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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. an 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. an 18-1151-35 AA, 2001-05 • p2 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. an 18-1151-35 AA, 2001-05 • p3 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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