Identification of neo-antigenic protein fragments by high resolution mass spectrometry

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

2.2.1.4 Identification of neo-antigenic protein fragments by high resolution mass spectrometry

The high resolution mass spectrometric analyses enabled the identification of several neo-antigenic protein fragments, such as Pmp21 with an N-terminal, central and a C-terminal domain, and the secreted chlamydial proteasome-like factor (Cpaf) present with an acidic 40 kDa and a basic (24 kDa) fragment.

Two polymorphic membrane proteins, Pmp6 and Pmp21, that contain an autotransporter domain and thus can pass the outer membrane of bacteria, were identified as full length proteins and as cleaved forms. Protein spot 45 (Figure 2.14) separated in 2D at ca. 130 kDa was identified previously as full length Pmp6 [170]. In addition, here, a 60 kDa C-terminal fragment of Pmp6 (spot 35 in Figure 2.14) was identified and characterised with antigenic properties, in full agreement with previously reported data [145,171]. For Pmp21, the largest of the 21 polymorphic membrane proteins expressed by C. pneumoniae, three fragments were detected and identified with molecular masses of ca. 70, 55 and 45 kDa. As shown in Figure 2.18, an N-terminal fragment (Pmp21-n; spot 36), a middle domain fragment m; spot 9) and a C-terminal fragment (Pmp21-c; spot 10) were found, which partially confirmed previously reported data [145,172]. Using purified RB, no C-terminal fragment of Pmp21 was found, while with purified EB only a C-terminal protein of ca. 47 kDa was previously identified [146,172]. In contrast, the present combination approach of imunoblotting and mass spectrometry (in which EB were used) provided the detection and identification of all three fragments of Pmp21 (see Figure 2.18). Identified peptides from all three fragments (8 peptides for N-terminus, 2 peptides for the middle part and 1 peptide for the C-terminal fragment) are marked in the amino acids sequence of Pmp21 (Figure 2.19).

Pmp21-n

Pmp21-m

Pmp21-c a

Pmp21-n

Pmp21-m

Pmp21-c a

Pmp21-n Spot 36 b

1400 1900 2400 m/z

[100 -108] [489 -499] [305 -323] [109 -126] [229 -246] [471 -488] [159 -180] [202 -225]

1104.5678 1264.5312 1963.0259 1978.8996 1983.0212 2105.1021 2302.1402 2432.1772

Pmp21-n Spot 36 b

1400 1900 2400 m/z

[100 -108] [489 -499] [305 -323] [109 -126] [229 -246] [471 -488] [159 -180] [202 -225]

1104.5678 1264.5312 1963.0259 1978.8996 1983.0212 2105.1021 2302.1402 2432.1772

1400 1900 2400 m/z

[100 -108] [489 -499] [305 -323] [109 -126] [229 -246] [471 -488] [159 -180] [202 -225]

1104.5678 1264.5312 1963.0259 1978.8996 1983.0212 2105.1021 2302.1402 2432.1772

Figure 2.18 (continued)

Figure 2.18: Mass spectrometric identification of Pmp21 protein fragments. (a), 2D gel insert with encircled protein spots corresponding to the identified Pmp21 fragments, Pmp21-n (N-terminal part, spot 36), Pmp21-m (middle part, spot 9), and Pmp21-c (C-terminal part, spot 10); (b), MALDI-FT-ICR mass spectrometric identification of Pmp21-n, spot 36; labelled peaks denote the identified peptides; (c), LC-MS/MS identification of Pmp21-m (spot 9), peptide ion m/z 775.8 (2+ ;

883TLTITGNSGDILFAK⁸⁹⁷); (d), LC-MS/MS identification of Pmp-c (spot 10), peptide ion m/z 600.3 (2+; ¹⁴²⁵GFIAGTSIDYR¹⁴³⁵). *: loss of NH3; ⁰: loss of H2O.

200 400 600 800 1000 1200 1400 m/z

215.2

200 400 600 800 1000 1200 1400 m/z

215.2

200 400 600 800 1000 1200 1400 m/z

215.2

300 400 500 600 700 800 900 1000 m/z

b₃

300 400 500 600 700 800 900 1000 m/z

b₃

Figure 2.19: Amino acids sequence of C. pneumoniae Pmp-21 protein. Identified peptides from spot 36 (N-terminal fragment) are marked in red, from spot 9 (middle part) in blue, and from spot 10 (C-terminal part) in green. For spot numbers see Figure 2.14.

