Integrated capillary zone electrophoresis-electrospray ionization tandem mass spectrometry system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate

Article abstract of DOI:10.1021/ac400523x

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE-ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE-ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity.

Full text of DOI:10.1021/ac400523x

Article
pubs.acs.org/ac
Integrated Capillary Zone Electrophoresis−Electrospray Ionization
Tandem Mass Spectrometry System with an Immobilized Trypsin
Microreactor for Online Digestion and Analysis of Picogram Amounts
of RAW 264.7 Cell Lysate
Liangliang Sun, Guijie Zhu, and Norman J. Dovichi*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
S
* Supporting Information
ABSTRACT: A capillary zone electrophoresis (CZE) electrospray ionization (ESI)
tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin
microreactor. The system was evaluated and then applied for online digestion and analysis
of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer
containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for
digestion, followed by CZE separation and MS/MS identification. The organic solvent
(50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein
sample preparation protocol. Neither protein reduction nor alkylation steps were
employed, which minimized sample loss and contamination. The integrated CZE−ESI-
MS/MS system generated confident identification of bovine serum albumin (BSA) with
19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded.
When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the
analysis of a standard protein mixture, the integrated system produced efficient protein
digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole
amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 1 and 7
2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading
corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE−ESI-
MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity.
We recently reported a protein sample preparation method
ottom-up proteomics is routinely used for characterization
Bof complex samples. As an extreme example of the depth based on methanol denaturation and immobilized trypsin
digestion; we employed that system for analysis of low-
nanogram amounts of RAW 264.7 cell lysate.11 In that
protocol, proteins were first denatured with 50% (v/v)
methanol. The denatured proteins were reduced in a buffer
containing 50% (v/v) methanol and then alkylated. Finally, the
treated proteins were digested using trypsin immobilized on
magnetic microspheres. The whole process was performed at
room temperature in 200 μL Eppendorf tubes. After ultra-
performance liquid chromatography (UPLC)−electrospray
ionization tandem mass spectrometry (ESI-MS/MS) analysis,
of sequencing, the identification of more than 10 000 proteins
from mammalian cell lysates has been reported.1 However,
most bottom-up sequencing protocols require microgram to
milligram amounts of sample, which limits its applications for
material-limited biological samples, such as circulating tumor
cells,2 where the sample size is only a few nanograms or less. In
order to improve the performance of bottom-up proteomics for
analysis of mass-limited biological samples, improvements in
both instrumentation and sample preparation are required for
highly efficient enzymatic digestion of trace amounts of
proteins, high-capacity peptide separation, and sensitive peptide
detection.
Several groups have developed efficient enzymatic digestion
of trace amounts of proteins from hundreds of cells,3−6 where
the initial protein sample amounts were on the order of 100 ng.
These protocols employ free trypsin to digest proteins. It is
likely that the system performance is limited by the
combination of low protein and trypsin concentrations.
Immobilized trypsin can generate better digestion performance
for proteins with very low concentration compared with free
trypsin,7,8 due to the high trypsin concentration and reduced
autodigestion of immobilized trypsin.9,10
2
1 and 23 2 proteins could be identified from only 6 and
30 ng of initial cell lysate, respectively. An additional 16
proteins could be detected from the 6 ng of cell lysate using an
accurate mass and time tag method. The results suggest that
organic solvent and immobilized trypsin based protocol may be
useful for ultratrace-level protein sample preparation.
As an example of high-capacity peptide separation and
sensitive peptide detection, Shen et al.12,13 coupled a highly
Received: February 18, 2013
Accepted: March 19, 2013
Published: March 19, 2013
© 2013 American Chemical Society
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efficient (peak capacities of ∼103) 15 μm i.d. packed LC
capillary column to a mass spectrometer for analysis of low-
nanogram amounts of proteins from complex samples.
Ultrahigh sensitivity (<75 zmol for individual proteins) was
obtained. Waanders et al.14 developed a high-sensitivity LC
system based on splitting gradient effluents into a capture
capillary and providing an inherent technical replicate for
nanogram complex sample analysis with high resolution. More
than 2400 proteins could be identified from kidney glomeruli
isolated by laser capture microdissection in a single analysis.
