Proteomics based on Mass

A compound or material that is not an analyte but is included in an unknown or standard to correct for issues in the processing or analysis of an analyte or analytes an internal standard is not a calibration standard. See Julka, S. and Regnier, F, Quantification in proteomics through stable isotope coding a review, J. Proteome Res. 3, 350-363, 2(X)4 Bronstrup, M., Absolute quantification strategies in proteomics based on mass spectrometry. Expert Rev. Proteomics 1, 503-512, 2004 Coleman, D. and Vanatta, L., Statistics in analytical chemistry, part 19-intemal standards, American Laboratory, December 2005. 

Proteomics Based on Mass Spectrometry —Identification of Proteins Based on Their Amino Acid Sequence... 

Bronstrup M. Absolute quantification strategies in proteomics based on mass spectrometry. Expert Rev Proteomics 2004 1(4) 503—512. 

Regnier, F. E. Riggs, L. Zhang, R. Xiong, L. Liu, P. Chakraborty A. Seeley E. Sioma, C. Thompson, R. A. Comparative proteomics based on stable isotope labeling and affinity selection. J. Mass. Spectrom. 2002,37,133-145. 

MS is the heart of proteomic analysis and the success of proteomic experiments depends largely on the sensitivity and accuracy of MS equipment used to identify peptide sequences. MS machines have three main components (Figure 2) a source, which generates peptide ions, a mass analyzer, which separates peptide ions based on mass to charge ratio (m/z), and a detector that detects the ion resolved by the mass analyzer. All the modern MS machines are computer controlled and assisted by highly intelligent software. 

Cruz-Monteagudo, M., Gonzalez Diaz, H., Borges, F., Dominguez, E.R. and Cordeiro, M.N.D.S. (2008) 3D-MEDNEs an alternative in silico technique for chemical research in toxicology. 2. Quantitative proteome-toxicity relationships (QPTR) based on mass spectrum spiral entropy. Chem. Res. Toxicol., 21, 619-632. 

After evaluation in toxicology studies, several enzymes have been discarded as having little or no additional diagnostic value, or perhaps due to the lack of suitable automated methods and available reagents. These findings are not dissimilar to those in human and veterinary medicine. Enzyme patterns obtained by proteomic techniques may lead to more sensitive assays based on mass rather than activity, and this may lead to biochip technology to further develop novel assays. 

In this chapter we will review proteomic investigations of cardiac proteins and focus on their application to the study of heart disease in the human and in animal models of cardiac dysfunction. The majority of these studies of the cardiac proteome have involved protein separation, visualisation and quantitation using the traditional 2-DE approach combined with protein identification by mass spectrometry. These essential technologies will be briefly described. However, there is increasing interest in using alternative gel-free techniques based on mass spectrometry or protein arrays for high throughput proteomics. These alternative approaches will be introduced, but further details can be found in Chapter 2 of this volume by Michel Faupel. 

Finally in this section, it must be emphasized that relative quantitative proteomics measurements currently use a range of methodologies other than those based on mass spectrometry. Recently (Unwin 2006) this range of techniques, including those summarized above plus protein arrays and flow cytometry, has been reviewed... 

Bottom-up proteomics based on two-dimensional (2D) gel electrophoresis followed by the tryptic digestion of spots and analysis by mass spectroscopy (MS). ... 

Methods based on liquid chromatography-mass spectrometry (LC-MS) and universally accepted search algorithms permit reliable identifications of low levels of proteins at high sensitivity [6]. Even semispecialized protein chemistry labs can readily identify proteins at the level of a few picomoles (10 pmol of a 50-kDa protein is 500 ng). Specialized groups with access to the latest advances in HPLC and mass spectrometry routinely work with subpicomolar quantities. Chemical proteomics as discussed here requires the more advanced equipment. 

Mass analyzers interrogate and resolve ions produced by an ion source based on their m/z ratios. Several types of mass analyzers are utilized for proteomic analysis including time-of-flight (TOF) quadrupoles, ion traps, and Fourier transform ion cyclotron resonance (FTICR). Mass analyzers may be assembled in hybrid configurations. MS instruments such as quadrupole TOF and quadra-pole ion trap-FTICR facilitate diversified applications and achieved great success. 

Figure 7.5. Simulation results that elucidate how the sensitivity and the selectivity of a proteomics experiment depend on various features (a) The choice of algorithm. The probity algorithm displays better sensitivity and selectivity than an algorithm that ranks strictly based on the number of matches, (b) The search conditions. Increasing the mass window of a search 10 times when searching with data that display small mass errors yields worse sensitivity and selectivitry. (c) The quality of the data. Data with less noise yields better sensitivity and selectivity.

The second step in proteomic research is to identify the separated proteins. This can be achieved by MS. Here, proteins are differentiated based on their mass-to-charge ratio (m/z). At first, the protein molecule is ionized. The resultant ion is propelled into a mass analyzer by charge repulsion in an electric field. Ions are then resolved according to their m/z ratio. Information is collected by a detector and transferred to a computer for analysis. The most commonly used ionization methods are... 

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