16 Current Trends in Mass Spectrometry
November 2006
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New Sample Fractionation
Strategies for Proteomic
Analyses by LC–MS
Mass spectrometry has long been a preferred tool for protein identification and biomarker discovery, but preparation of biological samples remains a challenge. Hindrances
include the wide range of protein concentrations, sample complexity, and loss or alteration of important proteins due to sample handling. This article describes recent developments in sample fractionation technologies that are overcoming these challenges in interesting ways and are enabling in-depth proteomic studies that were not possible in the
past.
Jerome Bailey, Peter Mrozinski, Tobias Preckel, Christine Miller, James Martosella, and
Robert Kincaid
P
roteomic analyses based upon liquid chromatography–mass spectrometry (LC–MS) are quite powerful,
but when protein digests are too complex, lowerconcentration peptides often escape detection. This is where
sample preparation plays a key role. For almost any proteomic
analysis today, robust and reproducible fractionation techniques are the key to ensuring that MS-MS can identify the
maximum number of proteins. At the same time, to avoid inadvertent loss or alteration of critical proteins, it is important
that fractionation steps minimize the number of sample
manipulations.
Successful fractionation of biological samples is crucial for
identification of diagnostic markers and therapeutic targets.
Some biological samples are excellent candidates for biomarker
study, but researchers have not yet fully explored them because of difficulties with sample preparation. Examples include plasma and serum analyses, in which highly abundant
proteins can mask the low-abundance proteins of interest,
and membrane-protein analysis, in which hydrophobicity
contributes to losses during sample handling. With new developments in sample fractionation techniques, these previously inaccessible samples are becoming accessible.
The Challenge of Plasma
Because plasma comes into contact with almost all of the tissues in the body and is easily obtainable, it is an important
source of new protein biomarkers for diagnostics and drug
discovery and development. However, plasma is a very complex sample that presents unique challenges, including the
fact that proteins are present over a wide concentration range
— from micrograms or milligrams per milliliter for the most
abundant proteins down to nanograms or picograms per milliliter for the less abundant species. Seven of the most abundant proteins in plasma — albumin, IgG, transferrin, haptoglobin, IgA, anti-trypsin, and fibrinogen — make up about
90% of the total protein mass. These proteins must be removed to enable detection of low-concentration biomarkers
by LC–MS.
Another challenge with plasma is the complexity of the
sample that remains after removal of the most abundant proteins. Sample complexity is an obstacle that is common to
many proteomic samples. The interesting proteins must be
fractionated to produce simpler mixtures with fewer coeluted
species. Only then is it possible to maximize the number of
proteins that can be identified in the search for important
protein biomarkers.
Reproducible Removal of High-Abundance Proteins
Multiple affinity removal (immunodepletion) is an excellent
method for eliminating highly abundant proteins. With this
technique, the sample is passed through a column or other
medium, where antibody–antigen interactions remove tar-
18 Current Trends in Mass Spectrometry
Normalized absorbance (mAU)
2500
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Bound fraction
Run 1
Run 40
Run 80
Run 120
Run 160
Run 200
2000
Flow-through fraction
1500
1000
500
0
2.5
5.0
7.5
10.0
12.5
Time (min)
15.0
17.5
20.0
Figure 1: Overlay of chromatograms from automated runs 1, 40, 80, 120, 160, and 200 from an Agilent Human Plasma 7 Multiple Affinity Removal column.
geted proteins. Immunodepletion requires optimized buffers that minimize
binding of nontargeted proteins, and
that ensure reproducibility and long column life. The method requires one
buffer to load, wash, and regenerate the
column, and a second buffer to elute the
bound high-abundance proteins.
Several factors contribute to successful immunodepletion. First and most
obvious, the process must be very selective. In other words, it must remove a
very high percentage of the undesired
proteins while allowing the other proteins to pass through. The removal of
abundant proteins can be optimized by
using antibodies that are specific to both
the protein and the species of interest,
and by mixing them in the proper (biological) ratios. Well-designed columns
can remove at least 98% of seven targeted high-abundance proteins, as
demonstrated by enzyme-linked immunosorbent assays (ELISA).
To maintain high recoveries of biomarkers, it is equally important to minimize the nonspecific binding of interesting low-abundance proteins to the
antibodies. The immunodepletion
process also must circumvent a tricky
problem — the association of many biomarkers with sticky carrier proteins such
as albumin. To avoid removing these
biomarkers along with the albumin, it
is important that buffers minimize protein–protein interactions.
