Identification of antigenic proteins by 2D gel eletrophoresis, immunoblotting and mass spectrometry

2 RESULTS AND DISCUSSION

2.2 Biomedical applications of high resolution mass spectrometric proteome analysis

2.2.2 Application of high resolution mass spectrometric methods to exosomes proteomics

2.2.2.2 Identification of antigenic proteins by 2D gel eletrophoresis, immunoblotting and mass spectrometry

The aim of this project was to identify and characterize antigenic proteins, specifically recognized by an exosomal antibody preparation from a human lung carcinoma cell line (A549). For this purpose, a combination of gel electrophoresis, immunoblotting and mass spectrometry was applied for the elucidation of immunologically important biomarkers for lung carcinoma. A second cancer cell line, a human melanoma cell line (624.38), was employed as a negative control in this experimental set-up. The exosomal antibody – ExoAb – was produced by a rat monoclonal hybridoma cell line; having as immunogen an exosomal preparation from A549 cells (see 3.2.4 and Figure 2.21).

Figure 2.21: Schematic representation of the conditions used for preparing an exosomal antibody (ExoAb) and cell lysates from two human cell lines. A solution containing 10 mM Tris pH 7.4, 150 mM NaCl, 1 % TritonX-100 and complete protease inhibitor cocktail was employed in the preparation of cell lysates from human lung carcinoma and human melanoma cell lines. Protein concentration was measured with BCA assay. The exosomal antibody (ExoAb) was produced by a rat monoclonal hybridoma cell line, having as immunogen an exosomal preparation from A549 cells. A549: human lung carcinoma cell line; 624.38: human melanoma cell line.

First, lysates from both cell lines (A549 and 624.38), and the ExoAb were characterized by 1D and 2D gel electrophoresis. Figure 2.22 shows the 2D gel separation of 30 µg ExoAb under denaturing, reducing and alkylating conditions.

For the IEF an IPG strip with the pH range 3 - 10 was used. Not only protein spots corresponding to heavy (around 50 kDa) and light chain (ca. 25 kDa) were observed in the gel, but also additional spots, indicating an oligoclonal character of the exosomal antibody, not a monoclonal one.

Monoclonal exosome antibody ExoAb

(protein G column purification) A549: a human lung carcinoma cell line

Cellular lysates (BCA assay)

10mM Tris pH 7.4; 150 mM NaCl; 1 % TritonX-100 and complete proteaseinhibitor cocktail

A549 exosomal preparation: immunogen

Rat hybridoma cell line

624.38: a human melanoma cell line

Monoclonal exosome antibody ExoAb

(protein G column purification) A549: a human lung carcinoma cell line

Cellular lysates (BCA assay)

10mM Tris pH 7.4; 150 mM NaCl; 1 % TritonX-100 and complete proteaseinhibitor cocktail

A549 exosomal preparation: immunogen

Rat hybridoma cell line

624.38: a human melanoma cell line

Figure 2.22: Coomassie stained 2D gel of 30 µg exosomal antibody (ExoAb) using a 3 – 10 IPG strip. Spots corresponding to heavy (around 50 kDa) and light chain (ca. 25 kDa) were observed in the gel, but also additional spots, indicating an oligoclonal character of the exosomal antibody.

Before Western blot experiments a control by dot blot was performed to check whether denatured, reduced and alkylated cell lysates samples from the two cell lines (A549 and 624.38) were giving response when treated with the exosomal antibody. In Figure 2.23 the dot blot result is shown. Both treated (denatured with a buffer containing 6M Urea, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5; reduced with dithiothreitol (DTT) and alkylated with iodacetamide (IAA)) and untreated samples were recognized by the exosomal antibody. This result suggested that cell lysates samples can be employed in future experiments in both forms - treated and untreated.

