Mass spectrometry peptide identification

Mass spectrometry peptide identification and quantification software (e.g., MaxQuant [5] or Proteome Discoverer Software, Thermo Scientific). 

The methods for each study are divided into the initial protein separation step, a second separation step if applicable, the type of mass analysis, and the software used for peptide identification. ID = one dimensional polyacrylamide gel electrophoresis, 2D = two dimensional polyacrylamide gel electrophoresis, MS = mass spectrometry (peptide mass fingerprinting), MS/MS = tandem mass spectrometry, MALDI-TOF = matrix assisted laser desorption/ionization-time of flight, MS FIT = software from Protein Prospector, http //prospector.ucsf edu/, ESI = electrospray ionization, Q-TOF = quadrupole-time of flight, PPSS2 =Protana s Proteomic Software Suite (ProtanaEngineering, Odense, Denmark), Mascot = Matiix Science, http //www.matrixscience.com/, TOF-TOF = MALDI plus TOF tandem mass spectrometry, RP-HPLC = reverse phase high performance liquid chromatography, Q-IT = quadrupole ion trap, LIT = linear ion trap. Bioworks = Thermo Electron Corporation. 

Medzihradszky, K.F., H. Leffler, M.A. Baldwin and A. Burlinghame. Protein identification by in-gel digestion, high-performance liquid chromatography and mass spectrometry peptide analysis by complementary ionization techniques. J. Am. Soc. Mass Spectrom. 12 215-221, 2001. 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

Li, J., Kelly, J.F., Chernushevich, I., Harrison, D.J., and Thibault P., Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectro-spray mass spectrometry, Anal. Chem. 72, 599, 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.

Dai, Y. Li, L. Roser, D. C. Long, S. R. Detection and identification of low-mass peptides and proteins from solvent suspensions of Escherichia coli by high performance liquid chromatography fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 1999,13,73-78. 

Wienkoop, S., Glinski, M., Tanaka, N., Tolstikov, V.V., Fiehn, O., Weckwerth, W. (2004). Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins. Rapid Commun. Mass Spectrom. 18, 643-650. 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

Cargile, B. J., Stephenson, J.L., Jr. (2004). An alternative to tandem mass spectrometry isoelectric point and accurate mass for the identification of peptides. Anal. Chem. 76, 267-275. 

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. 

Janini, G.M., Chan, K.C., Conrads, T.P., Issaq, H.J., Veenstra, T.D. (2004). Two-dimensional liquid chromatography-capillary zone electrophoresis—sheathless electrospray ionization-mass spectrometry evaluation for peptide analysis and protein identification. Electrophoresis 25, 1973-1980. 

The type of proteinaceous binder was correctly identified in all model samples. In only one case (S10), the animal glue was additionally identified, although the restorer who prepared these model samples declared that the sample contained only egg binder. It is possible that this sample was contaminated during its preparation or during laboratory treatment. The results indicate that this method does not allow reliable identification of the presence of individual egg yolk and egg white most probably it is caused by the presence of a trace of egg white that is always present in the egg yolk preparations (and vice versa) and can be detected by the highly sensitive PMM method. The identification of individual types of animal glues will never be reliable by MALDI-TOF mass spectrometry because of their similar composition the application of ESI (electrospray ionisation)-MS/MS (Section 6.5) could possibly overcome this problem. Only the fish glue, whose peptide... 

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