Download Improved detection and identification of low

application note
Ettan MALDI-ToF
Improved detection and identification of low-abundance
human bronchoalveolar lavage fluid proteins (BALF)
using 2-D electrophoresis and
Ettan MALDI-ToF mass spectrometry*
key words:
• Immobiline DryStrip • 2-D electrophoresis • BALF proteins
•
paper bridge sample application • Ettan MALDI-ToF
This application note describes an improved method for the
detection and identification of low-abundance human BALF
and plasma proteins, combining 2-D electrophoresis and
Ettan MALDI-ToF mass spectrometry. The method employs
paper bridge application of BALF and plasma proteins on
Immobiline™ DryStrip pH 4.5–5.5, 18 cm. The high sample
loading capacity of these narrow-range Immobiline DryStrip
gels used in the first dimension allowed detection and
identification of more spots on SDS PAGE gels in the second
dimension. BALF and plasma proteins excised from SDS gels
were run on an Ettan™ MALDI-ToF mass spectrometer. The
identified proteins were compared with existing human
BALF or plasma maps. Using paper bridge sample
application, narrow-range Immobiline DryStrip gels, and
high-resolution MALDI-ToF mass spectrometry with Ettan
MALDI-ToF, 12 BALF proteins (including several lowabundance proteins) and 2 plasma proteins, not previously
reported in human BALF and plasma maps respectively, were
successfully identified.
Introduction
Bronchoalveolar lavage fluid (BALF) proteins originating
from the peripheral epithelium and alveoli of the human lung
can be indicators of infectious and inflammatory disease
processes (1). Combining 2-D electrophoresis with matrixassisted laser desorption/ionization time-of-flight (MALDIToF) mass spectrometry provides a precise, rapid, and highly
sensitive method for the detection of BALF proteins by
peptide mass fingerprinting (PMF) (Fig 1). Previously
unidentified BALF proteins, present in low concentrations in
lung BALF can now be detected and identified using this
combined 2-D electrophoresis/MALDI-ToF approach.
In order to maximize detection of BALF proteins in 2-D
electrophoresis, narrow-range Immobiline DryStrip gels are
used to provide high resolution and sample loading capacity
* This application note is based on Sabounchi-Schütt, F., Åström, J., Eklund, A., Grunewald, J.
and Bjellqvist, B., Electrophoresis In Press (2001). Used with permission of the publisher.
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18-1151-35 AA, 2001-05 • p1
Fig 1a. Comassie-stained, micropreparative polyacrylamide
SDS gel of human lung BALF proteins. Circled spots were
identified as Coactosin (spot B 7), gamma actin (spot B 21),
and tubulin beta 2 (spot B 44), all of which have not
previously been reported in human BALF maps.
Intensity
752.57
1600
1400
1200
1000
800
600
806.57
865.49
400
1022.6
934.53
200
1337.8
1803.9
0
800
1 000
1 200
1 400
1 600
1 800
Mass (m/z)
Fig 1b. Ettan MALDI-ToF mass spectrum of spot B 7, identified
as Coactosin by peptide mass fingerprinting.
Ettan MALDI-ToF
(2). Using the paper bridge method (3) allows higher sample
loads on narrow-range Immobiline DryStrip gels enabling
detection of more spots on SDS gels.
High resolution, high sensitivity, and high-mass accuracy are
essential requirements of a MALDI-ToF mass spectrometer
for identification of low-abundance proteins.
Ettan MALDI-ToF, which utilizes an advanced quadratic
field reflectron (Z2 reflectron), produces high-resolution
spectra and is therefore ideal for the identification of lowabundance proteins.
This study describes the use of the paper bridge sample
application, narrow-range Immobiline DryStrip gels and
Ettan MALDI-ToF for the identification of low-abundance
BALF proteins in lung BALF of healthy individuals.
Detection and identification of human blood plasma
proteins using the combined 2-D electrophoresis/
Ettan MALDI-ToF approach is also described.
Products used
Amersham Biosciences products used in this
Application Note:
Immobiline DryStrip pH 4.5–5.5, 18 cm
IPG Buffer pH 3–10 NL
IPG Buffer pH 4.5–5.5
Immobiline DryStrip Kit
Cup Loading Strip Holder
Multiphor II Electrophoresis Unit
IPGphor
ImageScanner
ImageMaster 2D Elite Software
17-6001-85
17-6000-88
17-6002-04
18-1004-30
80-6459-43
18-1018-06
80-6414-02
18-1134-45
80-6350-56
Amersham Biosciences SE 600 Standard Vertical Unit 80-6171-58
Ettan MALDI-ToF 230 V
18-1145-00
Preparation
Bronchoscopy and collection of BALF from healthy, nonsmoking individuals were performed as described in reference
4. Collected lavage fluid was filtered and centrifuged at
400 × g for 10 min at 4 °C. Protein concentration of the
decanted supernatant was determined according to
procedures based on reference 5 with bovine serum albumin
as the protein reference marker. Blood samples were taken
from a separate group of healthy individuals and plasma
was obtained using standard procedures.
