For Research Use Only. Not for use in diagnostic procedures.
An Automated Sample Preparation System Based on the Mixing Driven by Oscillating Magnetic Fields
Chang Liu1; Anne-Sophie Wavreille2; Jonathan Sidibe3; Gérard Hopfgartner3; Amar Rida2; J. Larry Campbell1; Don Arnold4; Thomas R. Covey1
1SCIEX, Concord, ON, Canada; 2Spinomix, Lausanne, Switzerland; 3University of Geneva, Geneva, Switzerland; 4SCIEX, Redwood City, CA, USA
Mass spectrometry has historically been used to identify and quantify proteins and peptides. However, due to
the inherent complexity of the biological matrix, the necessary multi-step sample preparation process has been
limited to specialized laboratories with the training and expertise to perform these time-consuming assays in a
reproducible and robust manner. Magnetic particle technology is nowadays the gold standard for sample
processing, allowing high sensitivity and accuracy and also automation. This automation is however limited to
bulky and complex robotic systems. In this work, we introduce a new automatic sample processing platform with
significantly improved performances that is based on a unique way of magnetic particles handling and efficient
mixing within microfluidics environment.
INTRODUCTION
analysis. 1-3
Sample preparation methods using magnetic beads have been widely used for protein
However, the
magnetic property of particles is typically utilized only during the buffer exchange (supernatant removal)
process. The sample mixing with surface-functionalized beads is still achieved by mechanical agitation (e.g.
shaking, pipette mixing).4 With these methods, magnetic particles may aggregate and cluster in discrete areas
close to the walls of the container, greatly reducing mixing efficiency. On the sample preparation platform
introduced here, oscillating fields was used to disaggregate magnetic beads fully.
With the automatic control of pre-scripted program, surface functionalized magnetic beads can be fully
disaggregated and homogeneously mixed with the solution within the microfluidic chamber. We studied the
protein immunoprecipitation efficiency on this platform with prostate-specific antigen (PSA) as the model
protein. With a mixing time of 12s, almost all PSA was captured by anti-PSA coated magnetic beads,
comparable to the performance of the >30 min conventional protein on-bead capture in-tube.5 Protein digestion
was the most time-consuming step in the sample preparation process for the bottom-up protein analysis, which
typically required hours to complete.4 In our study, we chose five signature peptides to compare the digestion
kinetics of bovine serum albumin (BSA). A five-minute digestion on the platform based on magnetic fields was
comparable to the conventional overnight digestion in-tube. The information-dependent acquisition (IDA)
analysis suggested better peptide coverage for the BSA digested on the magnetic field-driven mixing platform,
due to the enhanced mixing of trypsin with BSA. The automatic control of the entire workflow and the fast
sample processing speed makes this platform an attractive system for protein sample preparation.
MATERIALS AND METHODS
Chemicals and reagents:
PSA, BSA, dithothreitol (DTT), iodoacetamide and formic acid were purchased from Sigma-Aldrich (Oakville,
ON, Canada). Biotin conjugated PSA antibody was obtained from Pierce (Burlington, ON, Canada), and trypsin
was purchased from Promega (Madison, WI). Streptavidin coated magnetic beads were obtained from
Chemicell (Berlin, Germany).
Sample preparation for the immunocapture test:
Streptavidin coated magnetic beads were mixed with biotin conjugated PSA for 2 h at room temperature. After
rinsing 3x with the PBS buffer, the beads were ready for the immunocapture test. After the capturing process,
supernatants were collected for the PSA identification with either SDS-PAGE analysis at the intact protein level,
or LC/MS/MS analysis at the peptide level after the standard denaturation, reduction, alkylation, and digestion
steps.
Sample preparation for the digestion test:
Pretreated BSA (denatured, reduced and alkylated) were mixed with trypsin at the 50:1 (protein:trypsin, w/w)
ratio, and the digestion temperature was set at 37 ºC. After incubation, 2% (v/v) formic acid was added to the
solution, and the samples were analyzed by LC/MS/MS.
LC/MS/MS conditions:
A Shimadzu 20A HPLC system with a Phenomenex Kinetex C18, 100x2.1mm, 2.6μm column at 40 ºC with a
gradient of eluent A water/acetonitrile (98/2) + 0.1% formic acid and eluent B water/methanol (2/98) + 0.1%
formic acid was used at a flow rate of 300 μL/min. The injection volume was set to 5 μL. A SCIEX QTRAP ®
5500 LC/MS/MS system with Turbo V™ source and Electrospray Ionization (ESI) probe was used for the
quantitative analysis of PSA and BSA. Signature peptides for both proteins were analyzed in the MRM mode
and every sample was injected three times in positive polarity. The IDA analysis was conducted on a research
prototype TOF system equipped with SCIEX cHiPLC® instrument.
