Identification of Chlamydia Pneumoniae antigens by high resolution FT-ICR-MS and LC-MS/MS

2 RESULTS AND DISCUSSION

2.2 Biomedical applications of high resolution mass spectrometric proteome analysis

2.2.1 Immunoproteomics for identification of protein biomarkers of Chlamydia Pneumoniae

2.2.1.3 Identification of Chlamydia Pneumoniae antigens by high resolution FT-ICR-MS and LC-MS/MS

For the identification of antigenic proteins derived from gels-immunoblots comparison, spots were excised from gels, digested with trypsin according to Mortz et al. [120] and the obtained fractions were purified using C18 OMIX^TM ZipTip pipette tips as previously described [88,169]. The resulting proteolytic peptides were analysed both by MALDI-FTICR-MS and by LC- tandem mass spectrometry.

Initially, proteins from 42 spots with consistent antigenic reactivity (> 2 %) were analysed (see Table 3). The remaining proteins (spots like 16, 47, 80, 89; see Figure 2.14) that were reactive in a few immunoblots towards positive and highly positive sera were analysed in a second step. To give one example of a low reactivity spot, Figure 2.16 shows the unambiguous MALDI-FT-ICR mass spectrometric identification of glyceraldehyde-3-phosphate dehydrogenase cytosolic protein (GAPDH) from protein spot 47 (see Figure 2.14).

Figure 2.16: MALDI-FT-ICR mass spectrometric identification of glyceraldehyde-3-phosphate dehydrogenase cytosolic protein (GAPDH) from spot 47 (see 2D gel in Figure 2.14). Six peptides were identified, and are assigned in the mass spectrum with labels and numbers (corresponding to the first and last amino acid of each sequence), and are in red in the amino acids sequence of the protein.

As already mentioned protein spots were investigated either by MALDI-FT-ICR-MS, or by LC-tandem mass spectrometry. For some of the proteins identified by MALDI-FTICR-MS, the data were complemented using liquid chromatography – tandem mass spectrometry. For example, the tryptic digestion mixture of protein spot 17 (see Figure 2.14) was measured by both MALDI-FT-ICR-MS (Figure 2.17a) and LC-MS/MS (Figures 2.17b, c). The major outer membrane protein (MOMP) was successfully identified in both cases. Sixteen peptides led to MOMP identification by LC-MS/MS. A very good coverage of b and y ions can be observed in Figure 2.17c for peptide ²⁹²ATFDADNIR³⁰⁰.

950 1050 1150 1250 1350 1450 1550 1650 m/z

892.4896 [ 71 –77 ] 998.5430 [ 78 –85 ] 1020.5853 [ 70 –77 ] 1500.7861[200 -214] 1540.8624[234 -247] 1658.7840 [309 -322]

911.4230 939.0633 1076.9602 1114.9051 1193.0400 1230.9882 1268.9355 1422.9633 1385.0185

850

1 MKVVINGFGR IGRLVLRQIL KRNSSVEVLA INDLVPGDAL TYLFKFDSTH 51 GRFPEDVRCE ADHLIVGKRK IQFLSERNVQ NLPWKDLGVD LVIECTGLFT 101 KKEDAEKHIQ AGAKRVLISA PGKGDIPTFV MGVNHKTFNP EKDFVISNAS 151 CTTNCLAPIA KVLLDNFGIT EGLMTTVHAA TATQLVVDGP SKKDWRGGRG 201 CLQNIIPAST GAAKAVTLCL PELKGKLTGM AFRVPIEDVS VVDLTVRLDK 251 STTYDDICKA MKQASETDLK GILDYTDEQV VSSDFIGSEY SSIFDALAGI 301 ALNDRFFKLV AWYDNETGYA TRIVDLLEYV EKNSK

Q9Z7T0 - glyceraldehyde-3-phosphate dehydrogenase cytosolic protein (GAPDH)

Chlamydophila pneumoniae (strain TW-183) Mass: 37.22 kDa; pI: 6.3

950 1050 1150 1250 1350 1450 1550 1650 m/z

892.4896 [ 71 –77 ] 998.5430 [ 78 –85 ] 1020.5853 [ 70 –77 ] 1500.7861[200 -214] 1540.8624[234 -247] 1658.7840 [309 -322]

911.4230 939.0633 1076.9602 1114.9051 1193.0400 1230.9882 1268.9355 1422.9633 1385.0185

850

Q9Z7T0 - glyceraldehyde-3-phosphate dehydrogenase cytosolic protein (GAPDH)

Chlamydophila pneumoniae (strain TW-183) Mass: 37.22 kDa; pI: 6.3

1000 1200 1400 1600 1800 2000 m/z

972.4999 [380 -388] 1022.4875 [292 -300] 1119.5670 [380 -389] 1309.5793 [ 63 -73] 1523.8158 [147 -161] 1631.8780[278 -291] 1719.8062 [132 -146] 1773.9255[241 -259] 2122.1042 [238 -259] 2204.1184 [219 -237]