Capillary electrophoresis (CE)−ESI-MS/MS has attracted
attention as an alternative tool for large-scale, high-sensitivity
proteomics analysis.15−22 This group has focused on the use of
an electrokinetically pumped sheath-flow electrospray interface.
This system has generated low-attomole peptide detection limit
with an LTQ-Orbitrap Velos as detector19,20 and yielded high-
zeptomole level peptide detection limit with triple-quadrupole
mass spectrometer employing multiple reaction monitoring.21
For analysis of low-nanogram and high-picogram amounts of
digest, CE−MS/MS generated more peptide and protein
identifications than LC−MS/MS.17,20 For mid-nanogram
samples, CE−MS/MS and LC−MS/MS produced comple-
mentary peptide identifications.17,22 In addition, CE−MS also
produced much higher peptide intensities than LC−MS.16
Online integration of an immobilized enzyme microreactor
with a CE−ESI-MS/MS system should be a facile method for
analysis of trace-level protein samples. Several research groups
have developed integrated microreactor−CE−MS systems for
online protein digestion, peptide separation, and detec-
tion.23−27 However, the sensitivity of those systems was limited
by the immobilized enzyme microreactor, the CE−MS
interface, and the mass spectrometer. In those reports, only
one or two small standard proteins were analyzed. In addition,
denaturation, reduction, and alkylation steps were performed
offline, followed by online protein digestion, separation, and
detection, which reduced the throughput and increased the
potential for protein sample loss and contamination.
Finally, an electrokinetically pumped sheath-flow electro-
spray interface18 was used to couple the CZE to an LTQ-
Orbitrap Velos mass spectrometer. The combination results in
highly sensitive peptide detection.
This integrated CZE−ESI-MS/MS system confidently
identified 2 1 protein groups from 300 pg of RAW 264.7
cell lysate; 7
2 protein groups were confidently identified
from 3 ng of the lysate.
EXPERIMENTAL SECTION
■
Materials and Chemicals. All chemicals were from Sigma-
Aldrich (St. Louis, MO, U.S.A.), unless specified. Poly(ethylene
glycol) (PEG, MW 10 000) was ordered from Polysciences, Inc.
(Warrington, PA, U.S.A.). Dimethyl sulfoxide (DMSO) was
ordered from Electron Microscopy Sciences (Hatfield, PA,
U.S.A.). Acetonitrile was purchased from Fisher Scientific
(Pittsburgh, PA, U.S.A.). Methanol was purchased from
Honeywell Burdick & Jackson (Wicklow, IE). Water was
deionized by a Nano Pure system from Thermo scientific
(Marietta, OH, U.S.A.). Fused capillaries (50 μm i.d. × 150 μm
o.d.) were purchased from Polymicro Technologies (Phoenix,
AZ, U.S.A.).
Dulbecco’s modified Eagle’s medium (DMEM) with L-
glutamine and fetal bovine serum (FBS) was purchased from
ATCC (Manassas, VA, U.S.A.). Mammalian cell−PE LB buffer
for cell lysis was purchased from G-Biosciences (St. Louis, MO,
U.S.A.). Complete, miniprotease inhibitor cocktail (provided in
EASYpacks) was purchased from Roche (Indianapolis, IN,
U.S.A.).
Preparation of the Immobilized Trypsin Microreactor.
A diagram outlining the preparation of the immobilized trypsin
microreactor is shown in Figure 1A. A fused capillary (50 μm
i.d. × 150 μm o.d., 50 cm) was successively washed with
methanol, sodium hydroxide, water, hydrochloric acid, water,
and methanol. The capillary was then dried with nitrogen at
As we show in this paper, integration of organic solvent
denaturation and immobilized trypsin digestion in a nanoliter
volume microreactor further improves sample preparation
efficiency. In this work, we integrate an immobilized trypsin
microreactor with a CZE−ESI-MS/MS system for online
digestion and analysis of picogram amounts of a RAW 264.7
cell lysate.
This system has a number of innovations.
First, the sample was prepared in a 50% acetonitrile (ACN)
buffer. Organic solvents are used to unfold the protein
structure28,29 and assist in tryptic protein digestion.30−32
Second, no protein reduction and alkylation steps were
performed. Eliminating these steps reduced potential sample
loss and contamination. Eliminating the steps also improved
the throughput.
