Molecular Techniques and Methods

Quick Identification of Unknown Proteins
by LC-Mass Spectrometer (LCQ DECA) Analysis

Copy Right © 2001/ Institute of Molecular Development LLC



INTRODUCTION

Target protein is purified by 2D gel electrophoresis. The purified protein is then partially digested by trypsin in gel to 1 kDa in size. The resulting peptides is analyzed by mass spectrometer (LCQ DECA). MS-data is analyzed by Sequest software.

The Finnigan LCQ DECA is an ultra-sensitive, ion trap LC mass spectrometer built upon the proven reliability of the company's classic LCQ. It provides unparalleled sensitivity and specificity among LC/MS analytical instruments for research applications. It has a completely redesigned API interface and ion optics which are responsible for ion formation, transmission and focussing. The resulting MS sensitivity is enhanced by an order of magnitude over its predecessor the LCQ. The instrument is coupled with the Finnigan Surveyor high-performance liquid chromatography system that provides a wide range of chromatographic options including nL/minute capillary experiments. Specified sensitivity for the DECA is 100:1 signal to noise ratio for a injection of 82 fmol of reserpine. A recent demonstration provided a reasonable MS/MS spectrum with just 7 fmol. Sub-picomole protein identification is common. Several features of the DECA make the instrument highly suited to proteomics applications. A central feature is data-dependent acquisition; as an LC peak elutes the mass spectrometer detects first the most abundant ion, it then performs a zoom scan that focuses on this ion alone and determines its charge state and thus the range of the scan required for the subsequent MS/MS experiment. The process is repeated for all the ions in the spectrum thereby generating exhaustive MS/MS data for all peptides in a given separation. Sequest software then takes data from the MS/MS experiments and challenges the database of choice for potential sequence matches. Throughput is maximized through use of the auto injector feature of Surveyor allowing multiple runs to be performed in the absence of the operator. A host of features ensure versatile operation and ensure that this instrument is the most dedicated proteomics machine available.




MATERIALS AND SOLUTIONS

Rehydration Solution-1 (100 ml)
10 mM DTT ------------------------------------------- 1 ml of 1 M DTT
100 mM NH4HCO3 ----------------------------------- 0.8 g NH4HCO3
Add distilled H2O to make a final volume of ---------- 100 ml


Rehydration Solution-2 (100 ml)
100 mM NH4HCO3 ----------------------------------- 0.8 g NH4HCO3
Add distilled H2O to make a final volume of ---------- 100 ml


Trypsin
Promega sequencing grade modified (V5111) - 100 ug


Trypsin Solution (1 ml)
Rehydration Solution-2 --------------------------------- 0.5 ml
5 mM CaCl2 ------------------------------------------- 5 ul of 1 M CaCl2
Trypsin ------------------------------------------------- 12.5 ug
Add distilled H2O to make a final volume of ---------- 1 ml


Incubation Buffer (1 ml)
Rehydration Solution-2 --------------------------------- 0.5 ml
5 mM CaCl2 ------------------------------------------- 5 ul of 1 M CaCl2
Add distilled H2O to make a final volume of ---------- 1 ml


Extraction Buffer (10 ml)
5 % Formic acid ---------------------------------------- 0.5 ml of 100% Formic acid
50 % Acetonitrile --------------------------------------- 5 ml of Acetonitrile
Distilled H2O ------------------------------------------- 4.5 ml


70% Acetonitrile: TFA buffer (100 ml)
70% Acetonitrile --------------------------------------- 70 ml of Acetonitrile
0.1% TFA --------------------------------------------- 100 ul of TFA
Distilled H2O ------------------------------------------- 30 ml


5% Acetonitrile: TFA buffer (100 ml)
5% Acetonitrile ---------------------------------------- 5 ml of Acetonitrile
0.1% TFA --------------------------------------------- 100 ul of TFA
Distilled H2O ------------------------------------------- 95 ml


SEP-PAK C18 Cartridge
Millipore-Waters Associates (Part No. 51910)




PROCEDURES

1. Run 2-D gel electrophoresis to purify the target protein.

2. Silver stain proteins in gel.

3. Carefully excise the target protein band.

4. Dehydrate the gel slice in acetonitrile (200 µl) for 30 min.

5. Remove liquid, dry in SpeedVac.

6. Add Rehydration solution to cover gel pieces.

7. Incubate 1 hr at 56°C

8. Cool to room temperature.

9. Add 1/10th vol. of 550 mM Iodoacetamide.

10. Incubate 45 min at room temperature in DARK.

11. Wash with 50-100 µL Rehydration solution-2 for 10 min.

12. Dehydrate with acetonitrile.

13. Swell the gel slice in 50-100 µL Rehydration solution-2 for 30 min.

14. Dehydrate with acetonitrile.

15. Remove liquid, and dry in SpeedVac.

16. Swell in Trypsin Solution (50-100 µl) on ice for 45 min.

17. Remove Trypsin Solution.

18. Add 5-10 µl Incubation Buffer.

19. Incubate at 37 °C, overnight.

20. Extract peptides in 200 ul Extraction Buffer for 20 min.

21. Repeat step 20 one more time.

22. Vacuum dry protein.

23. Resuspend the prortein pellet in 500 ul of 5% Acetonitrile: TFA buffer.

24. Clean the extracted protein by Sep-Pak C18 Cartridge as follow.


Preparation of Sep-Pak C18 Cartridge

25. Rinse Sep-Pak C18 Cartridge by 5 ml of methanol.

26. Rinse Sep-Pak C18 Cartridge by 5 ml of 70% Acetonitrile: TFA buffer.

27. Rinse Sep-Pak C18 Cartridge by 5 ml of 5% Acetonitrile: TFA buffer.

28. Load protein solution from step 23.

29. Wash protein-loaded Sep-Pak C18 Cartridge by 5 ml of 5% Acetonitrile: TFA buffer.

30, Repeat step 29, 2 x more.

31. Elute protein from Sep-Pak C18 Cartridge by 2 ml of 70% Acetonitrile: TFA buffer.

32. Vacuum dry the eluted peptides.

33. Continue to mass spectrometer (LCQ DECA) analysis.




NOTES




KIT INFORMATION




REFERENCES

  • le Coutre J., Whitelegge J.P., Gross A., Turk E., Wright E.M., Kaback H.R. and Faull K.F. (2000) Proteomics on Full-Length Membrane Proteins Using Mass Spectrometry. Biochemistry 39: 4237-4242.

  • Whitelegge J.P., le Coutre J., Lee J.C., Engel C.K., Privé G.G., Faull K.F., and Kaback H.R. (1999) Towards the Bilayer Proteome, Electrospray-Ionization Mass Spectrometry of Large Intact Transmembrane Proteins. PNAS 96: 10695-10698.

  • Blanco DR, Whitelegge JP, Miller JN and Lovett MA (1999) Demonstration by mass spectrometry that purified native Treponema pallidum rare outer membrane protein 1 (Tromp1) has a cleaved signal peptide. J. Bacteriol. 181(16): 5094-5098.

  • Whitelegge J.P., Gundersen C. and Faull K.F. (1998) Electrospray-Ionization Mass Spectrometry of Intact Intrinsic Membrane Proteins. Protein Science 7 (6): 1423-1430.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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