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Протеомика. PowerPoint PPT Presentation


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Протеомика. Цель и задачи протеомики. Конечная цель протеомики – установление и характеристика полного набора белков данного организма. Основные задачи протеомики: - идентификация белков клеток, тканей, биологических жидкостей организма; - определение структуры белков;

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Протеомика.

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:

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- ;

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:

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-

,

2D- - (MALDI),

--,

(SELDI),


( )


2D-

: :

~ 50 /

, MS ~1 /

Cy3/Cy5 ~ 0.5 /


: 2DE + MALDI-MS

2D

MALDI -

( )

TOF-TOF


2D

MALDI -


-


-


-.

, . - , .


: 1D - nanoLC + nanoESI-MS/MS

1D

-


:HPLC - nanoLC + nanoESI-MS/MS

HPLC + 1DnanoHPLC-nanoMS

-



-- -. - . , . , , , , .


20

(score)

20

  • N

  • gi|8486123 27875 88 reading frame [Influenza A virus]

  • gi|4996872 26451 74 M1 [influenza A virus (A/Taiwan/96/1769)]

  • gi|324260 27872 73 M1 subunit [Influenza A virus]

  • gi|75116 27866 73 M1 - influenza A virus (strain A/WSN/33)

  • -------------

  • 20. gi|6048806 27218 58 M1 [Influenza A virus (A/Duck/Hong Kong/698/79 (H5N3))]

35%

IVPSGPLKAE IAQRLEDVFA GKNTDLEVLM EWLKTRPILS PLTKGILGFV FTLTVPSERG LQRRRFVQNA LNGNGDPNNM

DKAVKLYRKL KREITFHGAK EIALSYSAGA LASCMGLIYN RMGTVTTEVA FGLVCATCEQ IADSQHRSHR QMVTTTNPLI RHENRMVLAS TTAKAMEQMA GSSEQAAEAM DIASQARQMV QAMRTIGTHH SSSAGLKDDL LENLQAYQKR MGVQMQRFK


(/ + )

m/z

Score

  • ,

  • m/z

  • 2. (MS/MS) :

  • 3. Score = S Score

Score


:2DE + MALDI-MS 1DE + LC-ESI-MS/MS

Protein NCBI mW score

conserved hypothetical lipoproteingi|31541381 85818119

conserved hypothetical lipoprotein gi|31541566100472 130 conserved hypothetical lipoprotein gi|31541571 120325 273

conserved hypothetical lipoproteingi|31541607130197 205 conserved hypothetical lipoprotein gi|31541608 133826 168

Protein NCBI mW score

conserved hypothetical lipoproteingi|31541381 85818127

conserved hypothetical lipoprotein gi|31541566100472 817

HepA/SNF gi|31541753 132618341

conserved hypothetical lipoprotein gi|31541571 120325 2152

conserved hypothetical lipoproteingi|31541607130197 1781

lipoprotein gi|45453612 18284 313

lipoprotein gi|4545363818339 362

lipoprotein gi|33327196 25889 436

conserved hypothetical lipoprotein gi|31541608 133826 1008


(PTMs)

  • - (serine, tyrosine, threonine or histidine)

  • -

  • ( N- )

  • -

  • (asparagine, hydroxylysine, serine,

  • threonine)

  • 100


- -

in vitro:

9

6

Cys + I

Cys+

Met

13

13-14

11

( )

in vivo:

S-S


.

Pro-Q Emerald (Mol. Prob.) Sypro Ruby M.gallisepticum

Pro-Q Diamond (Mol. Prob.) Sypro Ruby M.pneumoniae.

2DE M.gallisepticum, 18


2D

IPG-strip pH3-6

ClinProtMB-IMAC Fe (Bruker Daltonics)

MALDI -

- ()

/


- DnaK Mycoplasma gallisepticum

1 MTMSNNNGLI IGIDLGTTNS CVSVMEGAQK VVIENPEGKR TTPSVVSYKN GEIIVGDAAK RQMLTNPNTI VSIKRLMGTS KKVKINDKGV EKELTPEEVS

101 ASILSYLKDY AEKKTGQKIS RAVITVPAYF NDAERQATKT AGKIAGLTVE RIINEPTAAA LAYGIDKGHR EMKVLVYDLG GGTFDVSLLD IADGTFEVMA 201 TAGDNRLGGD DWDNKIIEWI IAEIKKDHPS LDLKSDKMAM QRLKEAAERA KIELSAQLET LISLPFIAVT PEGPVNAELT LSRAKFEELT KDLLERTRNP 301 IADVLKEAKV DPSQVDEILL VGGSTRMPAV QKLVESMIPN KAPNRTINPD EVVAIGAAVQ GGVLRGDVKD ILLLDVTPLT LAIETLGGVA TPIIKRNTTI 401 PVSKQIFSTA QDNQSESVDV SIYQGERPMA RENKSLGTFS LGGIQPAPKG KPQIEITFNI DANGILNVKA KDLTTGKENS ITISNSSELD ENEIQRMIRD 501 AEANKERDAI VKQRIEMRYE GEGIVNTINE ILGSKEAEAL PAQEKASLTK IVDGINGALK AEKWDELKEQ IDGFKKWRDD MSKKYGGGEA PAEPK

80


, .



.

  • HPLC-MS

  • GC-MS

  • Fingerprinting

  • Direct MS

  • HPLC-MS

  • GC-MS

  • CE-MS


, -


.

1. :

1.1. ,

1.2. ,

1.3. ,

1.4. ,

1.5. ,


.

2.

2.1.

(yeast two hybrid system)

(synthetic genetic array (SGA) analysis)

(correlated mRNA expression profile)

2.2.

-



.


6xHis- -.


-(Tandem Affinity Purification, TAP).


(. Surface plasmon resonance)- .

() , , , , .


. - . . , NOE (Nuclear Overhauser effect), , . .

, : . NOE. , . 15N,13C- X-.


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