Third, the immobilized microreactor was based on an
acrylamide-based monolithic. This material is hydrophilic and
tends to reduce nonspecific adsorption of peptides/proteins.
Fourth, the total volume of the prepared trypsin microreactor
was reduced to less than 40 nL. This small volume aids in
handling small volume protein samples.
Fifth, the 50% (v/v) ACN sample preparation buffer has
lower conductivity than the separation buffer of CZE. This low-
conductivity buffer results in sample stacking at the beginning
of CZE separation, which improved the CZE separation and
MS detection.
Figure 1. Procedure for preparation of the immobilized trypsin
microreactor (A) and schematic diagram of the integrated CZE−ESI-
MS/MS system (B).
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room temperature. Subsequently, an ∼10 cm portion of the
capillary was filled with 50% (v/v) 3-(trimethoxysilyl)-
propylmethacrylate in methanol and reacted at room temper-
ature for 24 h. After that, the capillary was washed with
methanol to remove the unreacted components and dried again
with nitrogen. The treated capillary was stored at room
temperature for use.
and 0.1 mg/mL protein samples were analyzed by the
integrated CZE−ESI-MS/MS system in triplicate.
Online Protein Digestion, Peptide Separation, and
Identification with the Integrated CZE−ESI-MS/MS
System. The integrated CZE−ESI-MS/MS system included
a separation capillary (50 μm i.d. × 150 μm o.d.) with an
immobilized trypsin microreactor at the injection end, an
electrokinetically driven sheath-flow electrospray interface,18
two Spellman CZE 1000R power supplies, and an LTQ-
Orbitrap Velos mass spectrometer (Thermo Fisher Scientific),
Figure 1B.
The details of the polymerization mixture and process were
similar to ref 33 with some modifications. A mixture of 20 mg
of acrylamide, 30 mg of N,N′-methylenebisacrylamide, and 30
mg of PEG was dissolved in 1 mL of 0.2 M sodium
bicarbonate/0.5 M sodium chloride (pH ∼ 8.0) buffer. The
mixture was vortexed for 30 s and then heated at ∼50 °C for 15
min to completely dissolve the monomers. Then, 2 μL of 20%
(v/v) TEMED (N,N,N′,N′-tetramethylethylene diamine) was
added into 0.5 mL of the prepared mixture, and the mixture
was degassed for 15 min using nitrogen. Subsequently, 7 μL of
N-acryloxysuccinimide (NAS) (140 mg/mL, dissolved in
DMSO) was added on top of the solution and degassed for
1 min. After that, 2 μL of 20% (w/v) ammonium persulfate
(APS) was added into the middle part of the solution to initiate
polymerization. After vortexing for several seconds, 190 μL of
the solution was mixed quickly with 10 μL of freshly prepared
trypsin (20 mg/mL in a buffer containing 0.5 M benzamidine,
pH 8.0). Finally, the vinylized part of the treated capillary (∼10
cm) was filled with the mixture through capillary action and
reacted at room temperature for 30 min.
After the reaction, ∼7 cm of microreactor was discarded. The
remaining portion of the capillary including a few centimeters
of the microreactor was successively washed with deionized
water, glycine (10 mg/mL in phosphate buffer, pH 8.0), and 5
mM NH4HCO3 (pH ∼ 8.0) for 30 min. The capillary was
stored at 4 °C before use.
Sample Preparation. Insulin chain b oxidized was
dissolved in 5 mM NH4HCO3 for analysis with the integrated
CZE−ESI-MS/MS system. Several standard protein standards
were dissolved in 50% (v/v) ACN and 5 mM NH4HCO3 for
analysis with the integrated CZE−ESI-MS/MS system. The
first sample was bovine serum albumin (BSA). The second was
a three-protein mixture of BSA, myoglobin (myo), and
cytochrome c (cyt c). The third was a seven-protein mixture
(BSA, myo, cyt c, α-casein, β-casein, β-lactoglobulin, and insulin
chain b oxidized).