1061.5714 1247.6329 1449.6551 2082.9860

1000 1200 1400 1600 1800 2000 m/z

1061.5714 1247.6329 1449.6551 2082.9860

1000 1200 1400 1600 1800 2000 m/z

1061.5714 1247.6329 1449.6551 2082.9860

16 identified peptides with LC-MS/MS

MKKLLKSALLSAAFAGSVGS LQALPVGNPSDPSLLIDGTI WEGAAGDPCDPCATWCDAIS LRAGFYGDYVFDRILKVDAP KTFSMGAKPTGSAAANYTTA VDRPNPAYNKHLHDAEWFTN AGFIALNIWDRFDVFCTLGA SNGYIRGNSTAFNLVGLFGV KGTTVNANELPNVSLSNGVV ELYTDTSFSWSVGARGALWE CGCATLGAEFQYAQSKPKVE ELNVICNVSQFSVNKPKGYK GVAFPLPTDAGVATATGTKS ATINYHEWQVGASLSYRLNS LVPYIGVQWSRATFDADNIR IAQPKLPTAVLNLTAWNPSL LGNATALSTTDSFSDFMQIV SCQINKFKSRKACGVTVGAT LVDADKWSLTAEARLINERA AHVSGQFRF

P27455- major outer membrane protein (MOMP) Chlamydophila pneumoniae (strain TW-183)

Mass: 42.10 kDa; pI: 7.5

16 identified peptides with LC-MS/MS

P27455- major outer membrane protein (MOMP) Chlamydophila pneumoniae (strain TW-183)

Mass: 42.10 kDa; pI: 7.5

Figure 2.17 (continued)

Figure 2.17: Identification of major outer membrane protein (MOMP) from spot 17 (for position of spot 17 in 2D gel see Figure 2.14) by MALDI-FT-ICR-MS (a) and LC-MS/MS (b, c). (a), MALDI-FT-ICR mass spectrometric identification of MOMP protein (ten peptides were identified, and they are labelled and shown in the spectrum with numbers corresponding to the first and the last amino acid of each sequence); (b), 16 peptides (in bold and red) led to MOMP identification using LC-tandem mass spectrometry (the fragmentation spectrum of the boxed peptide is shown in c);

(c), MS/MS of ion 511.8 (2+), with y and b ions assigned (*: loss of NH3; ⁰: loss of H2O).

As a result of this two-step analysis (first high reactive, and then low reactive protein spots), 22 proteins of C. pneumoniae (from 28 protein spots) and 6 human proteins were identified (spot 55, ATP synthase beta-subunit; 60, Porin-31-HM; 61, alpha-tubulin; 62, prohibitin; 70, pyruvate kinase; 71, elongation factor-Tu). Human proteins, also found in previously reported proteome maps [146], are likely resulting from contamination by the Hep-2 cells used in the C.

pneumoniae culture. For 17 proteins (contained in 20 protein spots) MALDI-FTICR-MS was the only tool used for protein identification, with an average mass accuracy < 9 ppm. In Table 4 proteins identified by MALDI-FT-ICR PMF are presented, together with position number on 2D map (see Figure 2.14), short name, accession number from database, molecular weight, isoelectric point and average mass accuracy.

Table 4: Identification of C. pneumoniae antigenic proteins by MALDI-FT-ICR peptide mass

aProtein short name.

bAccession numbers are from SWISS-PROT or TrEMBL database.

cThe database search was perfomed with the ProFound engine.

dSpot 38 is separated in the 2D map at an acidic pH (4.5 – 4.7) and spot 17 appears in a more basic area (pH 6.0 – 7.5).

eSpot 35 represents a fragment of Pmp6 protein which is separated at smaller molecular mass (~

60 kDa) and slightly basic pH (~ 7).

fThe molecular mass and the pI are for the entire Pmp 21 protein. The N-terminal part has around 70kDa (spot 36), the middle part ~ 55 kDa (spot 9) and the C-terminal domain ~ 45 kDa (spot 10).

gCpaf-n fragment has around 24 kDa and Cpaf-c approximately 40 kDa and a pI between 5 and 5.1.

hThe monoisotopic masses were looked against NCBInr database using the MASCOT search engine allowing 2 missed cleavages.

In addition, seven proteins (from eight protein spots) were identified by LC-MS/MS analysis (Table 5). For two of the seven proteins identified (Pmp21-c and Cpaf-c), only single peptide masses provided individual ion scores exceeding the identity or extensive homology threshold of unambiguous identification.

Table 5: Identification of C. pneumoniae antigenic proteins by LC- tandem mass spectrometry.

Individual ions scores > 41 indicated identity or extensive homology (p < 0.05). For spot numbers see Figure 2.14.