The chlamydial protease/proteasome-like activity factor protein (Cpaf) was established as a further example in which no intact protein, but only neo-antigenic fragments were isolated by 2D gel electrophoresis and identified. Only a few secreted proteins have been identified for C. pneumoniae, of which Cpaf is a protease that cleaves host cell transcription factors essential for MHC class I

1 MSAGIAFEAH SLHSSELDLG VFNKQFEEHS AHVEEAQTSV LKGSDPVNPS 51 QKESEKVLYT QVPLTQGSSG ESLDLADANF LEHFQHLFEE TTVFGIDQKL 101 VWSDLDTRNF SQPTQEPDTS NAVSEKISSD TKENRKDLET EDPSKKSGLK 151 EVSSDLPKSP ETAVAAISED LEISENISAR DPLQGLAFFY KNTSSQSISE 201 KDSSFQGIIF SGSGANSGLG FENLKAPKSG AAVYSDRDIV FENLVKGLSF 251 ISCESLEDGS AAGVNIVVTH CGDVTLTDCA TGLDLEALRL VKDFSRGGAV 301 FTARNHEVQN NLAGGILSVV GNKGAIVVEK NSAEKSNGGA FACGSFVYSN 351 NENTALWKEN QALSGGAISS ASDIDIQGNC SAIEFSGNQS LIALGEHIGL 401 TDFVGGGALA AQGTLTLRNN AVVQCVKNTS KTHGGAILAG TVDLNETISE 451 VAFKQNTAAL TGGALSANDK VIIANNFGEI LFEQNEVRNH GGAIYCGCRS 501 NPKLEQKDSG ENINIIGNSG AITFLKNKAS VLEVMTQAED YAGGGALWGH 551 NVLLDSNSGN IQFIGNIGGS TFWIGEYVGG GAILSTDRVT ISNNSGDVVF 601 KGNKGQCLAQ KYVAPQETAP VESDASSTNK DEKSLNACSH GDHYPPKTVE 651 EEVPPSLLEE HPVVSSTDIR GGGAILAQHI FITDNTGNLR FSGNLGGGEE 701 SSTVGDLAIV GGGALLSTNE VNVCSNQNVV FSDNVTSNGC DSGGAILAKK 751 VDISANHSVE FVSNGSGKFG GAVCALNESV NITDNGSAVS FSKNRTRLGG 801 AGVAAPQGSV TICGNQGNIA FKENFVFGSE NQRSGGGAII ANSSVNIQDN 851 AGDILFVSNS TGSYGGAIFV GSLVASEGSN PRTLTITGNS GDILFAKNST 901 QTAASLSEKD SFGGGAIYTQ NLKIVKNAGN VSFYGNRAPS GAGVQIADGG 951 TVCLEAFGGD ILFEGNINFD GSFNAIHLCG NDSKIVELSA VQDKNIIFQD 1001 AITYEENTIR GLPDKDVSPL SAPSLIFNSK PQDDSAQHHE GTIRFSRGVS 1051 KIPQIAAIQE GTLALSQNAE LWLAGLKQET GSSIVLSAGS ILRIFDSQVD 1101 SSAPLPTENK EETLVSAGVQ INMSSPTPNK DKAVDTPVLA DIISITVDLS 1151 SFVPEQDGTL PLPPEIIIPK GTKLHSNAID LKIIDPTNVG YENHALLSSH 1201 KDIPLISLKT AEGMTGTPTA DASLSNIKID VSLPSITPAT YGHTGVWSES 1251 KMEDGRLVVG WQPTGYKLNP EKQGALVLNN LWSHYTDLRA LKQEIFAHHT 1301 IAQRMELDFS TNVWGSGLGV VEDCQNIGEF DGFKHHLTGY ALGLDTQLVE 1351 DFLIGGCFSQ FFGKTESQSY KAKNDVKSYM GAAYAGILAG PWLIKGAFVY 1401 GNINNDLTTD YGTLGISTGS WIGKGFIAGT SIDYRYIVNP RRFISAIVST 1451 VVPFVEAEYV RIDLPEISEQ GKEVRTFQKT RFENVAIPFG FALEHAYSRG 1501 SRAEVNSVQL AYVFDVYRKG PVSLITLKDA AYSWKSYGVD IPCKAWKARL 1551 SNNTEWNSYL STYLAFNYEW REDLIAYDFN GGIRIIF