Figure 2.23: Dot blot experiment with cell lysate samples shows that both treated and untreated samples are recognized by the exosomal antibody. In the dot blot experiment the first antibody (ExoAb) was used in a dilution of 1:1,000, while the detection antibody (goat anti-rat horse radish peroxidase (HRP) labelled antibody) was diluted 1:5,000 in PBS-Tween (80 mM Na2HPO4 × 2H2O, 20 mM NaH2PO4 × H2O, 100 mM NaCl, pH 7.5; 0.5 % Tween). (a) untreated sample; (b) treated sample (denatured with a solution of 6M Urea, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5;

reduced with DTT; alkylated with IAA); A549: human lung carcinoma cell lysate; 624.38: human melanoma cell lysate.

1D and 2D gel separations of both cell lysates, followed by immunoblotting experiments (using the ExoAb) were carried out in order to detect the presence of antigenic protein structures. First, 1D protein separations were performed using 12 % SDS-PAGE. Each of the two gels prepared contained one lane of A549 cell lysate, and one lane of 624.38 cell lysate (12 µg total protein amounts were used for each gel lane). One gel was used further for transfer on nitrocellulose membrane and immunoblotting with the exosomall antibody. The second gel was stained with Coomassie and was used for mass spectrometric investigation. Immunoblotting following 1D gel separations resulted in three protein bands detected only in A549 cell lysate gel, after subtracting common bands appearing in both cell lysates (A549 and 624.38). Upon protein bands excision, proteolytic digestion, mass spectrometric analysis and database search in each band several proteins were identified. One example is shown in Figure 2.24. For the band EXO1 database search provided six protein hits.

A549^(a

Figure 2.24: Comparison between the immunoblot and the corresponding Coomassie stained 1D gel resulted in the detection of three protein bands present only in human lung carcinoma cell lysate: bands EXO1, EXO2, EXO3 (a). Band EXO1 from the Coomassie stained gel (a) was excised from gel (see gel insert in b), digested with trypsin, and analysed by LC-MS/MS. Upon database search six human proteins were found. The first three proteins are: 1 – annexin A1; 2 – ANXA2; 3 – annexin A2, isoform 2 (c). *. 12 µg sample were used for each lane. MWM: molecular weight marker; A549: human lung carcinoma cell lysate: 624.38: human melanoma cell lysate.

Given the complexity of the cell lysate protein mixtures, 1D could not resolve proteins efficiently, so in the next step, two-dimensional gel electrophoresis was employed as a separation tool. For each cell lysate (A549 and 624.38) two 2D gels were prepared, one for Western blot, the other one for Coomassie staining and mass spectrometric identification. In a first step the electronic images of the two immunoblots were compared, and after subtraction of spots present in both cell lysates, five spots were found only in the human lung carcinoma blot (spots 1, 2, 3, 4, 5 in Figure 2.25a). A second comparison (gel-immunoblot alignment) was performed between the immunoblot and the corresponding Coomassie stained gel of the human lung carcinoma (A549) cell lysate. Only four out of five

spots identified earlier could be localized in the Coomassie gel (spots 1, 2, 3, 5 in Figure 2.25b). These spots were excised from the Coomassie 2D gel and digested with trypsin. Digestion mixtures were measured by LC-MS/MS and resulted masses were used for database search by Mascot, employing the MS/MS ion search engine.

Figure 2.25: (a) The electronic images of the immunoblots for lung carcinoma (A549) and melanoma (624.38) cell lysates were compared. In green are highlighted common spots; with numbers from 1 to 5 are assigned protein spots recognized by the exosomal antibody only in the lung carcinoma immunoblot. (b) Gel-immunoblot alignment for lung carcinoma cell lysate (A549) resulted in the localization of spots 1, 2, 3 and 5 in the Coomassie stained gel (spot 4 could not be found).

b b

Spots 1, 2, 3 and 5 were excised from the Coomassie gel of the lung carcinoma cell lysate (Figure 2.25b), digested with trypsin, and investigated by liquid chromatography – tandem mass spectrometry. Each protein spot delivered more than one protein hit. Table 9 summarizes the major proteins found with this approach.

