that possesses advantages of high
sensitivity, multiplexed quantitation, low sample
consumption and convenient operation was established. A
series of extensible rhodamine-based mass tags that
ensured quantification of multiple proteins with ultrahigh
sensitivity through two-stage signal amplification were
developed. Thrombin was detected at zeptomole sensitivity
in 2 μL serum/plasma, as was free cancer antigen 125
However, the specialized instrumentation and labeling
elements greatly increase the operating costs and limit their
application. Compared with elements, organic molecules
1
2
enable considerably more possibilities for tag design.
Combining organic tags with ambient mass spectrometry
AMS) can enable simple, patient-friendly and low cost
(
13
detection. Thus far, stable and extensible tag development,
sensitive and multiple-protein detection, and flexible and
convenient operation remain challenging in MS
immunoassays.
(
CA125).
Three
protein
biomarkers
(CA125,
carcinoembryonic antigen, and epithelial cell adhesion
molecule) were simultaneously detected in situ on ca. 20
cells. This platform is promising for multiple protein
detection in a single drop of sample or at the single-cell scale
for clinical diagnosis and therapy.
Herein, an ultrasensitive and multiplexed AMS
immunoassay (Figure 1a) was developed based on the new
design of mass tags and immune-recognition on chips. New
series of rhodamine-based mass tags (RMTs, Figure 1b)
were designed with two-stage MS signal amplification and
excellent distinguishability. The use of multiple mass tags,
signal amplification and a convenient and high-throughput
Most clinical biomarkers and therapeutic targets are
membrane proteins or secreted proteins; thus, monitoring
their expression levels in bodily fluids or on cell surfaces is
1,2
of great physiological value. Immunoassays have been
3
successfully used in protein detection for decades, evolving
4
from enzyme-linked immunosorbent assays (ELISA) to
5
6
fluorescence-, electrochemistry-, and mass spectrometry
7
(MS) -based assays. Immunofluorescence assays are
generally applicable due to their ultrahigh sensitivity, but
band overlap and unavoidable background interference
limit multiplexed detection. Although the latest iterative
8
staining strategies have increased their throughput,
efficient multiplexed measurements on one spot are still
needed. Electrochemical immunosensors also suffer from
restricted electrochemical probes and electrodes.
As a readout of immunoassays, MS overcomes the
multiplexed detection limitation because of its high mass
resolution. Direct MS analysis of proteins provide sequence
and quantitative information, but the detection of large
biomolecules is often hampered by limited sensitivity.
Indirect protein measurement through labeling strategies
Figure 1. (a) Schematic of an AMS immunoassay platform and its
workflow for protein detection in bodily fluids and on cell surfaces:
protein or cell capture, protein recognition in a sandwich-type
structure, mass tag dissociation and MS analysis. (b) Chemical
9
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structure of three RMTs (RMT443, RMT415, and RMT387) and the
two-step cleavage of mass tags during MS detection.
regulation and nanoparticle catalysis (Figures S12-S14)
contributed to the efficient tag dissociation. The second
amplification came from the cleavage beside the ester bond
induced by in-source CID, resulting in the accumulation of
the distributed signals from various RMT derivatives. All
RMT443 derivatives generated the same fragment at m/z
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AMS technique allowed the ultrasensitive and efficient
detection of multiple protein biomarkers in single drops of
bodily fluids or on a few cells in situ.
In detail, the AMS immunoassay platform was established
based on an array-type electrospray accelerated chip spray
ionization (eCSI) setup (Figure S2). The target protein
capturing and labeling and the mass tag dissociation and
ionization were all realized on an indium tin oxide glass chip
4
43.2 (Figure S7), corresponding to a conjugated structure
of rhodamine terminal. An in-source CID energy of 100 V
afforded the maximum 3-fold enhancement of signal
intensities (Figure 2a and 2b and Figure S8). Benefiting
from the noise reduction after fragmentation, an
approximate 6-fold higher signal-to-noise (S/N) ratio was
achieved for m/z 443.2 than for m/z 611.2 before
fragmentation (Figure 2c). Similar cleavages and S/N gains
were achieved for RMT415 (Table S2, Figure S10) and
RMT387 (Table S3, Figure S11) with final MS reporters at
m/z 415.2 and m/z 387.2, respectively. These three peaks
were easily and clearly identified in mass spectra (Figure
2d), demonstrating the advantage of MS in overcoming the
spectral band overlap. The two-stage signal amplification
and high mass resolution make the highly sensitive and
multiplexed protein detection possible and extendable.
