Protein tandem mass spectrometry

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Tandem mass spectrometry (MS/MS) produces precise structural or sequence information by selective and specific induced fragmentation on samples up to several thousand Daltons. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for sequencing by mass spectrometry. The smaller sequences can be used to deduce the sequence of the whole protein. 

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

The liquid chromatography - tandem mass spectrometry (LC/MS/MS) technique was proposed for the determination of corticosteroids in plasma and cerebrospinal fluid (CSF, liquor) of children with leucosis. Preliminai y sample prepai ation included the sedimentation of proteins, spinning and solid-phase extraction. MS detection was performed by scanning selected ions, with three chai acteristic ions for every corticosteroids. The limit of detection was found 80 pg/ml of plasma. 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

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. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

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 2.6. LC-tandem mass spectrometry to examine complex mixtures. The mixture of many different proteins is digested to yield peptides and the peptides are resolved into fractions hy cation exchange chromatography followed by reverse phase chromatography. The fractionation steps resolve the peptides into fractions that he processed hy tandem mass spectrometry to yield sequence information suitable for database searching.

One attempt to overcome these disadvantages has been to use multidimensional liquid chromatography (LC) followed directly by tandem mass spectrometry to separate, fragment and identify proteins (Link et al., 1999). In this process, a denatured and reduced protein mixture is digested with a protease to create a collection of peptides (Fig. 2.6). The peptide mixture is applied to a cation exchange column and a fraction of these peptides are eluted based on charge onto a reverse-phase column. The... 

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.

Neubauer, G., and Mann, M. (1999). Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry potentials and hmitations. Anal. Chem. 71, 235-242. 

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. 

Demirev, P. A. Ramirez, J. Fenselau, C. Tandem mass spectrometry of intact proteins for characterization of biomarkers from Bacillus cereus T spores. Anal. Chem. 2001, 73, 5725-5731. 

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. 

Peng, J., Elias, J.E., Thoreen, C.C., Licklider, L.J., Gygi, S.P. (2003). Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC—MS/MS) for large-scale protein analysis the yeast proteome. J. Proteome Res. 2, 43-50. 

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. 

Von Haller, P.D., Yi, E., Donohoe, S., Vaughn, K., Keller, A., Nesvizhskii, A.I., Eng, J., Li, X.J., Goodlett, D.R., Aebersold, R., Watts, J.D. (2003). The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application of Statistical Tools for Data Analysis and Interpretation. Mol. Cell. Proteomics 2, 428 -42. 

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

Ducret, A., Van Oostveen, I., Eng, J.K., Yates, J.R., 3rd, Aebersold, R. (1998). High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci. 7, 706-719. 

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. 

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