Proteome Analysis by Mass Spectrometry

MS-MS can provide enhanced information on the individual peptide contained in a proteolytic digest, facilitating the identification of the proteins and offering the possibility of de novo sequencing (Kinter and Sherman, 20(X)) when no representative entry is in a database. Typically in a first pass, the introduced peptides are separated according to m/z by the mass spectrometers (MS spectrum). A list of peptides with signals above a pre-established threshold is created. In a second pass, a mass window centered on a selected peptide is isolated by the mass spectrometer and the kinetic energy of the selected peptide is increased. The collision of the peptide with small gas molecules (CID) transfers 

Additional analyses known as orthogonal methods have been developed to increase the confidence of peptide-mass assignments. The orthogonal information restricts the number of peptides that match the isobaric (same mass) peptides, which are identical in the original database search based on peptide masses. The orthogonal methods include the 

TABLE 16.15 Resource sites with MS-based protein identification tools 

CBRG http //cgrg.inf.ethz.ch/MassSearch.html 

Proteome, ExPASy http //www.expasy.ch/tools/ proteome 

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Leitner, A., and Lindner, W. (2004) Current chemical tagging strategies for proteome analysis by mass spectrometry./. Chrom. B813, 1-26. 

Proteome analysis by mass spectrometry. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 399-424. 

Griffin, T.J., Goodlett, D.R., Aebersold, R. (2001). Advances in proteome analysis by mass spectrometry. Curr. Opin. Biotechnol., 12(6), 607-612. 

Griffin TJ, Lock CM, Li XJ, Patel A, Chervestsova I, Lee H, Wright ME, Ranish JA, Chen SS, Aebersold R. Abundance ratio-dependent proteomic analysis by mass spectrometry. Anal Chem 2003 75 867-874. 

Figure 3.42 Proteomic analysis by mass spectrometry. This mass spectrum was obtained by analy7ing a trypsin-treated band in a gel derived from a yeast nuclear-pore sample. Many of the peaks were found to match the masses predicted for peptide fragments from three proteins (Nupl2Gp. Kapl22p. and Kapl20p) within the yeast genome. The band corresponded to an apparent molecular mass of 100 kd. [From M. P. Rout, J. D. Aitchison, A. Suprapto,...

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Joubert-Caron R et al. Protein analysis by mass spectrometry and sequence database searching a proteomic approach to identify human lymphoblastoid cell line proteins. Electrophoresis 2000 21 2566— 2575. 

Steen H, Jebanathirajah JA, Rush J, Morrice N, Kirschner MW. Phosphorylation analysis by mass spectrometry Myths, facts, and the consequences for qualitative and quantitative measurements. Mol. Cell. Proteomics 2006 5 172-181. 

The miscellaneous modifications described in this section involve only a small number of proteins and have not been subjected to throughput analysis by proteomic analysis and mass spectrometry. 

Over the years, many different approaches based on these two basic principles have been developed.4 9 We decided to focus on developing an approach to transfer analytes by coupling capillary tubing with electrospray ionization devices. From this basic design principle, we were able to develop a simple three-position device for the analysis of proteomic samples by mass spectrometry.7,10 We developed this principle further into an automated nine-position device,6 and to perform frontal analysis separations of peptides.11 This chapter reviews these early developments in coupling microfabricated devices to mass spectrometers. 

Joubert-Caron, R., Le Caer, J.P., Montandon, F., Poirier, F., Pontet, M., Imam, N., Feuillard, J., Bladier, D., Rossier, J., and Caron, M., 2000, Protein analysis by mass spectrometry and sequence database searching a proteomic approach to identify human lymphoblastoid cell line proteins [In Process Citation]. Electrophoresis 21 2566-2575. 

Gauss, C., Kalkum, M., Lowe, M., Lehrach, H., and Klose, J. (1999). Analysis of the mouse proteome. (I) Brain proteins Separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575-600. 

Griffin, T. J. Han, D. K. Gygi, S. R Rist, B. Lee, H. Aebersold, R. Parker, K. C. Toward a high-throughput approach to quantitative proteomic analysis Expression-dependent protein identification by mass spectrometry. J. Am. Soc. Mass. Spectrom. 2001,12,1238-1246. 

Adkins, J.N., Vamum, S.M., Auberry, K.J., Moore, R.J., Angell, N.H., Smith, R.D., Springer, D.L., Pounds, J.G. (2002). Toward a human blood serum proteome analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics 1,47-955. 

However, IHC as a practical method continues to evolve with increasing demands for standardization, and for true quantification of protein analytes by weight, in the context of their cellular microenvironment. Further studies combining proteomics by mass spectrometry and IHC are likely to lead to the refinement of both methods in the analysis of FFPE tissues. The end result may be the creation of a broader field that defines and quantifies protein expression at a cellular level, incorporating the advantages of the wide spectrum of proteins demonstrable by mass spectrometry and the precise localization offered by IHC. 

Zhou, H., Ranish, J.A., Watts, J.D., and Aebersold, R. (2002) Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat. Biotechnol. 20, 512-515. 

Mann, M., Hendrickson, R.C., and Pandey, A. 2001. Analysis of proteins and proteomes by mass spectrometry. Annual Review of Biochemistry 70, 437-473. 

Beranova-Giorgianni, S. Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry strengths and limitations. TrAC 2003, 22, 273-281. 

Ha GH, Lee SU, Kang DG et al. Proteome analysis of human stomach tissue separation of soluble proteins by two-dimensional polyaerylamide gel eleetrophoresis and identification by mass spectrometry. Electrophoresis 2002 23 2513-2524. 

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