Q9Z6U5 – Polymorphic outer membrane protein (Pmp21)

and II antigen presentation [173-174]. Secreted in the cytosol of Chlamydia infected cells, the full length protein has around 70 kDa (in the case of C.

pneumoniae and C. trachomatis) [174-175]., Both C- and N-terminal antigenic fragments of Cpaf were identified in the present study: a basic part with ca. 24 kDa and pI 7 (Cpaf-n), and an acidic part with ca. 40 kDa and a pI of 5 (Cpaf-c);

the latter fragment had not been previously found [174], possibly due to its co-migration with the RpoA protein (spot 14 in Figure 2.14). Figure 2.20 (a and b) shows the 2D gel insert with encircled spots of the Cpaf fragments, spot 26 (Cpaf-c) and spot 44 (Cpaf-n), together with the MALDI-FT-ICR-MS analysis of Cpaf-n; the calculated and experimental peptide masses and corresponding mass accuracies are given in Table 7. The MS results for the C-terminal part of Cpaf (spot 26) were complemented by LC-MS/MS data that are shown in Figure 2.20c (see also Table 5).

Cpaf-n Cpaf-c

Figure 2.20 (continued)

Figure 2.20: Identification of Cpaf (Chlamydial protease/proteasome like-activity factor) protein fragments. (a), 2D gel section containing encircled spots 26 (Cpaf-c) and 44 (Cpaf-n); (b), MALDI-FT-ICR-MS of Cpaf-n tryptic peptides from spot 44 (see Table 7 for peptides identified and mass accuracies); (c), LC-MS/MS identification of Cpaf-c fragment (spot 26): MS/MS of ion 638.8 (2+), with y and b ions assigned (*: loss of NH3; ⁰: loss of H2O).

800 1000 1200 1400 1600 1800 2000 2200 m/z

[85 -91] [105 -115] [92 -104] [53 -68] [69 -84] [29 -45]

850.4770 1283.6858 1556.7502 1919.9652 1964.0126 1985.0357

800 1000 1200 1400 1600 1800 2000 2200 m/z

[85 -91] [105 -115] [92 -104] [53 -68] [69 -84] [29 -45]

850.4770 1283.6858 1556.7502 1919.9652 1964.0126 1985.0357

Cpaf-n Spot 44

1059.5545 1127.5621 1902.9556

800 1000 1200 1400 1600 1800 2000 2200 m/z

[85 -91] [105 -115] [92 -104] [53 -68] [69 -84] [29 -45]

850.4770 1283.6858 1556.7502 1919.9652 1964.0126 1985.0357

800 1000 1200 1400 1600 1800 2000 2200 m/z

[85 -91] [105 -115] [92 -104] [53 -68] [69 -84] [29 -45]

850.4770 1283.6858 1556.7502 1919.9652 1964.0126 1985.0357

Cpaf-n Spot 44

1059.5545 1127.5621 1902.9556

MS/MS, m/z 638.8 (2+)

200 300 400 500 600 700 800 900 1000 1100 m/z

215.0

200 300 400 500 600 700 800 900 1000 1100 m/z

b₂

200 300 400 500 600 700 800 900 1000 1100 m/z

215.0

200 300 400 500 600 700 800 900 1000 1100 m/z

b₂

Table 7: FT-ICR mass spectrometric identification of Cpaf-n (spot 44): calculated and experimental m/z values of identified peptides together with the corresponding mass accuracies are shown.

Identified peptide m/zcalc m/zexp ∆m (ppm)

85VLSNYVR⁹¹ 850.4781 850.4770 1.3

105TESAYIPYVLK¹¹⁵ 1283.6882 1283.6858 1.9

92SLNDYHAGITFYR¹⁰⁴ 1556.7492 1556.7502 0.6

53ELLFGWDLSQQTQQAR⁶⁸ 1919.9610 1919.9652 2.2

69LQLVLEEKPTTNYCQK⁸⁴ 1964.0157 1964.0126 1.6

29DNAYQDLNVIEHLISLK⁴⁵ 1985.0338 1985.0357 0.9

2.2.1.5 Interpretation of the results obtained in the C. pneumoniae

Im Dokument Analytical development, biochemical and biomedical applications of high resolution mass spectrometric proteome analysis (Seite 75-81)