Proteomics analytical strategies

With apologies to authors whose relevant work was excluded because of space constraints, we conclude that the use of photoaffinity/ proteomic strategies in connection with drug function has gained significant momentum from the availability of proteomic analytical methods. 

The use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays essential to solve the proteome complexity. Currently, more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) combined with the everyday more powerful mass spectrometers. Two fundamental analytical strategies can be employed the bottom-up and the top-down approach. 

Yasui Y, Pepe M, Thompson ML, Adam BL, Wright GL, Qu Y, Potter JD, Winget M, Thornquist M, Feng Z (2003) A data-analytic strategy for protein biomarker discovery profiling of high-dimensional proteomic data for cancer detection. Biostatistics 4 449-463... 

The study of protein structure, function, quantity, and interactions during maturation and progression of disease is referred to as proteomics. Analytical approaches that use a combination of two-dimensional (2-D) gel electrophoresis for protein separation and MS analysis for protein identification followed by database searches is a widely practiced proteomics strategy.The tryptic peptides extracted from gels are analyzed by MALDI-TOF MS and microcolunm or capillary LC tandem mass spectrometry (MS/MS) techniques. Typically, the MALDI-TOF MS techniques are used to quickly identify peptide fragments and confirm the presence of known proteins. Nano-scale capillary LC/MS/MS techniques (using 50-100 pm diameter columns, operating at flow rates of 20-500 nL/min) are... 

Tiller et al. demonstrated an analytical strategy with on-line LC/UV/MS and LC/MS/MS to rapidly obtain structural information for leachables from a drug-delivery device. Similar to proteomics-based applications, the analysis strategy makes use of data-dependent analysis, wherein the mass spectrometer first obtains molecular ions using full-scan techniques, and makes real-time decisions about MS/MS product-ion spectra that must be obtained. In this way, molecular weight and substructural information are both obtained for many components during a single HPLC run. 

No current analytical strategy is capable of fully resolving complex biological samples. For this reason, orthogonal separation techniques are often combined to maximize peptide separation before the mass spectrometric analysis. The aim is to reduce as much as possible the number of coeluting peptides introduced into the MS at any given time, so as to maximize peptide identification and proteome coverage. 

Proteomics is a rapidly expanding field whose aim is to systematically study protein structure, function, interactions, and dynamics. Much effort is presently directed at the development of analytical strategies to analyze many proteins simultaneously. This developing technology is also influencing the design of critical medical studies and questions in biochemistry and bioorganic chemistry. 

Analytical Strategies to Overcome the Mixture Complexity and Dynamic Range Limitations Associated with Proteome Analysis... 

Fig. 11.20. Analytical strategies in (a) protein biochemistry requiring highly purified proteins and (b) proteomics addressing the entire proteome of a Uving ceU. Adapted from Chap. 3 by Hjemp and Jensen in Ref. [15] by permission. Wiley-VCH, Weinheim, 2007.

Thingholm TE, Jensen ON, Larsen MR (2009) Analytical strategies for phosphopro-teomics. Proteomics 9 1451-1468... 

Due to the lack of suitable standard reference material for quantification purposes in phospho-proteomics and metallomics, reliable calibration strategies were developed for the direct microlocal analysis of phosphorus and metals in protein spots and in thin sections of brain tissue using LA-ICP-MS.16,17,116 For quantification of analytical data, the application of a solution based calibration strategy was proposed with LA-ICP-MS6 and the simultaneous determination of P, S, Si, Al, Cu and Zn concentrations in human brain proteins (Alzheimer s disease) or for imaging thin... 

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. 

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. 

In this chapter, we focus on the strategies that have been developed during the past few years to increase the proteome coverage in shotgun proteomics studies (Figure 1). The analytical techniques that might increase the fragmentation rate are outlined briefly in Section 2 (for a comprehensive review,... 

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