Quantitative proteomics, strategies

Wang, S., Zhang, X., Regnier, F. E. (2002). Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates. 

Domon, B. and Aebersold, R. (2010) Options and considerations when selecting a quantitative proteomics strategy. Nat Bioteclmol 28, 710-721. 

Zappacosta, F., and Annan, R.S. (2004) N-terminal isotope tagging strategy for quantitative proteomics Results-driven analysis of protein abundance changes. Anal. Chem. 76, 6618-6627. 

In quantitative proteomics, two alternative strategies have been developed. The first one is based on 2-D PAGE combined with mass spectrometry for protein identification (Haynes and Yates, 2000). This method and current advances in the differential display of proteins by 2-D PAGE are discussed at length in another chapter of this book. The second approach exploits mass spectrometry in combination with stable isotope labeling for gaining accurate quantitative information on proteins. Quantitative MS via stable isotope labeling of proteins... 

Lottspeich, E. (2005). A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5, 4-15. 

Quantitative proteomics requires global approaches to the proteome, as prefractionation of the proteome complicates quantification due to distribution of proteins over various fractions and difficulties in determining the recovery of proteins in complex analytical procedures. However, after labelling and tryptic digestion, the tryptic peptides relevant to the study are preferentially selectively isolated from the very complex digest of the proteome. In the ICAT procedure, this is performed by avidin AfC (Ch. 18.4.1). Alternative strategies are applied, in the liquid phase based on signature peptides, or in FT-ICR-MS. 

The potential of AMT in FT-ICR-MS was already discnssed [31-33], If an AMT can be isotope-coded, the same strategy can be applied to quantitative proteomics. One of the problems of FT-ICR-MS in quantitative proteomics is the space charging constraint of the FT-ICR cell. Data-dependent selective ejection of highly abundant ions and prolonged accumulation of low-abundant ions in the external quadrapole, prior to ion transfer to the FT-ICR cell, helps in extending the dynamic range of the method [108],... 

In the previous sections, comparative quantitative proteomics is discussed. This is aiming at relative quantification of protein levels in various cell states. In some applications, absolute rather than relative quantification of proteins is relevant, e.g., as an alternative to immunoaffinity methods for quantitative bioanalysis of protein dmgs. This requires different strategies. 

In addition to protein identification and characterization, another major goal of proteomics research is to quantify protein expression levels. However, MS is not inherently quantitative. Thus, the intensity of a peptide ion introduced to the mass spectrometer via ESI or MALDI does not reflect necessarily the amount of peptide present in the sample, due to the strong dependence of ionization on the physical and chemical nature of the analyte. To overcome this challenge, numerous quantitative analysis strategies have been developed to measure the differences in protein abundances between two different cellular states of a biological system (for example, normal and diseased cells). 

The objective of quantitative proteomics is to identify differentially expressed proteins in a biological sample. Differential expression of proteins is caused by a disease state, stress due to external factors (drugs, toxins, etc.), or experimental manipulation. Quantitative proteomics can help identify biomarkers of a particular disease and aid in an early diagnostic intervention and prevention of a disease. Several strategies have been developed for quantitative proteomics some are exclusively gel-based approaches, and others require mass spectrometry measurements [66]. 

In another variant for quantitative proteome analysis by 2D PAGE, one could exploit the technique of stable isotope tagging. In this approach, the labeling strategy involves light/heavy forms of the same tagging molecule. An example of such an approach... 

Figure 4 Overview of MS-based strategies for quantitative proteomics. Depending on the point at which the label is introduced, most procedures are classified as (A) in vivo labeling, (B) predigestion labeling in vitro, or (C) postdigestion labeling in vitro.

Table 4.4 Some applications of chemical labeling strategies for quantitative proteomics.

Ji, J. Chakraborty A. Geng, M. Zhang, X. Amini, A. Bina, M. Regnier, F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B Biomed. Sci. Appl. 2000, 745,197-210. 

Zhang, H., Yan, W., and Aebersold, R. (2004) Chemical probes and tandem mass spectrometry A strategy for the quantitative analysis of proteomes and subproteomes. Curr. Opin. Chem. Biol. 8, 66-75. 

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