RAW 264.7 cells were cultured in a T25 flask at 37 °C and
5% CO2 in DMEM with L-glutamine and 10% FBS. The cells
were lysed with 1 mL of mammalian cell−PE LB buffer (pH
7.5) supplemented with complete protease inhibitor for 30 min
on ice. The cell lysate was centrifuged at 18 000g for 15 min,
and the supernatant was collected for measurement of protein
concentration with the BCA method. After that, a 300 μL
aliquot of the cell lysate was precipitated with 1.2 mL of cold
acetone at −20 °C for 24 h, followed by centrifugation at 18
000g for 15 min. The protein pellet was washed with cold
acetone again and dried at room temperature. A 60 μg aliquot
of the proteins was dissolved in 30 μL of 0.5% (w/v) sodium
deoxycholate (SDC) and 10 mM NH4HCO3 buffer (pH 8.0).
After centrifugation, a 15 μL aliquot of the protein solution was
mixed with 15 μL of ACN, which resulted in an ∼1 mg/mL
protein solution dissolved in 50% ACN, 0.25% SDC, and 5 mM
NH4HCO3 buffer (pH 8.0). In addition, 5 μL of the 1 mg/mL
protein solution was further diluted to 0.1 mg/mL with 50%
ACN and 5 mM NH4HCO3 buffer (pH 8.0). The 1 mg/mL
The protocol had three steps. Protein samples were injected
into the microreactor end of the separation capillary by air
pressure. Proteins were then digested by the microreactor at
room temperature. Finally, the digests were separated with
CZE and analyzed by the LTQ-Orbitrap Velos.
For oxidized insulin chain b analysis, a 34 cm long separation
capillary with a 1.5 cm long microreactor was used, and the
digestion time was 1 min. For analysis of BSA and the standard
protein mixtures, a 34 cm long separation capillary with a 2 cm
microreactor was used, and the digestion time was 2 min. For
analysis of the RAW 264.7 cell lysate, a 34 cm separation
capillary with 2 cm microreactor and 3 min digestion time was
used. In all cases, the separation buffer was 5 mM NH4HCO3
(pH 8.0), and the sheath-flow buffer was 50% (v/v) methanol
and 0.05% (v/v) FA. For standard proteins analysis, 19.5 kV
was applied at the injection end of the capillary for separation
and 1.5 kV was applied for electrospray. For RAW 264.7 cell
lysate analysis, 15 kV was applied at the injection end of the
capillary for separation, and 1 kV was applied for electrospray.
The size of the tip for electrospray was ∼8 μm.
For LTQ-Orbitrap Velos analysis, the ion transfer tube
temperature was held at 300 °C. The mass spectrometer was
programmed in data-dependent mode. Full MS scans were
acquired in the Orbitrap mass analyzer over m/z 395−1900
range with resolution 60 000 (m/z 400). Ten most intense
peaks (for standard protein analysis) and 20 most intense peaks
(for cell lysate analysis) with charge state ≥2 were selected for
sequencing and fragmented in the ion trap with a normalized
collision energy of 35%, activation q = 0.25, activation time of
20 ms, and one microscan. For standard protein analysis, peaks
selected for fragmentation more than twice within 60 s were
excluded from selection for 60 s. For the standard protein
mixtures and cell lysate analysis, peaks selected for
fragmentation more than once within 30 s were excluded
from selection for 30 s.
Data Analysis. Database searching of raw files was
performed in Proteome Discoverer 1.3 with MASCOT 2.2.4.
For standard proteins from bovine, the insulin chain b sequence
was added into ipi.bovin.v3.68.fasta to form the database. For
myoglobin, equine.fasta was used for database searching. For
RAW 264.7 cell lysate, Swiss-Prot database with taxonomy as
Mus. (16 252 sequences) was used. The database searching
parameters included up to four missed cleavages allowed for full
tryptic digestion, precursor mass tolerance 20 ppm, and
fragment mass tolerance 1.0 Da. For analysis of standard
proteins from bovine, oxidation of cysteine (+47.9847 Da) was
set as dynamic modification. Database searching against the
corresponding decoy database was also performed to evaluate
the false discovery rate (FDR) of peptide identification.