The electromagnetic mixer:
The sample preparation platform was composed of a fluid pumping system, a set of four electromagnets, and a
disposable microfluidic cartridge containing several fluid delivery channels and a diamond-shaped sample
mixing chamber. This chamber was placed in the middle of facing electromagnets, and the real-time localized
magnetic field gradient can be controlled by adjusting the magnitude, frequency, and phase of waveforms
applied to the magnets. During the buffer exchange process, a steady state magnetic field was used to trap
beads at corners of the chamber.
MW PSA input SN1 SN2 SN3
Signature peptide IVGGWECEK from input PSA
kDa
49
37
In addition, the peptide sequence coverage were compared for BSA samples digested with the conventional
method (incubation with a thermoshaker) and with the electromagentic mixer. Due to the enhanced mixing of
trypsin with BSA, better peptide sequence coverage (Figure 6A), and less peptides with missed cleavage sides
(Figure 6B) were observed for the digestion process with a electromegnetic mixer.
(A)
26
Signature peptide IVGGWECEK from the supernatants
collected after the immunocapture
19
15
(B)
25
100
90
20
80
70
15
60
50
Figure 3. The efficiency of immunocapture with a electromagnetically actuated mixer, determined both at the
intact protein level (left) and the peptide level (right).
10
40
30
5
20
10
Digestion
Electromagnet
0
Tryptic digestion was the most time-consuming step in the sample preparation process for the bottom-up protein
analysis, which typically required hours to complete. As shown in Figure 4, the digestion yield was low for the 5
min incubation time with the conventional digestion protocol (incubation with a thermoshaker). However, the
digestion kinetics can be significantly improved with the electromagnetic mixer. As shown in Figure 5, a 5-min
digestion time is enough to achieve a higher yield than the 2-h digestion mixed with a thermoshaker.
LVNELTEFAK
AEFVEVTK
0
1-h digestion - mixing
with a thermoshaker
overnight digestion mixing with a
thermoshaker
5-min digestion mixing with an
electromagnetic mixer
1-h digestion - mixing
with a thermoshaker
overnight digestion mixing with a
thermoshaker
5-min digestion mixing with an
electromagnetic mixer
Figure 6. (A) The peptide sequence coverage (%), and (B) the number of peptides identified with a missed
cleavage sites for three different digestion conditions: 1-h digestion on a thermoshaker, overnight digestion on
a thermoshaker, and 5-min digestion with an electromagnetic mixer.
CONCLUSIONS
Figure 1. The chip-based electromagnetically actuated mixer.
An automated sample preparation system has been introduced in this study. The use of RF driven oscillating
magnetic fields could form a homogeneous “dynamic fog” suspension of magnetic beads, allowing an optimal
exposure and enhanced mixing with surrounding liquid medium. The reaction kinetics can be significantly
improved. Here, we demonstrated the complete immunocapture within 12 s, and the fast digestion within 5 min
with this electromagnetic mixer. The entire process of the protein sample preparation could be shortened from
an entire day to 10-15 min. This platform could be potentially used as an automated protein sample preparation
device for the real-time LC/MS/MS analysis.
RESULTS
Immunoprecipitation
PSA was used as the model protein to demonstrate the fast immunocapture kinetics. As shown in Figure 2,
longer than 10 min was necessary to complete the immunocapture if the sample was placed on a shaker. On
the electromagnetically actuated mixer, almost all PSA was captured on anti-PSA beads within 12 s (Figure 3).
5 min
10 min
30 min
1h
2h
5 min
10 min
30 min
1h
2h
Figure 4. The digestion kinetics studied by comparing the intensity of two signature peptides from BSA.
Sample were incubated with a thermoshaker.
Percentage of PSA captured on beads
ABSTRACT
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Figure 2. The efficiency of immunocapture when
the sample was placed on a shaker. Supernantants
were collected after the immunocapture step for the
quantification of un-captured PSA.
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TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.
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© 2015 AB Sciex.
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2 min
5 min
10 min
Figure 5. The digestion yield comparison with five signature peptides of BSA.