To enable detection of low-abundance proteins by LC–MS, it is very important for the affinity removal column
to have sufficient sample capacity. Currently, commercially available 100 mm
3 4.6 mm columns for human fluids
allow loading of 70 µL of plasma, while
100 mm 3 10 mm columns allow loading of 300 µL of plasma. This high loadability permits LC–MS-MS detection
of biomarkers in the nanogram-permilliliter range.
Finally, the multiple affinity removal
process should be easily automated, fast,
and reproducible. Figure 1 shows an overlay of six chromatograms obtained during the automated removal of seven highly
abundant proteins from 200 human
plasma samples. A conventional LC system was used to automate the sample injection, as well as the introduction of the
two buffers that the immunodepletion
process requires. The figure shows reproducible results for over 200 injections. In
addition, one-dimensional gel patterns of
the flow-through fractions from multiple
runs (data not shown) indicated consistency and robustness of the method.
New Methods for Protein-Level
Fractionation
To achieve the greatest number of protein
identifications with LC–MS analyses, samples typically must be fractionated in multiple dimensions. Fractionation can occur
either before or after protein digestion. At
the peptide level, researchers often successfully reduce sample complexity via a combination of strong cation exchange and reversed-phase LC. Fractionation can occur
instead (or in addition) at the protein level.
Macroporous reversed-phase C18 (mRPC18, Agilent Technologies) columns constitute one relatively new way to achieve
reproducible, high-resolution separations
of proteins with high recoveries.
OFFGEL electrophoresis (pI-based
fractionation system developed jointly by
Agilent and DiagnoSwiss S.A., Monthey,
Switzerland) is another new technique for
protein fractionation (1,2). It separates either proteins or peptides by isoelectric
point (pI), with sample recovery in the
solution phase. The solution-phase, pIbased fractionation system has the advantage of providing the protein or peptide
pI, which scientists can then use to validate the results of protein database
searches that are based upon MS-MS data.
Both macroporous reversed-phase C18
columns and the solution-phase, pI-based
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Current Trends in Mass Spectrometry 19
100
Control serum
80
Absorbance (mAU)
Cortisol-deficient serum
Serum with elevated rheumatoid factor
60
40
20
0
10
15
20
25
30
35
40
Time (min)
Figure 2: Chromatograms from separation of immunodepleted serum on an Agilent mRP-C18 column.
fractionation system allow one to load
sufficient sample so that low-level proteins can be detected in subsequent
LC–MS analyses. These techniques are
discussed in detail below.
The process is faster than with spin concentrators and avoids the molecular
weight cutoff that typically is associated
with that technique.
Columns for High Protein
Recoveries
Immunodepletion in Combination
with Macroporous ReversedPhase C18 Fractionation
The use of macroporous reversed-phase
C18 columns is a relatively new method
for protein fractionation. These columns
exhibit excellent resolution, loadability,
and reproducibility. Conventional reversed-phase LC columns often lack the
resolution needed for complex samples
like cell lysates, and protein recoveries
are typically only 30–80%. The macroporous columns provide much better
resolution. They also dramatically improve recoveries because a proprietary
surface treatment, in combination with
the large-pore bonded-silica material,
prevents irreversible protein adsorption.
Under optimized conditions, protein recoveries are typically greater than 95%
and have been measured at 98% for immunodepleted serum (3). High recoveries eliminate sample cross-contamination and allow direct sample
comparison, as shown in Figure 2.
In addition, macroporous reversedphase columns are ideal for desalting
and concentrating protein samples (4).
For biomarker discovery in human
plasma, it is very powerful to combine
multiple affinity removal of highly abundant proteins (discussed earlier) with
macroporous reversed-phase fractionation of the remaining proteins. The
transition between the two techniques
is very easy; after denaturation with urea,
researchers apply the immunodepleted
plasma directly onto the macroporous
reversed-phase C18 column (5).
A recent study took advantage of the
combination of immunodepletion and
macroporous reversed-phase C18 fractionation (6). After affinity removal,
the researchers concentrated, desalted,
and fractionated the plasma with the
macroporous reversed-phase C18 column at 80 °C. The optimized LC conditions provided high protein recoveries, enhanced peak resolution, and
reproducible fractionation.
After macroporous reversed-phase fractionation, the researchers digested the proteins in solution with trypsin. They ana-
lyzed the peptides by LC–MS-MS using a
polymeric-based microfluidic device in
combination with an ion trap. The overall workflow minimized sample manipulations; for example, it was not necessary to perform dialysis, buffer exchange,
or precipitation. The combination of
affinity removal with high-capacity
macroporous reversed-phase C18 fractionation and subsequent peptide analysis allowed the identification of hundreds
of low-abundance plasma proteins.