For standard protein analysis, MASCOT significance thresh-
old 0.05 (95% confidence) was used to filter the peptide
identification. For RAW 264.7 cell lysate data, MASCOT
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significance threshold of 0.01 (99% confidence) was applied to
filter the peptide identification. In addition, manual evaluation
was also used for the cell lysate peptide identifications. At the
protein level, protein grouping was enabled, and each protein
group had at least one unique peptide. Leucine and isoleucine
were considered as equal, and the strict maximum parsimony
principle was applied.
resulting peaks are sharper and of higher amplitude, which
assists detection. This protocol also has potential limitations for
analysis of highly structured proteins with many disulfide bonds
due to the elimination of reduction and alkylation steps. The
50% (v/v) ACN helps to denature non-cross-linked proteins,
but the denaturation performance will be limited for highly
structured proteins with many disulfide bonds.
Evaluation of the Integrated CZE−ESI-MS/MS System.
To evaluate the system for protein analysis, we used two model
proteins, insulin chain b oxidized and BSA. Insulin chain b
o x i d i z e d ( 3 0 a m i n e a c i d s w i t h s e q u e n c e
FVNQHLCGSHLVEALYLVCGERGFFYTPKA and 3494 Da
molecular weight) was first used to evaluate the system.
Because it is a small protein, the protein was directly dissolved
in the separation buffer (5 mM NH4HCO3) for analysis. After
online digestion, separation, and identification with the
integrated CZE−ESI-MS/MS system, 100% sequence coverage
was obtained with a 7 fmol sample. Two identified peptides,
GFFYTPKA and FVNQHLcGSHLVEALYLVcGER, were
extracted from the raw files of triplicate runs with mass
tolerance as 5 ppm, Figure 2. The two peptides were well-
RESULTS AND DISCUSSION
■
Integrated CZE−ESI-MS/MS System. The system was
composed of a separation capillary with an immobilized trypsin
microreactor, an electrokinetically driven sheath-flow CE−MS
interface,18 two high-voltage (HV) supplies, and an LTQ-
Orbitrap Velos mass spectrometer, Figure 1B.
The microreactor was synthesized in situ at the injection end
of the separation capillary. The integrated system does not
require an additional interface to connect the microreactor and
separation capillary. The trypsin microreactor was prepared
from an acrylamide monolithic due to its good permeability,
fast mass transfer, high stability, and easy modification.34 The
hydrophilic nature of the acrylamide microreactor reduced
nonspecific adsorption of proteins/peptides. The total volume
of the trypsin microreactor used in this work was less than 40
nL, which was efficient for trace protein sample digestion. In
order to keep high electroosmotic flow (EOF) in the separation
capillary, only a short portion of the separation capillary was
vinylized for preparation of the microreactor, Figure 1A. Excess
succinimide groups on the monolithic material were blocked
with glycine. Glycine’s amine group reacted with the
succinimide groups, leaving the carboxyl groups of glycine
exposed to the separation buffer, which was useful for
improving the EOF in the separation capillary.
An electrokinetically driven sheath-flow interface, which was
developed by our group,18 was used due to its very high
sensitivity for peptide detection.19,20 Two power supplies were
used in the experiment. HV supply I was applied at the
injection end of the capillary. HV supply II was applied to the
interface to produce electrospray. The voltage difference
between the power supplies drove the capillary electrophoretic
separation, while the potential provided by the second power
supply drove the electrospray interface. An LTQ-Orbitrap
Velos mass spectrometer (Thermo Fisher Scientific) was used
for peptide identification.
In conventional studies of microgram or larger sample
amounts, proteins are denatured, reduced, and alkylated,
followed by trypsin digestion. However, these sample pretreat-
ment steps can lead to sample losses that are significant when
dealing with low-nanogram or picogram protein homogenates.
We applied a simplified sample preparation protocol when
analyzing samples smaller than a few nanograms. We used a
buffer containing 50% (v/v) ACN and 5 mM NH4HCO3 to
dissolve the proteins, which was then directly loaded into the
integrated CZE−ESI-MS/MS system for protein digestion in
the trypsin microreactor, peptide separation in the capillary,
and identification by tandem mass spectrometry.