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(
Figure 1a). Briefly, gold was deposited on the front of a chip
and modified with recognition units such as
aptamers/antibodies for protein or cell capture. 2 μL
sample was sufficient for detection, which is patient-
friendly for clinical diagnosis. Target proteins/cells were
captured on the chips and an immune sandwich were
formed after the addition of gold nanoparticle (GNP) probes
functionalized with self-assembly recognition aptamers and
RMTs. After incubation and washing, the in situ dissociation
and detection of multiple RMTs were achieved directly by
eCSI MS within 2 minutes. Samples can be screened
efficiently using an automatic sampling rail (Figure S5). The
platform was successfully applied to the detection of
zeptomole proteins in serum/plasma and the simultaneous
detection of multiple membrane proteins on approximately
20 cells in situ.
Mass tags, acting as MS reporters and signal amplifiers, play
key roles in ultrasensitive and multiplexed protein
quantification. Serial and homologous molecules with high
dissociation efficiency and good MS response are the best
candidates of mass tags for multiplexed detection. Three
analogous molecules, named RMT443, RMT415, and
RMT387 according to the different terminals (Figure 1b),
were synthesized as mass tags for the first time through
universal synthesis methods (Figure S1). Stable storage for
more than 6 months after synthesis was proven which is
crucial for their practical application. Rhodamine
derivatives were selected due to their good response and
Figure 2. Mass tag dissociation. (a) Mass spectrum of RMT443
dissociation under eCSI (M: tag residue of C28
spectrum of dissociated RMT443 with the source fragmentation energy
of 100 V (R: tag residue of C28 ). (c) Signal-to-noise ratios of m/z
28.4, m/z 611.4, and m/z 443.2 under different fragmentation energy
0-100 V). (d) MS spectrum of three RMTs after dissociation and
30 2 3 2
H N O (CH )11). (b) Mass
1
4
stability in MS and their fluorescence which could be
15
31 2 3
H N O
exploited in imaging. The thiol group and undecyl chain
6
(
enabled their self-assembly on GNPs and efficient
dissociation during MS detection. Unlike other tags that
have to be dissociated with lasers, high temperature or
fragmentation, with m/z 443.2, m/z 415.2, m/z 387.2 as the final MS
reporters.
1
2,13,16,17
special reagents,
these mass tags can be dissociated
Quantitation of proteins at ultralow concentrations in
biofluids and on cell surfaces was achieved with the help of
crystal violet (CV, Figure S15) dissolved in the electrospray
solvent at certain concentrations as internal standard (IS).
The intensity ratios of the MS reporter (m/z 443.2, Figure
and handled easily under atmospheric conditions with
general electrospray solvent.
The MS detection of RMTs underwent two-step cleavages
(Figure 1b), which resulted in the two-stage signal
amplification and spectral simplification. The efficient
dissociation of RMTs from GNPs during eCSI obtained the
primary amplification, and the subsequent fragmentation
by the in-source collision-induced dissociation (CID)
provided the secondary signal amplification and the
simplification of the MS spectrum. In detail, abundant mass
tag derivatives (Table S1) were identified in the mass
spectra of RMT443 after primary cleavage of the Au-S bonds
3
b, red bar) and fragment ion of CV (m/z 340.2, Figure 3b,
blue bar) were calculated for quantitation. The IS showed
good signal stability during the whole detection (Figure 3a,
RSD = 6.64%, n = 15). Standard thrombin was quantified
over a wide range with ultrahigh sensitivity when spiked in
PBS and serum (Figure 3c). The LOD for thrombin spiked in
PBS was 5.45 fmol/L (S/N = 3, 2 μL sample), corresponding
to 10.9 zmol, and an LOD of 35.1 zmol was obtained for
thrombin spiked in serum, which is superior to most of the
sandwich-type thrombin detection assays with either
(
(
Figure 2a). Experiments investigating the eCSI parameters
Figure S3) suggested that chemical/electrochemical
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electrochemical or optical signal amplifiers.18,19 Compared
microscope through the fluorescent signal from RMTs
(Figures S28, S38 and S39), which helped to guide the MS
analysis. Since fluorescent imaging did not damage the mass
tags, subsequent MS quantitation was obtained on the same
chip. CA125 on cell surfaces was successfully detected alone
with high sensitivity (Figure S31). An obvious MS signal
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to the LOD of 0.4 pmol for direct detecting thrombin using
7
eCSI-MS, 10 times higher sensitivity was achieved (Figure
3b) which attributed to the new RMT design and the signal
amplification strategy. Quantitative accuracy was
confirmed by the recoveries (95.9%-118%, RSD < 11.8%, n
2
1
=
6, Table S4). Good selectivity was proven under the
optimized molar ratio of GNPs/aptamers/RMTs
1/120/5000, Figure S19) by almost no signals of MS
from the mass tags was observed in 25 OVCAR-3 cells. Few
2
2
signals were detected from a large number of MCF-7 cells,
(
indicating the high selectivity and low nonspecific
adsorption of GNP probes for cell analysis. A good linear
response was found between MS intensity ratios and cell
numbers, and the LOD was 13 OVCAR-3 cells (S/N = 3,
Figure S33).