Fractionation of Difficult
Membrane Proteins
The macroporous reversed-phase C18
columns have proven extremely useful for
studies of membrane proteins. Although
membrane proteins are an excellent source
of potential biomarkers, they have received
less study because their hydrophobicity
poses recovery challenges. On both gels
and most reversed-phase LC columns, they
resolve poorly and are difficult to recover.
Standard C18 reversed-phase columns irreversibly bind hydrophobic proteins,
causing shifts in retention time and irreproducible peak areas. The latest well-engineered macroporous reversed-phase
columns overcome these problems, making the macroporous reversed-phase C18
columns excellent tools for biomarker discovery with these difficult samples (7).
20 Current Trends in Mass Spectrometry
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hv
Starts with micro-wells filled
with sample diluted in buffer
Electrode
IPG gel
pH gradient
hv
Electrode
pH = pl
Protein A
pH = pl
Protein B
IPG gel
pH gradient
using an optimized gradient on a 50 mm
3 4.6 mm macroporous reversed-phase
C18 column. After combining, 17 fractions underwent in-solution digestion.
The peptides in the digests were fractionated with 11 salt steps on a capillary
strong cation exchange column, and the
resulting fractions were analyzed using the
Agilent HPLC-Chip/MS system in combination with an ion trap. The MS-MS
spectra were searched against the SwissProt database (Swiss Institute of Bioinformatics, Geneva, Switzerland) using the
Spectrum Mill MS Proteomics Workbench
software (Agilent Technologies), and results were validated using conservative criteria. The result was identification of an
unprecedented number of proteins.A total
of 954 proteins were identified, including
470 membrane proteins, of which 337
were integral membrane proteins.
A number of factors enabled the identification of such a large number of membrane proteins in this difficult sample. The
unique surface treatment of the macroporous reversed-phase C18 column allowed very high protein recoveries. The
column, in combination with the optimized gradient and elevated temperature,
enabled excellent separation. Third, the
high loadability of the macroporous reversed-phase C18 column, combined with
the sensitivity of the peptide analysis system, allowed detection of numerous lowlevel peptides.
hv
pI-Based Fractionation for Sample
Recovery in the Liquid Phase
Electrode
pH = pl
Protein A
pH = pl
Protein B
IPG gel
pH gradient
Figure 3: Schematic of solution-phase, pI-based protein and peptide fractionation.
A recent study showed the advantage
of a macroporous reversed-phase column
for fractionation of membrane proteins
from HeLa cells (8). The workflow incor-
porated high-resolution fractionation steps
but required a minimum of sample manipulations. The membrane proteins were
solubilized and then were fractionated
The solution-phase, pI-based fractionation system is a relatively new separation
sytem that fractionates either proteins or
peptides by pI. It achieves the same resolution as immobilized pH gradient isoelectric focusing (IPG IEF), but it removes
the requirement for tedious post-IEF
sample handling. After IEF, the IPG gel
strips must be cut into sections and the
peptides extracted and purified (9,10).
With solution-phase, pI-based fractionation, the proteins or peptides end up in
the liquid phase, which makes this technique directly compatible with LC–MS.
There is no need for additional sample
extraction or purification — steps that
could lead to sample losses.
Figure 3 shows the principle of solution-phase, pI-based fractionation. The
22 Current Trends in Mass Spectrometry
3
Reversed-phase
retention time (min)
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November 2006
pl
10
10
20
30
Figure 4: Visualization of color-coded protein concentrations from a HeLa cell lysate fractionated by a combination of pI (Agilent 3100 OFFGEL
Fractionator, 24 fractions, pH 3–10) and reversed-phase LC (Agilent mRP-C18 column, 1536 fractions).
sample is diluted in focusing buffer and
is loaded into a series of isolated wells.
The well forms a liquid-tight seal against
a rehydrated conventional IPG gel strip.
Upon application of an electric field, the
proteins or peptides are forced to migrate along the electric field lines through
the liquid-IPG gel strip system until they
reach the well where the pH of the gel is
equal to the pI of the peptide or protein.
As the electric field extends into the wells,
the majority of proteins or peptides are
conveniently recovered in the liquid
phase, which contains no detergents, surfactants, or other species that interfere
with LC–MS.