Figure 2. Extracted ion electropherogram of two peptides of insulin
chain b oxidized after analysis by the integrated CZE−ESI-MS/MS
system in triplicate with 7 fmol injection amount per run. Five-point
Gaussian smoothing was applied to the spectra, and the peptides were
extracted with mass tolerance of 5 ppm.
This protocol has at least two advantages. First, the one-step
sample preparation reduces the sample loss and also avoids the
contamination from the reducing and alkylating reagents.
Second, the sample buffer (50% (v/v) ACN and 5 mM
NH4HCO3) has lower conductivity than the separation buffer
(5 mM NH4HCO3). This difference in buffer conductivity
results in sample stacking at the beginning of the
separation,35,36 which improves separation efficiency. The
separated, and their migration time was reasonably reproducible
(relative standard derivation (RSD) less than 5%) in triplicate
runs. In addition, RSD of the peptide intensity in triplicate runs
was less than 30% for 7 fmol loading amount and was about
20% for 70 fmol loading amount. These results indicate that the
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integrated CZE−ESI-MS/MS system is efficient and reprodu-
cible for analysis of low-femtomole amounts of proteins.
We further determined the relationship between the injection
amount (A) of insulin chain b oxidized and the peptide
intensity (I), Figure 3A. When the sample injection amount
chain b oxidized ranged from 0.01 to 0.1 mg/mL, resulting in
7−70 fmol injection amounts. The digestion time was fixed, so
the generated peptide amount directly related to the trypsin
digestion rate. The peptide intensity from the mass
spectrometer is a good parameter to estimate the peptide
amount. Protease digestion can be described by the Michaelis−
Menten kinetics. We linearlized the classical Michaelis−Menten
equation, which was expressed in terms of peptide intensity (I),
the Michaelis constant (Km), and loaded amount (A) (eq 1):
Km
1
I
1
Imax
=
+
Imax
A
(1)
A plot of 1/I versus 1/A followed this linearlized equation (R =
0.99), Figure 3B.
To check the system for the analysis of larger and more
complex proteins, BSA with 66 kDa molecular weight and 17
disulfide bonds was also analyzed by the system. BSA was
dissolved in 50% (v/v) ACN and 5 mM NH4HCO3 (pH 8.0)
and directly injected into the microreactor end of the
separation capillary and digested at room temperature for 2
min without reduction and alkylation, followed by CZE−ESI-
MS/MS analysis in duplicate. When about 4 fmol BSA was
injected and analyzed by the system, two peptides and 4.6%
sequence coverage were consistently produced, which suggest
that the integrated system is also efficient for the analysis of
proteins with large molecular weight and many disulfide bonds
without reduction and alkylation. When the injection amount
was increased to 20 fmol, the peptide number and sequence
coverage dramatically increased to an average of 14 and 19%,
respectively.
No peptides were detected from the tandem spectra when 1
fmol of BSA was injected. However, we generated an extracted
ion electropherogram corresponding to a BSA peptide
(KVPQVSTPTLVEVSR, m/z 820.4744, +2) with mass
tolerance of 2 ppm. This extracted ion electropherogram had
an average intensity of 1.6 × 104, which is consistent with the
average intensity produced by 4 fmol sample (7.55 × 104). In
addition, the charge state of the peptide from 1 fmol was the
same as that from 4 fmol, and the migration times of the
peptide between the 1 and 4 fmol results were reasonably
reproducible. These results indicate that BSA could be detected
from injection of only 1 fmol.
Application for Analysis of Standard Protein Mix-
tures. A three-protein mixture (BSA, 0.12 mg/mL, cytochrome
c, 0.01 mg/mL, and myoglobin, 0.01 mg/mL) dissolved in 50%
(v/v) ACN and 5 mM NH4HCO3 (pH 8.0) was analyzed by
the integrated CZE−ESI-MS/MS system in triplicate. Injection
amounts were 7 fmol (BSA), 3 fmol (cytochrome c), and 2
fmol (myoglobin) for each run. The three proteins could be
confidently and reproducibly identified by the system with the
number of peptide identifications (IDs) as 10 2 (cytochrome
c), 11 1 (myoglobin), and 1 1 (BSA), Table 1. Besides the
three proteins, transthyretin was also identified with the
Figure 3. Relationship of the injection amount of insulin chain b
oxidized and the peptide intensity (GFFYTPKA and
FVNQHLcGSHLVEALYLVcGER) (A) and the relationship between
the reciprocal of the injection amount and the reciprocal of the peptide
intensity, eq 1 (B), produced by the integrated CZE−ESI-MS/MS
system in triplicate. The peptide intensity was obtained by peak
extraction with mass tolerance of 5 ppm. Gaussian smoothing with five
points was applied.