reporters from either negative control (Figure 3b) or
nontarget proteins (Figure S23), and good interday
repeatability was achieved (RSD = 10.1%, n = 6, Figure 3d).
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Three important cancer biomarkers localized on the cell
surface—CA125, carcinoembryonic antigen (CEA), and
epithelial cell adhesion molecule (EpCAM)—were analyzed
simultaneously in situ. Three individual GNPs modified with
distinct mass tag-aptamer pairs were prepared for each
protein, mixed and incubated with cells together. After
incubation and washing, obvious red light was observed
under a fluorescence microscope for both cell lines (Figure
4a, inset). Then, the three labeled mass tags were
dissociated and analyzed simultaneously by eCSI MS, and
the corresponding reporters clearly appeared in the
spectrum without any cross-interference (Figure 4a). The
MS results revealed that CA125 and EpCAM were
overexpressed on OVCAR-3 cell surfaces, while CEA and
EpCAM were overexpressed on MCF-7 cell surfaces (Figure
2
3-25
4
b).
These results were confirmed at cell numbers as
Figure 3. Thrombin analysis. (a) Schematic of thrombin detection and
the total ion chromatogram (TIC) and extracted ion chromatograms
low as 25 (Figure S37), illustrating the ultrasensitivity and
multiplexed detection of these platforms.
(
EICs) of the MS reporter at m/z 443.2 and the IS at m/z 340.2 for
thrombin detection. (b) MS spectra of the direct detection of thrombin
LOD: 0.4 pmol, S/N = 3) and the detection of 0.02 amol thrombin and
(
a blank control using RMT443 and the IS. (c) Calibration curves of
thrombin spiked in PBS (20 zmol to 200 amol) and serum (100 zmol to
200 amol), and standard addition results in plasma. (n = 3, error bar:
standard deviation, SD) (d) Repeatability of thrombin detection
(
interday RSD = 10.1%, n = 6).
The application of this versatile AMS immunoassay was
expanded to the detection of cancer antigen 125 (CA125)
biomarker in serum (LOD of 0.2 U/mL, Figures S24-27,
Table S5). Antibodies were immobilized on chips for
recognition, confirming that this assay can be flexibly
extended to other recognition units. Serum samples from
ovarian/breast cancer patients were examined using this
assay. Consistent results with human ELISA kit (Table S6)
demonstrated the high possibility for clinical diagnosis.
Figure 4. In situ membrane protein analysis on cell surfaces. (a) Mass
spectra of EpCAM (m/z 387.2), CEA (m/z 415.2) and CA125 (m/z
443.2) on MCF-7 (up) and OVCAR-3 (bottom) cells. Inset: merged
fluorescence and bright-field photographs of captured MCF-7 and
OVCAR-3 cells incubated with three GNP probes (scale of 20 μm). (b)
Multiplexed detection of CA125, CEA, and EpCAM on different numbers
of OVCAR-3 and MCF-7 cells captured on the chips. (n = 3, error bar:
SD)
1
Since most biomarkers are localized on the cell surface, the
mass tag and AMS immunoassay were assessed for the in
situ detection of multiple proteins on living cells. Ovarian
cancer cells (OVCAR-3) and breast cancer cells (MCF-7)
were chosen as models. These cells were captured on chips
In summary, an ultrasensitive and multiplexed AMS
immunoassay was established for the measurement of
proteins in single drops of bodily fluids or on a few cells in
situ. Rhodamine-based molecules were verified to be ideal
mass tags that presented high MS intensities and excellent
distinguishability. Compared with expensive inorganic MS
tags and fluorescent tags with unavoidable band overlap,
organic mass tags will provide abundant possibilities for
immunoassays. Representative proteins were detected
2
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by mucin 1 protein aptamer when 2 μL cell suspension
solutions were deposited on chips, followed by the
incubation with GNP probes for target protein recognition
in situ. The incubation time was optimized and controlled to
ensure the efficient labeling of surface protein and minimize
the interference from cell internalization of probes (Figure
S29). Target proteins on cell surface were observed under a
7
with a 10 -fold sensitivity enhancement, with a zeptomole
LOD in a 2 μL sample, and three membrane proteins were
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simultaneously detected in situ without cross-interference
on ca. 20 cells. In addition, fluorescent imaging of labeled
cells enables the guidance of MS detection by RMTs. All of
these characteristics make this versatile platform a
promising technique for the multiplexed detection of
dozens of protein biomarkers in a single drop of sample or
at the single-cell scale for clinical diagnosis and therapy.