To remove salt and low molecular weight contaminants from
the BALF samples, extracted BALF supernatants were
purified according to reference 6. The purified BALF samples
were lyophilized using a vacuum centrifuge and the resulting
pellet resuspended in a solution containing 8 M urea, 4%
CHAPS, 65 mM DTT, and 1% IPG Buffer pH 3–10 NL
prior to electrophoresis.
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Method
Micropreparative 2-D electrophoresis of BALF and plasma
proteins
Samples were focused on Immobiline DryStrip pH 4.5–5.5,
18 cm. The immobilized pH gradient (IPG) strips were
rehydrated overnight at room temperature in 8 M urea, 2%
CHAPS, 1% IPG Buffer pH 3–10 NL, 19 mM DTT, and
bromophenol blue tracking dye.
Sample loading to the IPG strips was performed using a
novel paper bridge application method (3). A series of
preliminary trials revealed that optimal spot resolution and
number of detected spots could be achieved using the paper
bridge application method. Based on these findings, 3 mg
BALF or 5 mg plasma were separated using Immobiline
DryStrip pH 4.5–5.5, 18 cm gels. Samples were loaded onto
paper bridges positioned between the electrodes from the
Immobiline DryStrip Kit and the IPG strips; BALF samples
(3 mg protein) were loaded at the anode of the IPG strips
while plasma samples (5 mg protein) were applied at the
cathode. The IPG strips were focused on
Multiphor™ II Electrophoresis Units for a total of 70 kVh
over 20 h. IPG strips can also be run on
IPGphor™ electrophoresis unit using paper bridges on Cup
Loading Strip Holder, which achieves equally high spot
resolution.
All chemicals and reagents used for the second dimension of
2-D electrophoresis are described in reference 6. Initial
equilibration of the Immobiline DryStrip gels was performed
for 15 min in 19 mM DTT, 50 mM Tris, 6 M urea, 30%
glycerol, 2% SDS, and tracking dye. Equilibration continued
for a further 15 min in a solution containing all of the
components described except DTT, which was replaced by
0.2 M iodoacetamide. Second-dimension gel electrophoresis
was performed using 14 cm × 14 cm × 1.5 mm 9–18%
gradient, self-cast polyacrylamide SDS gels, with a modified
Laemmli buffer composition (7). Immobiline DryStrip gel
length was cut to fit the Amersham Biosciences™ SE 600 vertical
electrophoresis units†. Electrophoresis was performed with a
current of 50 mA/gel for 150 min.
Gels were stained with Coomassie™ Blue (6) and scanned in
ImageScanner™. Spots were evaluated using
ImageMaster™ 2-D Elite software, version 3.0 (7). BALF
and plasma proteins were compared using
ImageMaster 2D Elite software in order to identify BALFspecific proteins. BALF spot maps were also compared to the
SWISS-2DPAGE human plasma map.
† Modification of Immobiline DryStrip lengths is not necessary if Ettan DALT II
electrophoresis unit is used. The unit was not available at the time this experiment was
performed.
Ettan MALDI-ToF
Mass spectrometry of detected protein spots on Ettan MALDI-ToF
Plugs were punched out manually from the gels, washed
twice in 150 µl 0.2 M NH4HCO3, dissolved in 50%
acetonitrile (ACN), and incubated at 30 °C for 1 h. The gel
plugs were dried under vacuum and digested in 5 µl trypsin
(0.5 µg) overnight at 30 °C. Peptides were eluted twice in
100 µl 50% ACN/ 0.45% TFA at 30 °C for 1 h and the
resulting extracts were pooled and lyophilized. The
lyophilized samples were dissolved in 5 µl MALDI-ToF
matrix (=-cyano-4-hydroxy-cinnamic acid in 50% ACN/
0.45 % TFA) containing ILE7 Angiotensin III and hACTH
18-39 peptide reference markers (Ettan chemicals range).
Combined sample/matrix mixtures were applied in 1 µl
volumes to the target slide using the dried-droplet method (8).
