Tandem mass spectrometry protein identification

A general protocol for mass spectrometric analysis of phosphoproteins is illustrated in Figure 15 various steps of this protocol are cleavage of purified phosphoproteins, isolation and preferential enrichment of phosphopeptides, selective detection of phosphopeptides in the digest, identification of the phosphorylation sites using tandem mass spectrometry, and identification of phosphopeptides/proteins through a database search. 

Kinter, M. and Sherman, N.E., Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley, Chichester, U.K., 2000. 

Gatlin, C.L., Eng, J.K., Cross, S.T., Detter, J.C., and Yates, J.R III, Automated Identification of amino acid sequence variations in proteins by HPLC/mi-crospray tandem mass spectrometry, Anal. Chem., 72, 757, 2000. 

Tandem mass spectrometry (MS/MS) is another common approach used for protein identification. In this method, proteins are digested and the resulting peptides are ionized directly from the liquid phase by... 

The major advantage of the tandem mass spectrometry approach compared to MALDI peptide fingerprinting, is that the sequence information obtained from the peptides is more specific for the identification of a protein than simply determining the mass of the peptides. This permits a search of expressed sequence tag nucleotide databases to discover new human genes based upon identification of the protein. This is a useful approach because, by definition, the genes identified actually express a protein. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Figeys, D. Aebersold, R. High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry inital comparison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer. Electrophoresis 1997,18, 360-368. 

Mawuenyega, K.G., Kaji, H., Yamuchi, Y., Shinkawa, T., Saito, H., Taoka, M., Takahashi, N., Isobe, T. (2003). Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography—tandem mass spectrometry. J. Proteome Res. 2, 23-35. 

Qian, W.J., Liu, T., Monroe, M.E., Strittmatter, E.F., Jacobs, J.M., Kangas, L.J., Petritis, K., Camp, D.G., 2nd, Smith, R.D. (2005b). Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis the human proteome. J. Proteome Res. 4, 53-62. 

Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Nat. Acad. Sci. USA 102, 9463-9468. 

The above-mentioned method is effective in identifying the molecules of detected ions. However, because PVDF film is not permeable to light, it is difficult to observe tissue sections. To resolve this problem, we developed a method to fix tissue sections on transparent film, and then performed MS on those sections.6 We used a conductive film because we expected the ionization efficiency would increase when the electric charge accumulation on the sample was reduced. The film used for this purpose was a polyethylene terephthalate (PET) film with a thickness of 75-125 pm, having a 5 15-nm-thick layer of evaporated oxidation indium tin (ITO) upon it (ITO film). This film is used in touch-panel displays because of its high transparency and superior conductivity. We used it to perform MS/MS for tissue sections and succeeded in identifying multiple proteins from mass spectra.6 Therefore, the further development of this method will enable the application of the mass-microscopic method to observe tissue by optical microscope and to perform tandem mass spectrometry (MSn) at the observation part, simultaneously, enabling the identification of molecules included the part. 

C. Protein Identification Via Electrospray Tandem Mass Spectrometry. . 13... 

Even though the MALDI peptide mass mapping technique is very powerful, it has limitations. It requires well-separated proteins, is less sensitive than identifications based on electrospray tandem mass spectrometry, can only identify proteins whose complete sequences are available in databases, and does not produce redundant information. 

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

Figeys, D., Van Oostveen, I., Ducret, A., and Aebersold, R. (1996). Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level. Anal. Chem. 68, 1822—1828. 

Figeys, D., Lock, C., Taylor, L., and Aebersold, R. (1998). Microfabricated device coupled with an electrospray ionization quadrupole time-of-flight mass spectrometer protein identifications based on enhanced-resolution mass spectrometry and tandem mass spectrometry data. Rapid Commun. Mass Spectrom. 12, 1435 — 1444. 

Coon, J.J., Ueherheide, B., Syka, J.E., Dryhurst, D.D., Ausio, J., Shahanowitz, J. and Hunt, D.E. (2005) Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 102, 9463-9468. 

Dongre, A.R., Eng, J.K., Yates, J.R. Ill (1997) Emerging tandem-mass-spectrometry techniques for the rapid identification of proteins. Trends Biotechnol. 15, 418-425. 

Fig. 17.9. Schematic diagram of MuDPIT proteomics (adapted from Yates, 1998). Protein mixtures of interest are cleaved via proteolytic activity and separated via a series of liquid chromatography separations. Identification of the proteins present relies on sequence tags produced from tandem mass spectrometry. Even low-abundance proteins should be represented at least on one occasion.

D. Figeys, A. Ducret and R. Aebersold, Identification of proteins by capillary electrophoresis-tandem mass spectrometry , Evaluation of an on-line solid-phase extraction device , J. Chromatogr. A 763 295-306 (1997). 

D. Figeys, A. Ducret, J. R. Yates-III and R. Aebersold, Protein identification by sohd phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry , Nat. Biotechnol. 14 1579-1583 (1996). 

D. Figeys, I. van Oostveen, A. Ducret, and R. Aebersold, Protein identification by capillary zone electrophoreis/microelectrospray ionisation-tandem mass spectrometry at the subfemto-mole level, Anal. Chem., 68 (1996) 1822-1828. 

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