Protein^a/Acc. no.^b Spot

no. Identification

score No. of identified peptides

Mass (kDa) pI

Pmp21-m/Q9Z6U5 9 112 2^c 169.41^d 4.8

Pmp21-c/Q9Z6U5 10 85 1^c 169.41^d 4.8

CpB1072/Q9Z6M7 16 62 2^c 21.87 5.6

YscL/Q9Z780 24 65 2^e 25.94 5.0

Cpaf-c/Q9Z6P3 26 76 1^c 69.34^f 5.5^f

PorB/Q9Z752 53 112 2^c 37.59 5.7

ClpP_1/Q9Z832 80 104 2^c 21.06 5.6

CpB0756/Q9Z7H7 89 75 3^e 68.18 4.9

aProtein short name.

bAccession numbers from SWISS-PROT or TrEMBL database.

cPeptide matches above identity threshold.

dMolecular mass and pI of full length Pmp 21. The N-terminal part has ca. 70 kDa (spot 36), the middle part ~ 55 kDa (spot 9) and the C-terminal domain ~ 45 kDa (spot 10).

ePeptide matches above homology or identity threshold.

fCpaf-n fragment has ca. 24 kDa and Cpaf-c ca. 40 kDa and pI 5- 5.1.

All identified C. pneumoniae proteins are summarised in the Table 6 together with their molecular weight, isoelectric point, the mass spectrometric method used and the protein accession number from SWISS-PROT or TrEMBL database. The functional classes of the identified antigens included "difficult"

proteins such as cell envelope (Omp2, MOMP), outer membrane proteins (PorB, Pmp6, Pmp21), and proteins involved in translation (RpsA, EF-Tu, RpIL) and transcription (RpoA, transcription termination factor rho - Rho). In addition, antigenic proteins with mixed functions (Cpaf) and chaperones (molecular chaperone HSP 70, DnaK; chaperonin GroEL, GroEL; endopeptidase Clp1, ClpP_1) were identified. Antigenic proteins that were either unknown or previously detected only in other Chlamydia species, such as Chlamydia trachomatis, were identified in 28 protein spots derived from 22 C. pneumoniae proteins.

Table 6: Antigenic C. pneumoniae proteins were identified by MALDI-FT-ICR peptide mass fingerprinting (A) and LC-tandem mass spectrometry (B) (spot numbers are according to Figure 2.14).

Short name Spot no. Mass pI MS Acc. no.^a (kDa) method

Omp2 1, 3 61.63 6.0 A P23700

GroEL 4 58.45 5.3 A P31681 RpsA 5 65.17 5.2 A Q9Z8M3 DnaK 6, 7 71.42 5.0 A P27542 Pmp21-m 9 169.41^b 4.8 A, B Q9Z6U5 Pmp21-c 10 169.41^b 4.8 A, B Q9Z6U5 Pmp21-n 36 169.41^b 4.8 A Q9Z6U5

EF-Tu 13 43.28 5.4 A Q9Z9A7

RpoA 14 41.95 5.0 A Q9Z7S8

CpB1072 16 21.87 5.6 A, B Q9Z6M7 MOMP 17, 38 42.10 7.5^c A, B P27455 YscL 24 25.94 5.0 A, B Q9Z780 Cpaf-c 26 69.34^d 5.5^d A, B Q9Z6P3 Cpaf-n 44 69.34^d 5.5 A Q9Z6P3

Rho 28 51.90 6.8 A Q9Z7U4

CpB0704 30 40.59 7.1 A Q7VPW6

RecA 31 38.38 7.7 A Q9Z7E4

Pmp6 35 145.97^e 5.3^e A Q9Z899

DhnA 39 38.35 7.2 A Q9Z8Q7

GAPDH 47 37.22 6.3 A Q9Z7T0

RpIL 51 13.45 5.0 A Q9Z9A1

PorB 53 37.59 5.7 A, B Q9Z752

PyrH 56 27.42 5.4 A Q9Z7K7

ClpP_1 80 21.06 5.6 A, B Q9Z832 CpB0756 89 68.18 4.9 A, B Q9Z7H7

aAccession numbers are from SWISS-PROT or TrEMBL database.

bThe molecular mass and the pI are for the entire Pmp 21 protein. The N-terminal part has around 70kDa (spot 36), the middle part ~ 55 kDa (spot 9) and the C-terminal domain ~ 45 kDa (spot 10).

cSpot 38 is separated in the 2D-map at an acidic pH (4.5 – 4.7) and spot 17 appears in a more basic area (pH 6.0 – 7.5). Spot 17 was identified by MALDI-FT-ICR-MS and LC-MS/MS, spot 38 by MALDI-FT-ICR.

dCpaf-n fragment has around 24 kDa and Cpaf-c approximately 40 kDa and a pI between 5 and 5.1.

eSpot 35 represents a fragment of Pmp6 protein which is separated at smaller molecular mass (~

60 kDa) and slightly basic pH (~ 7).

2.2.1.4 Identification of neo-antigenic protein fragments by high resolution

Im Dokument Analytical development, biochemical and biomedical applications of high resolution mass spectrometric proteome analysis (Seite 68-75)