Mass spectrometry was performed using Ettan MALDI-ToF
in quadratic field reflectron mode. Ionization was initiated
by pulsed shots from the 337 nm nitrogen laser
incorporated in the Ettan MALDI-ToF instrument. Ions were
accelerated into the time-of-flight analyser at 18kV with a
time lag of 100–200 ns in the pulsed extraction ion source.
Ions were detected on double micro-channel plates and a
500 M sample/s analogue-to-digital converter. Spectra were
calibrated using internal reference peptide markers.
Protein identification was achieved by PMF of the spectral
data using the search program ProFound version 4.04 at
http://www.proteometrix.com. Protein search parameters
included species of origin, (all species or Homo sapiens
only), protein Mr range 5 000–3 × 106, and isolectric point
(pI) 1–14. Only mono-isotopic peptide masses were used in
PMF.
Intensity
1790.9
600
500
400
300
1954.0
200
945.48
1516.8
100
1132.6
1198.7
0
800
1 000
1 200
1 400
1 600
1 800
2 000
Mass (m/z)
Fig 2. Ettan MALDI-ToF mass spectrum of gamma actin. The
protein, corresponding to spot B 21 in Figure 1, was identified
by peptide mass fingerprinting.
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Intensity
1130.6
200
150
1040.6
1246.7
1245.7
100
1039.5
1271.8
1229.7
50
0
1 050
1 100
1 150
1 200
1 250
1 300
Mass (m/z)
Fig 3. Ettan MALDI-ToF mass spectrum of tubulin beta 2. The
protein, corresponding to spot B 44 in Figure 1, was identified
by peptide mass fingerprinting.
Results
The combination of paper bridge sample application and
Immobiline DryStrip pH 4.5–5.5, 18 cm on Multiphor II
electrophoresis unit in the first dimension of 2-D
electrophoresis enabled detection of 250 lung BALF spots in
the pH range 4.5–5.2 on SDS gels. High sample loads of up
to 3 mg BALF could be applied using the micropreparative
method described without detectable streaking or loss of
proteins from the gels. Of the 250 BALF spots detected by
2-D electrophoresis, 49 spots (corresponding to 28 different
proteins) were selected and identified by PMF from
Ettan MALDI-ToF spectra. Furthermore, 28 spots
(17 proteins) of the 49 BALF spots identified could not be
matched to the corresponding plasma SDS gels and are
hereafter referred to as BALF-specific proteins. A total of 12
of the BALF-specific proteins identified were previously
unidentified in human lung BALF 2-D protein maps. Spectra
for two of these proteins, gamma actin and tubulin beta 2
are shown in Figures 2 and 3 respectively.
For the plasma samples, 200 plasma spots were detected on
SDS gels and 18 spots, corresponding to 9 different proteins
were identified by PMF. Two of the 9 identified proteins,
CD 5 antigen-like receptor and Kinogen HMW heavy chain
protein, had not previously been identified in the 2-D plasma
map at the SWISS-2DPAGE website (7).
Ettan MALDI-ToF
Conclusions
Acknowledgement
Combined 2-D electrophoresis with Ettan MALDI-ToF mass
spectrometry was performed on BALF proteins from the
epithelial lining of the lungs, as well as, proteins from blood
plasma of healthy patients. Using the paper bridge method
and narrow-range Immobiline DryStrip gels for focusing
facilitated detection of low-abundance BALF and plasma
proteins in 2-D electrophoresis. Moreover,
Ettan MALDI-ToF, when used in quadratic field reflectron
mode, provided high-resolution spectra, which facilitated
identification of low-abundance BALF and plasma proteins
by PMF.
This work was conducted in collaboration with the Dept. of
Medicine, Lung research Laboratory, Karolinska Hospital,
Stockholm, Sweden.
References
1.
Hermans, C. and Bernard, A. Am. J. Respir. Crit. Care Med. 159, 646–678 (1999).
2.
Application Note: Improved spot resolution and detection of proteins in 2-D
electrophoresis using 24 cm Immobiline DryStrip Gels, Amersham Biosciences,
code number 18-1150-23, edition AA (2001).
3.
Sabounchi-Schutt, F. et al., Electrophoresis 21, 3649–3656 (2000).
4.
Eklund, A. and Blashcke, E. Thorax 41, 629–634 (1986).
5.
Bradford, M. Anal. Biochem. 72, 248–254 (1976).
6.
Sabounchi-Schütt, F. et al., Electrophoresis In Press (2001).
7.
2-D Electrophoresis using Immobilized pH Gradients, Amersham Biosciences,
code number 80-6429-60 (1998).
8.
Karas, M. and Hillenkamp, F. Anal. Chem. 60, 2299–2317 (1988).
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