One very useful aspect of solutionphase, pI-based fractionation is that it
can be applied twice in the same experiment — it can fractionate at both the
protein level and the peptide level. Another convenient feature is that it requires
a minimum of sample handling. Fractionated proteins can proceed directly to
the digestion step, and fractionated peptides can proceed directly to LC–MS.
A unique advantage of solution-phase,
pI-based fractionation is that researchers
can adjust the separation by using IPG
gels with various pI ranges. By using IPG
gels with sufficient resolution (less than
0.2 pI units), it is possible to resolve isoforms of a protein (such as charged posttranslational modifications) and to recover them in individual solutions.
Likewise, it is possible at the peptide level
to identify amino acid mutations (11).
Comparison of Solution-Phase,
pI-Based Fractionation with
Strong Cation Exchange
A recent experiment that compared fractionation of peptides with either strong
cation exchange or solution-phase, pIbased fractionation showed the advantage of solution-phase, pI-based fractionation for increased protein identifications.
Immunodepleted human serum was digested tryptically. The resulting peptides
were fractionated either into 24 fractions
by solution-phase, pI-based fractionation
(pH 3 to 10), or into 50 fractions by strong
cation exchange. The individual fractions
from both experiments were then analyzed using the Agilent HPLC-Chip/MS
combined with an ion trap. The separation by solution-phase, pI-based fractionation produced twice the number of protein identifications and about three times
the number of peptide identifications as
the strong cation exchange technique.
Use of pI Values from SolutionPhase, pI-Based Fractionation for
Validation of Database Search
Results
Another advantage of solution-phase, pIbased fractionation is that the experimentally determined protein or peptide pIs
can be used as an additional validation
tool when reviewing results of protein
database searches. When the software
used for a protein database search also
calculates and displays peptide pIs, it is
easy to compare the calculated pIs with
those from the solution-phase, pI-based
separation. Researchers can use the experimentally derived pIs to rule out false
positive matches, or they can validate additional matches with slightly lower scores
when the pI values correspond as well. In
a recent study, use of pI as an additional
validation criterion led to 19–22% more
peptide identifications (12).
Very High-Resolution
Fractionation for Complex
Samples
For complex samples such as cell lysates,
it is possible to separate proteins first by
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solution-phase, pI-based fractionation and
second by macroporous reversed-phase
LC. The transition between the two techniques is seamless, with no sample manipulation in between. The combination can
provide up to 1500 individual fractions,
for an extremely high-resolution separation. With a macroporous reversed-phase
LC column of smaller internal diameter,
it is possible to introduce the fractions directly into a time-of-flight MS for measurement of molecular mass. Alternatively,
with visualization software, such as the experimental software shown in Figure 4, it
is possible to compare samples and pick
selected fractions for digestion and MSMS analysis. Relative to two-dimensional
gel electrophoresis, the combination of solution-phase, pI-based fractionation and
macroporous reversed-phase LC provides
a more information-rich, faster, more reproducible, and easier-to-use alternative.
Conclusion
Successful execution of LC–MS-based proteomic studies is highly dependent upon
well-designed, seamless sample preparation schemes. For plasma and serum samples, affinity chromatography is proving
to be a robust, reproducible method for
removing highly abundant proteins that
mask low-level proteins of interest. For
many complex proteomic samples, fractionation at the protein level helps to reduce sample complexity so that it is possible to identify more proteins. While
historically, researchers have used gels to
fractionate proteins, new techniques such
as solution-phase, pI-based fractionation,
and reversed-phase separations with
macroporous LC columns provide high
loadability, resolution, and reproducibility, and are easier to automate. These new
methods also are enabling the successful
fractionation of difficult samples such as
membrane proteins, as well as opening up
rich possibilities for biomarker discovery.
References
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Josserand, H.H. Girault, and J.S. Rossier,
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Current Trends in Mass Spectrometry 23
L-S. Yang, C. Szafranski, and G. Nicol,
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Jerome Bailey is Program Manager,
Bioseparations, Agilent Technologies, Little
Falls, Delaware. Peter Mrozinski is an
Application Scientist, Bioseparations, Agilent
Technologies, Little Falls, Delaware. Tobias
Preckel is Product Manager, OFFGEL
Fractionator, Agilent Technologies,
Waldbronn, Germany. Christine Miller
is an Application Scientist, LC–MS, Agilent
Technologies, Santa Clara, California.
James Martosella is an R&D Scientist,
Bioseparations, Agilent Technologies, Little
Falls, Delaware. Robert Kincaid is a
Senior Research Scientist, Agilent
Laboratories, Santa Clara, California. n
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