Methods and reagents to label bacteria and virus and identify their interacting proteins
Disclosed herein is a chemo-proteomic probe for labelling and monitoring a live microbe interacting with a host cell and for qualitative and quantitative analyses of those proteins involved during a microbe infects the host cell. This probe comprises a functional group for conjugating to a surface protein of a live microbe under a physiological condition; a photo-reactive group for covalent cross-linking to an interacting cell protein of a host; and a tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting protein of a host cell for qualitative and quantitative proteomics analyses. The probe may further comprise a visualization tag. This technology takes advantage of the high throughput feature of mass spectrometry analysis and combines it with a uniquely designed chemistry to achieve high efficient isolation and analysis of host cell proteins interacting with a pathogen at different stages of an infection.
1. A chemo-proteomic probe comprising:
a) a functional group for conjugating to a surface protein of a live microbe under a physiological condition;
b) a photo-reactive group for covalent cross-linking to an interacting protein of a host cell; and
c) a tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting protein of a host cell for qualitative and quantitative analyses.
2. The chemo-proteomic probe according to claim 1 further comprising a visualization tag for viewing the interactions between the microbe and a host cell in real time.
3. The chemo-proteomic probe according to claim 1 , wherein the live microbe is a pathogen to human or an animal comprising a bacterium or a virus.
4. The chemo-proteomic probe according to claim 1 , wherein the probe is suitable for studying the temporal interactions between a pathogen and its host cell.
5. The chemo-proteomic probe according to claim 1 , wherein the photo-reactive group is a photocrosslinker or a photocleavable caging group.
6. The chemo-proteomic probe according to claim 5 , wherein the photocrosslinker is a diazirine or an aryl azide.
7. The chemo-proteomic probe according to claim 1 , wherein the probe has a formula:
or an acceptable salt thereof, wherein X is said functional group selected from the group consisting of N-hydroxysuccinimide (NHS), maleimide (MAL), or aminooxy (ONH 2 ).
8. The chemo-proteomic probe according to claim 7 , wherein said X is
9. The chemo-proteomic probe according to claim 1 , wherein the tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting protein of a host cell for qualitative and quantitative analyses is selected from the group consisting of biotin, thiobiotin, azide-alkyne, aldehyde-hydrazine, aldehyde-hydroxylamine, thiol-iodoacetyl, and a thiobiotin-based affinity tag.
10. The chemo-proteomic probe according to claim 1 , wherein the live microbe is a bacterium or a virus and the docking surface reactive group of a host cell for covalent conjugation is a reactive group of glycan.
11. The chemo-proteomic probe according to claim 1 , wherein the live microbe is a bacterium or a virus and the docking surface reactive group of a host cell for covalent conjugation is a functional group selected from the group consisting of amino (NH 2 ) and thiol (SH).
12. A method of identifying proteins interactions between a host and a pathogen microbe in real time, comprising the steps of:
a) providing a chemo-proteomic probe comprising: a functional group for conjugating to a surface protein of a live microbe under a physiological condition; a photo-reactive group for covalent cross-linking to an interacting protein of a host cell; and a tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting protein of a host cell for qualitative and quantitative analyses;
b) allowing the microbe to infect the host;
c) crosslinking at fixed time points so that the interacting proteins of the host cells have sufficiently interacted with the surface proteins of the pathogen microbe;
d) isolating the cross-linked complex of the surface proteins of said pathogen microbe and the interacting cell proteins of a host utilizing the tag; and
e) performing qualitative and quantitative analyses of the isolated cross-linked protein complex.
13. The method according to claim 12 , wherein the pathogen microbe is a virus or a bacterium.
14. The method according to claim 12 , wherein the qualitative and quantitative analysis is to sequence the isolated cross-linked protein complex by mass spectroscopy.
15. The method according to claim 12 , wherein the chemo-proteomic probe further comprising a visualization tag to view the microbe-host cell interaction in real time.
16. The method according to claim 15 , wherein the photo-reactive group is a diazirine or an aryl azide.
17. The method according to claim 15 , wherein the chemo-proteomic probe has a formula:
or an acceptable salt thereof, wherein X is said functional group selected from the group consisting of N-hydroxysuccinimide (NHS), maleimide (MAL), or aminooxy (ONH 2 ).
18. The method according to claim 17 , wherein said X is
19. The method according to claim 12 , wherein the tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting cell protein of a host for qualitative and quantitative analyses is selected from the group consisting of biotin, thiobiotin, azide-alkyne, aldehyde-hydrazine/hydroxylamine, thiol-iodoacetyl, and a thiobiotin-based affinity tag.
20. A kit for identifying interactions between a host cell and a pathogen microbe in real time, comprising:
a) a chemo-proteomic probe comprising: a functional group for conjugating to a surface protein of a live microbe under a physiological condition; a photo-reactive group for covalent cross-linking to an interacting cell protein of a host; and a tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting cell protein of a host for qualitative and quantitative proteomics analyses;
and
b) reagents for enabling said conjugation and crosslinking.