Table 9: Summary of LC-MS/MS based protein identification for spots 1, 2, 3 and 5 (see Figure 2.25b). The major proteins found are given with position in gel, the identification score, molecular weight and pI value.

Protein/Acc. no.^a Spot

no. Identification

score Mass

(kDa) pI

Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH)/P04406 1 223 36.20 8.3

Aldo-keto reductase family 1, member C3/

gi:24497583 1 188 37.23 8.1

Aldo-keto reductase family 1, member

C2/P52895 2 256 36.74 7.4

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/P04406

2 66 36.20 8.3

Chain A Fidarest bound to human aldose

reductase/gi:13096112 3^b 2925 36.10 6.6

Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH)/P04406 3^b 434 36.20 8.3

Aldehyde dehydrogenase 1A1/Q5SYR1 5 970 55.45 6.3 Glucose-6-phosphate 1-dehydrogenase (G6PD)/

P11413 5 232 59.68 6.4

aAccession numbers from SWISS-PROT or TrEMBL database.

bThe digestion mixture of spot 3 was measured by LC-MS/MS using the LTQ-Orbitrap mass spectrometer. The other spots were measured by LC-MS/MS using an Esquire 3000+ ion trap instrument.

The mass spectrometric identification of glyceraldehyde-3-phosphate dehydrogenase protein (GAPDH) from spot 1 is shown in Figure 2.26 (see Figure 2.25b for spot position in 2D gel, and Table 9 for identification score). The fragmentation spectrum of the peptide (235 – 248) is given in this figure, together with the amino acids sequence of GAPDH, in which the identified peptides are highlighted in red.

Figure 2.26: Mass spectrometric identification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from spot 1 (see Figure 2.25b and Table 9) by LC-tandem mass spectrometry. In the amino acids sequence of the protein, the identified peptides are shown in red. The fragmentation mass spectrum of ion 765.9 (2+) (with y and b ions assigned), which led to the identification of peptide (235 – 248)is also displayed. Peptide (235 – 248) is in red and underlined in the amino acids sequence.

MGKVKVGVNG FGRIGRLVTR AAFNSGKVDI VAINDPFIDL NYMVYMFQYD STHGKFHGTV KAENGKLVIN GNPITIFQER DPSKIKWGDA GAEYVVESTG VFTTMEKAGA HLQGGAKRVI ISAPSADAPM FVMGVNHEKY DNSLKIISNA SCTTNCLAPL AKVIHDNFGI VEGLMTTVHA ITATQKTVDG PSGKLWRDGR GALQNIIPAS TGAAKAVGKV IPELNGKLTG MAFRVPTANV SVVDLTCRLE KPAKYDDIKK VVKQASEGPL KGILGYTEHQ VVSSDFNSDT HSSTFDAGAG IALNDHFVKL ISWYDNEFGY SNRVVDLMAH MASKE

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 949.5

Table 9 shows that glyceraldehyde-3-phosphate dehydrogenase protein (GAPDH) was identified in three of the analysed spots: spot 1, 2 and 3 from the Coomassie stained gel of the human lung carcinoma cell lysate (see Figure 2.25b). Together with this protein other proteins were identified also (see Table 9), due to a possible co-migration with the antigenic proteins. Protein spots 1, 2, and 3 were separated in the 2D gel at similar molecular weights, but different pI values (Figure 2.25b). The pI shift can be explained by the presence of post-translational modifications (PTMs). Moreover, literature data show that GAPDH’s expression was found to be strongly enhanced in human lung cancer tissues [200] (A549 is a human lung carcinoma cell line).

2.2.2.3 Affinity-mass spectrometry and two-dimensional gel

Im Dokument Analytical development, biochemical and biomedical applications of high resolution mass spectrometric proteome analysis (Seite 86-94)