Using the Agilent 2100 bioanalyzer for
quality control of protein samples prior to
MS-analysis
|
 |
Whereas in the past mass spectrometers (MS) were confined to the
realm of small molecules, the development of soft ionization methods
such as matrix-assisted laser desorption/ionization (MALDI) and electrospray
ionization (ESI) revolutionized the analysis of large biomolecules1,
2. Today, the analysis of proteins up to 300 kDa using MS has
become a routine method to gain molecular weight information with
high accuracy and structural information. However, sample purity
greatly influences the results of MS analysis. In MS service facilities,
scientists have to rely on the information provided by the customers
and the quality of the sample preparation they have done. Bad results
are often the consequence of customers being too optimistic about
their own samples. Especially concentration and purity are often overestimated.
To avoid unproductive MS runs, a fast and simple precheck
is valuable. In this Application Note we show that the Agilent 2100
bioanalyzer can be used for quality control of protein samples prior to
MS analysis. A corresponding data set analyzed with LC/MS and with
the Agilent 2100 bioanalyzer using the Protein 200 Plus LabChip® kit is
shown.
|
Experiments
|
All samples were adjusted to a
concentration of 1 mg/mL by dilution
with the appropriate buffer
prior to the analysis (based on
information given by the customer).
|
LC-MS analysis
|
The experimental setup comprised:
|
• Micro-HPLC-Pump (ABI 140C)
• Autosampler (PE200)
• HPLC column (Nucleosil C8
125 x 2 mm, 5 µ, 120 Å from
Macherey & Nagel)
• UV detector (ABI 785A) set to
214 nm, in row with
• Single quadrupole mass spectrometer
(API165 from Applied
Biosystems) with a 1:10 flow
splitter
|
The analysis was performed as
follows:
|
10 µL sample was injected and the
proteins were separated with a
flow of 250 µL/min. A linear gradient
was applied using eluent A
(0.1 % TFA in water) and eluent B
(0.1 % TFA in acetonitrile) from 5 %
eluent B within 15 miutes to 95 %
eluent B. The gradient was started
with 20 % eluent B when analyzing
samples with low salt or very
hydrophobic proteins. For MS
detection an ESI-spray source was
used in positive ion mode with an
ionization voltage of 5000 V and
an orifice potential of 40 V. The
analysis was carried out with a
full scan between 600 and 1800
m/z (2.7 s/scan). For deconvolution
the “Biotoolbox” software
was used.
|
|
Agilent 2100 bioanalyzer and the
Protein 200 Plus assay
|
Samples and chips were prepared
according to the protocol provided with the Protein 200 Plus LabChip
kit. The kit includes 25 chips, spin
filters and all reagents needed for
the experiments including the
Agilent Protein 200 Plus ladder
and the upper and lower marker
premixed in the sample buffer.
The chip-based separations were
performed on the Agilent 2100 bioanalyzer using the dedicated
Protein 200 Plus software assay.
The software automatically determines
the size of each protein in
kDa, its relative concentration to
the upper marker and the percent
total of the protein. Latter gives
direct information on the purity of
a particular protein.
|
Results and discussion
|
Several protein samples were analyzed
both with the LC/MS method
(optimized for robust online
desalting and rough separation of
impurities) as well as the Agilent
2100 bioanalyzer and the Protein
200 Plus assay. A summary of the
results is shown in table 1.
|
|
The protein sample 9-15 for example
did not cause any problem
during MS analysis (figure 1). It
was purified by the customer
using a gel-column and was provided
in a low salt buffer (50 mM
Tris/HCl, 1 mM EDTA). A molecular
weight of 33484 Da was
expected. The UV-chromatogram
showed a single peak at 6.3 minutes
(figure 1B), which corresponds to
the peak at 6.7 min in the TIC
(figure 1C). After deconvolution
of the correlated mass spectrum
(figure 1D) the protein mass was
found to be exactly as expected
(figure 1E). Also, the 2100 bioanalyzer
electropherogram (figure 1A)
showed a main peak at 30.6 kDa
with a purity of 85.0 %, which corresponds
nicely to the MS results.
For protein sample 16 the results
were already more difficult to
interpret (figure 2). The protein
was purified with a Ni-NTA-column
and was provided in a buffer
with relatively high ionic strength
compared to the protein sample
9-15 (50 mM NaH2PO4, 250 mM
imidazole, 300 mM NaCl). A molecular
weight of approximately
30 kDa was expected. The UV
chromatogram showed a main peak
at 4.7 minutes and some minor
peaks at 7.4 minutes and 11.2
minutes However, the TIC showed
two prominent signals (figure 2C) –
one at 5.1 minutes, which corresponds
to the 4.7 minutes signal in the UV, and a second peak at
11.5 minutes, which seemed to be
only a minor impurity in the UV
but was obviously easy to ionize.
Both TIC signals were analyzed,
where the first mass spectrum
turned out to be very noisy (figure
2D), which is usually an indication
for inhomogenious samples and/or
a high tendency for aggregation.
As expected, the deconvolution
yielded several mass peaks of which
the most intense around 30 kDa
could only be considered as a hint
that the expected protein is present.
The second mass spectrum showed
a polymer pattern between 600
and 900 m/z (data not shown).
However, the data from the analysis
with the Agilent 2100 bioanalyzer
already showed that only a
small peak of the expected 30 kDa protein is visible in the
electropherogram (figure 2A).
Instead, the analysis revealed
two main peaks at 65.9 kDa and
131.9 kDa.
|
|
Conclusion
|
The presented data showed that
there is a good correlation
between the results determined
with the Agilent 2100 bioanalyzer
and MS-analysis. Since the Agilent
2100 bioanalyzer provides a fast,
standardized method for protein
analysis with automated and
detailed data analysis, it is an ideal
tool for quality control prior to MS
analysis. In this case, the LC/MS
method lasted approximately 20
minutes and additional 10 minutes
are needed for column equilibration and sample preparation.By
comparison, the Agilent 2100 bioanalyzer
can analyze 10 samples
within 45 minutes including sample
preparation. Depending on the
kit, proteins from 5-200 kDa can
be analyzed with a resolution of
10 %, or better, throughout the size
range3, 4. However, sizing accuracy
is strongly dependent on the protein
characteristics, which is also
the case for other methods such
as traditional gel-electrophoreses
(SDS-PAGE) or size exclusion
chromatography. Sizing reproducibility
is in the range of 0.5–5 %.
In contrast, excellent mass accuracy
of 0.01 % and very high sensitivity
can be obtained by mass spectrometry
if the protein sample fulfills
the purity criteria needed for
the analysis. Comparing the sizing
results from the Agilent 2100 bioanalyzer
with those obtained by
MS, the absolute error of size
determination with the Agilent
2100 bioanalyzer ranges from
6–14 % for the proteins analyzed in
this study. With the help of the
Agilent 2100 bioanalyzer “dirty”
samples can be identified and only
“clean” samples with the right
concentration will be subjected to
MS analysis. This pre-screening
reduces costs and significantly
saves time. Furthermore, the
Agilent 2100 bioanalyzer can
reveal additional information, such
as concentration determination or
the formation of multimers, which
was not possible with the LC/MS
method described.
|