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ASSOCIATED CONTENT
Supporting Information
This information is available free of charge via the Internet at
http://pubs.acs.org.
Reagents, instruments, mass tag synthesis (Figure S1), chips
preparation and eCSI mechanism (Figures S2-S6), mass tags
dissociation (Tables S1-S3, Figures S7-S14), protein detection
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(
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Derivatization for liquid chromatography-mass spectrometry. TrAC,
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Revyakin, A.; Patel, R.; Macklin, J. J.; Normanno, D.; Singer, R. H.;
Lionnet, T.; Lavis, L. D. A general method to improve fluorophores for
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AUTHOR INFORMATION
Corresponding Author
2
50.
*
*
Phone: +86-10-62758198 (Y. B.).
Email: yu.bai@pku.edu.cn (Y. B.).
(
16) Du, R.; Zhu, L.; Gan, J.; Wang, Y.; Qiao, L.; Liu, B. Ultrasensitive
detection of low-abundance protein biomarkers by mass
spectrometry signal amplification assay. Anal. Chem. 2016, 88, 6767-
6772.
Author Contributions
All authors have given approval to the final version of the
manuscript.
(
17) Lee, J. R.; Lee, A.; Kim, S. K.; Kim, K. P.; Park, H. S.; Yeo, W. S. Mass
spectrometry signal amplification method for attomolar detection of
antigens using small-molecule-tagged gold microparticles. Angew.
Chem., Int. Edit. 2008, 47, 9518-9521.
Notes
(
18) Deng, B.; Lin, Y.; Wang, C.; Li, F.; Wang, Z.; Zhang, H.; Li, X. -F.; Le,
The authors declare no competing financial interest.
X. C. Aptamer binding assays for proteins: the thrombin example—a
review. Anal. Chim. Acta 2014, 837, 1-15.
(
19) Ju, H. Functional nanomaterials and nanoprobes for amplified
biosensing. Appl. Mater. Today 2018, 10, 51-71.
20) Zhang, J. -J.; Cheng, F. -F.; Zheng, T. -T.; Zhu, J. -J. Versatile
ACKNOWLEDGMENT
We thank Ms. Xue Zhao for her help in part of the experiment
of CA125 detection. This work was financially supported by the
National Natural Science Foundation of China (21874003,
(
aptasensor for electrochemical quantification of cell surface glycan
and naked-eye tracking glycolytic inhibition in living cells. Biosen.
Bioelectron. 2017, 89, 937-945.
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Obermair, A.; Zeillinger, R. Expression of mucins and cytokeratins in
ovarian cancer cell lines. Cancer Lett. 1999, 145, 133-141.
2
1527809, 21575007, and 21322505) and the Ministry of
Science and Technology of the People's Republic of China
(2016YFF0100303) and National Key R&D Program of China
(
2017YFC0906800).
(
22) Morgado, M.; Sutton, M. N.; Simmons, M.; Warren, C. R.; Lu, Z.;
Constantinou, P. E.; Liu, J.; Francis, L. L. W.; Conlan, R. S.; Bast, R. C., Jr.;
Carson, D. D. Tumor necrosis factor-α and interferon-γ stimulate
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cancers through NFκB. Oncotarget 2016, 7, 14871-14884.
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(8) Gut, G.; Herrmann, M. D.; Pelkmans, L. Multiplexed protein maps
link subcellular organization to cellular states. Science 2018, 361,
eaar7042.
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Mass tag dissociation. (a) Mass spectrum of RMT443 dissociation under eCSI (M: tag residue of
C28H30N2O3(CH2)11). (b) Mass spectrum of dissociated RMT443 with the source fragmentation energy of
1
00 V (R: tag residue of C28H31N2O3). (c) Signal-to-noise ratios of m/z 628.4, m/z 611.4, and m/z 443.2
under different fragmentation energy (0-100 V). (d) MS spectrum of three RMTs after dissociation and
fragmentation, with m/z 443.2, m/z 415.2, m/z 387.2 as the final MS reporters.
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