increased from 7 to 70 fmol, the peptide intensity also
increased, but the relationship is not linear. In our experiment,
the injection volume was fixed, and the concentration of insulin
Table 1. Results of the Triplicate Analysis of the Three-Protein Mixture
MW (kDa)
pI
injection amount (pg)
injection amount (fmol)
peptide IDs
sequence coverage (%)
69
74 17
cytochrome c
myoglobin
BSA
11.7
16.9
69.5
15.7
9.5
7.8
6.2
6.3
40
40
3
2
10
11
1
2
1
1
1
2
480
7
2
6
2
5
a
transthyretin
Un
Un
1
a
The injection amount of transthyretin was unknown (Un).
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number of peptide IDs as 1 1, Table 1. Transthyretin might
be an impurity from BSA, because transthyretin was also
identified from the 20 fmol BSA data with six peptides.
Interestingly, we also identified transthyretin as an impurity in
BSA using top-down analysis of intact proteins (data not
shown).
We further analyzed a seven-protein mixture (BSA, 0.11 mg/
mL, cytochrome c, 0.009 mg/mL, myoglobin, 0.009 mg/mL, α-
casein, 0.015 mg/mL, β-casein, 0.015 mg/mL, β-lactoglobulin,
0.015 mg/mL, and insulin chain b oxidized, 0.01 mg/mL)
dissolved in 50% (v/v) ACN and 5 mM NH4HCO3 (pH 8.0)
in triplicate. The injection amount was 6 fmol (BSA), 3 fmol
(cytochrome c), 2 fmol (myoglobin), 2 fmol (α-casein), 2 fmol
(β-casein), 3 fmol (β-lactoglobulin), and 12 fmol (insulin chain
b oxidized) for each run. Six proteins (all but β-lactoglobulin)
were identified from triplicate runs, and their sequence
coverage ranged from 6% to 100% and peptide IDs ranged
from 2 to 16, Table 2. Besides the identification of the six
respectively. Information of the identified protein groups is
listed in the Supporting Information. The protein groups
(except one protein group, glial fibrillary acidic protein)
obtained from the 300 pg sample were also identified from
the 3 ng sample. For glial fibrillary acidic protein, one unique
peptide was identified, and its Exp value and mass error were
1.4 × 10−6 and 1 ppm, respectively, which confirmed the
protein group identification. Most of the proteins identified
from the 300 pg and 3 ng samples were relatively highly
abundant in RAW 264.7 cell lysate according to the protein
spectral count information from the large-scale proteome data
generated previously in our group. In addition, the peptide
identifications from the 300 pg and 3 ng samples were also
manually evaluated, and the annotated tandem spectra of those
peptides are listed in the Supporting Information. We also
analyzed the molecular weight and pI of proteins generated by
triplicate analysis of the 3 ng RAW 264.7 cell lysate. The
molecular weight ranged from 5.7 to 58.8 kDa, and pI ranged
from 4.5 to 11.0, which suggest that the integrated CZE−ESI-
MS/MS system is efficient for small and large proteins, and also
acidic and basic proteins. We further checked the disulfide
bonds of the identified proteins from 3 ng RAW 264.7 cell
lysate, and the identified proteins tend to have few or no
disulfide bonds, which is expected due to the much easier
denaturation of these proteins with 50% (v/v) ACN. The result
indicates that the integrated CZE−ESI-MS/MS system might
have potential limitations for identification of highly structured
proteins with many disulfide bonds. On the other hand, the
system might be useful for selective identification of the
proteins with few disulfide bonds in complex samples.
