Detection of antigenic proteins by two-dimensional gel eletrophoresis and immunoblotting

The aim of the present study was to identify relevant antigenic structures for C.

pneumoniae serodiagnosis. For this purpose a proteomics approach previously used for direct protein identification from biological material [161], was developed and applied. High resolution FT-ICR mass spectrometry and HPLC-MS have been recently shown to be powerful tools in fast and unambiguous protein identifications [88,162-166]. Therefore, in this work the combination of 2D, immunoblotting (with sera characterized by the MIF assay [167]), FT-ICR-MS and LC-tandem mass spectrometry was successfully employed in the identification of several unknown antigens and neo-antigenic protein fragments (see Figure 2.13).

Figure 2.13: A schematic representation of the affinity-mass spectrometric methods involved in the identification and characterization of C. pneumoniae antigenic structures.

Cultivation and preparation of C. pneumoniae

2D

Western Blot, Immunoblots evaluation

In-gel-digestion of antigenic proteins

Database search, BLAST

PROTEIN IDENTIFICATION

MALDI-FTICR-MS LC-MS/MS

MALDI-FTICR-MS

Cultivation and preparation of C. pneumoniae

2D

Western Blot, Immunoblots evaluation

In-gel-digestion of antigenic proteins

Database search, BLAST

PROTEIN IDENTIFICATION

MALDI-FTICR-MS LC-MS/MS

MALDI-FTICR-MS

As shown in Figure 2.13 the starting point of the experimental design consisted in the cultivation and preparation of C. pneumoniae. For cultivation, C. pneumoniae TW-183 (from Prof. Jens Kuipers, Department of Rheumatology, Medical School of Hannover, Germany) were propagated in HEp-2 cells as described in detail in the Experimental part (see 3.2.1.1). After cultivation and protein extracts preparation the concentration of the samples was determined using Uptima biochinonic acid assay (Interchim, Montlucon, France). Samples of 550 µg total protein were applied on nonlinear IPG strips (3-10, 17 cm) and were run in the first dimension (IEF). After equilibration, the IPG strips were applied on the top of 10 % SDS polyacrylamide gels for the second separation step. For each experiment four 2D gels were run simultaneously and were processed under identical conditions. Three gels were further subjected to immunoblotting and the fourth gel was used for mass spectrometric analysis. In this way, approximately 600 protein spots could be separated within a mass range from 15 to 130 kDa in one gel, in agreement with previously reported data [145]. A typical 2D gel electrophoretic separation of C. pneumoniae TW-183 is shown in Figure 2.14.

Gels that were used later for protein identification were stained with colloidal Coomassie blue according to Neuhoff et al. [48] and scanned using a GS-710 calibrated imaging densitometer (Bio-Rad). For the immunoblotting procedure, sera from a total of 39 human donors were analysed by MIF for the prevalence of antibodies against C. pneumoniae, C. trachomatis and C. psittaci. 27 sera were collected from healthy volunteers (donors from the University of Konstanz), and 12 sera originated from a previous study, from patients who underwent heart transplantation [168]. One serum from the second group tested positive for C.

trachomatis antibodies, and thus was excluded from the study.

After gels-immunoblots alignment 89 spots were detected as immunoreactive among the tested sera. Figure 2.14 shows all these spots assigned with a number in a gel stained with sensitive Coomassie colloidal blue (the underlined numbers correspond to the later identified antigenic proteins). For each spot the

reactivity was estimated based on the recognition frequency and corresponding intensity among all sera (see Table 3).

Figure 2.14: 2D gel of Chlamydia pneumoniae protein extract. 550 µg total protein were loaded on a 3-10 NL IPG strip (17 cm) and separated proteins were visualised with sensitive colloidal Coomassie staining. 89 antigenic protein spots were revealed by 2D and immunoblotting and are assigned on the gel with a number. The underlined numbers correspond to the identified C.

pneumoniae antigenic proteins.

Table 3: The reactivity of antigenic proteins was calculated based on the recognition frequency and corresponding intensity among all sera (only identified proteins with reactivities > 2 % are listed in this table). Spot numbers correspond to antigenic proteins shown in the 2D gel from Figure 2.14.

Spot no. Frequency^a Reactivity (%)^b

1 27 25.6

3 25 22.6

4 23 19.5

5 7 4.9

6 15 11.3

7 3 2.6

9 13 9.4

10 10 8.3

36 6 3.8

13 28 25.2

14 13 10.9

17 13 9.0

38 10 9.4

24 9 4.9

26 7 7.9

44 4 2.3

28 7 4.9

30 27 30.1

31 7 6.0

35 7 3.4

39 5 2.3

51^c 3 4.9

53 7 5.3

56 3 2.3

aNumber of sera (from the total 38 sera used in this study) with positive reaction.

bSum of the intensity classes (0 - 7) for the 38 sera given in % of the maximal reachable value (38

× 7).

cProtein not resolved in all gels (the separation was in the lower molecular weight range): could be that the intensity is underestimated.

Assignments of antigenic proteins were based on the MIF analyses of the sera, as illustrated by the immunoblots in Figure 2.15. Of the 38 sera tested, 11 sera were negative for C. pneumoniae antibodies (antibody titer < 64), 17 sera were positive (titer 64 - 256) and ten were highly positive (titer ≥ 512). The negative sera revealed a significantly smaller number of reactive spots than the

seropositive sera (higher antibody titers are directed against a larger number of antigens). A good correlation between antibody titers determined by MIF and the number of the identified immunoreactive spots was observed (Figure 2.15).

Figure 2.15: Immunoblots showing antigenic proteins identified by high resolution mass spectrometry after incubation with sera presenting different antibody titers as determined by the MIF assay (for mass spectrometric protein identification see Tables 4, 5,

6). (a), serum from a MIF-negative donor (antibody titer

< 64); (b), serum from a MIF-positive donor (titer > 64). (c), serum from a highly MIF-positive donor (titer ≥ 512).

a b c a b c

The intensities from immunoblot for each spot were determined with a Luminiscent Image Analyser. The maximal intensity for each spot was determined (within the range 0 - 64,000 events) using the AIDA software package (Raytest/Fuji) and only spots with intensities ≥ 500 were considered as immunoreactive towards the used sera. The measured maximal intensities of the spots were classified into eight categories based on a log2-scale (0= <500, 1= <1,000, 2= <2,000, 3= <4,000, 4= <8,000, 5= <16,000, 6= <32,000, 7= <64,000 events/pixel). To compare the frequency and the intensity of immunoreactive spots, the overall reactivity was calculated in percent of the maximal reachable value for each spot. For this purpose, the sum of the measured intensity categories for a number of donors (n) was divided by the product of (n) × (the highest achievable intensity of 7) and multiplied with 100 % (see Table 3).

2.2.1.3 Identification of Chlamydia Pneumoniae antigens by high