Table 2. Results of the Triplicate Analysis of the Seven-
Protein Mixture
injection
amount
(pg)
injection
amount
(fmol)
sequence
MW
(kDa)
peptide
a
coverage
a
IDs
(%)
BSA
69.5
11.7
16.9
24.5
25.1
3.4
440
36
36
60
60
40
6
3
3
16
13
2
6
73
cytochrome c
myoglobin
α-casein
β-casein
2
96
2
20
2
4
19
insulin chain b
oxidized
12
4
100
We further extracted the electropherograms from a peptide
from macrophage migration inhibitory factor, which was
identified from both 300 pg and 3 ng samples, Figure 4. The
integrated CZE−ESI-MS/MS system yielded reasonably
reproducible migration time for the peptide for analysis of
both 300 pg and 3 ng samples. In addition, the peptide intensity
from triplicate analysis of the 3 ng cell lysate was quite
consistent (RSD = 8%), and the peptide intensity from the 300
pg cell lysate was reasonably reproducible (RSD = 30%). In
addition, when the injection amount was increased from 300 pg
to 3 ng, the average peptide intensity generated by the system
increased from 7.5 × 104 to 7.6 × 105. The reproducible
peptide migration time and peptide intensity indicate that the
integrated CZE−ESI-MS/MS system is reproducible for trace
complex cell lysate analysis. The peptide intensities generated
for 300 pg and 3 ng cell lysates reasonably reflect the difference
in sample amounts, which suggests that the system might be
useful for further quantitative protein analysis.
We also extracted two peptides of 10 kDa heat shock protein.
One peptide (VLQATVVAVGSGGK) was only identified from
0.3 ng cell lysate, S-Figure 1 in the Supporting Information, and
another peptide (VLQATVVAVGSGGKGK) was only identi-
fied from 3 ng cell lysates, S-Figure 2 in the Supporting
Information. These two peptides, extracted with mass tolerance
as 2 ppm, generated reasonable signal intensities from all the
300 pg and 3 ng cell lysate data. The migration time and
peptide intensity of these two peptides from 300 pg and 3 ng
cell lysates were also reproducible, which further confirmed the
reproducibility of the integrated CZE−ESI-MS/MS system.
The intensity of these two peptides also increased with loading
amounts from 300 pg to 3 ng.
β-lactoglobulin
18.4
15.7
18.3
60
3
Un
Un
0
1
4
0
7
b
transthyretin
Un
uncharacterized
protein
Un
27
BARX2 protein
31.0
Un
Un
1
2
a
Peptide IDs and sequence coverage were from the combined
b
triplicate runs. The injection amounts of these proteins were
unknown (Un).
proteins, three additional proteins (transthyretin, uncharac-
terized protein, and BARX2 protein) were also identified with
the number of peptide IDs as 1, 4, and 1, respectively, Table 2.
These proteins might be impurities present in the seven
standard proteins.
The protein mixtures analyzed in the experiments contained
basic and acidic proteins, large and small proteins, and
phosphorylated and unphosphorylated proteins, which suggest
that the integrated CZE−ESI-MS/MS system is of high
sensitivity and efficiency for analysis of complex mixtures of
proteins.
Application for Analysis of Picogram Range of RAW
264.7 Cell Lysate. The integrated system was used to analyze
RAW 264.7 cell lysate in triplicate. Two buffers were used to
prepare the protein samples. In one case, a sample containing
0.1 mg/mL proteins was dissolved in 50% (v/v) ACN, 0.025%
(w/v) SDC, and 5 mM NH4HCO3. In a second case, a 1 mg/
mL protein sample was dissolved in 50% (v/v) ACN, 0.25%
(w/v) SDC, and 5 mM NH4HCO3. The injection amounts per
run for the 0.1 and 1 mg/mL samples were ∼300 pg and ∼3 ng,
respectively.
After triplicate analysis, 2 1 and 7 2 protein groups were
identified from the 300 pg and 3 ng RAW 264.7 cell lysates,
One obvious limitation of our analysis is that the protein
homogenate was prepared from a large volume and only a small
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Analytical Chemistry
Article
AUTHOR INFORMATION
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. William Boggess in the Notre Dame Mass
Spectrometry and Proteomics Facility for his help with this
project. This work was funded by the National Institutes of
Health (R01GM096767).
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ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
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