Sampling for proteomics

Biological Samples for Proteomic Analysis 15.2.2.1 Protein Isolation... 

Parasitic flatworms pose several problematic areas when preparing samples for proteomic analysis. For example, parasites of target species... 

Although liquid chromatography techniques have become quite popular in the separation of peptides in complex protein digests, they are yet to make an impact for the separation of protein samples for proteome-wide applications. It is envisioned that in the future their application for protein separation will increase. Various combinations of reversed-phase (RP)-HPLC with ion-exchange, size-exclusion, chromato-focusing (CF), IEF, and capillary electrophoresis (CE) have emerged for 2D separation of complex mixtures of proteins and peptides. A recent addition in this field is the use of CF as the first dimension and RP-FIPLC as the second-dimension separation device.14 CF is a column-based liquid-phase separation technique, in which proteins are fractionated on the basis of differences in their p/values in a weak ion-exchange column. 

Badock V, Steinhusen U, Bommert K, et al. (2001). Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography. Electrophor. 22 2856-2864. 

The body tissue that is most often sampled for proteomics analyses is the blood, partly because of its relatively easy access and partly because, during its circulation through all other organs, it picks up leakage proteins and thus reflects to some extent all the physiological processes occurring within the body. Of course the downside of this aspect is the consequent complexity... 

Chen, J., Gao, J., Lee, C.S. (2003b). Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies. J. Proteome Res. 2, 249-254. 

Olivova, P., Gilar, M., Dorschel, C. A., Gebler, J.C. (2005). Improved peptide identification and protein coverage for proteomic samples using alternative 2D-HPLC MS/MS approaches. ASMS, 2005, San Antonio, TX Poster TP29. 

Wang, H., Hanash, S. (2005). Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry. Mass Spectrom. Rev. 24, 413 126. 

In one study by Hood et al., 282 of 1153 identified proteins were identified by at least 2 unique tryptic peptides from FFPE prostate cancer (PCa) tissue.9 According to the gene ontology classification of the proteins identified, -65% of proteins were predicted to be intracellular proteins, while -50% of the total human proteome is predicted to be located in the intracellular compartment. Additionally, 20% of the proteins identified in the PCa tissue were classified as membrane proteins, which is significantly less than the predicted 40% for the human proteome. This relative disparity is not unexpected, considering the Liquid Tissue sample preparation kit lacks specific protocols for membrane protein extraction. The Liquid Tissue method has also been used for proteomics studies of a variety of FFPE tissue samples, including pancreatic tumors,28 squamous cell carcinoma,4 and oral human papillomavirus lesions.27... 

The use of mass tagging reagents to analyze proteomic data has greatly improved the ability to compare samples for protein expression differences. However, a major limitation of the ICAT procedure (Section 1, this chapter) is that it can only compare two samples simultaneously, usually a test and a control. Even with the ECAT design (Section 2) using multiple lanthanide metals to make a series of different mass tag signatures, it is difficult to extend the... 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

Bergkvist, J., Ekstrom, S., Wallman, L., Lofgren, M., Marko-Varga, G., Nilsson, J., and Laurell, T. (2002). Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation. Proteomics 2, 422—429. 

Mass spectrometry provides a wealth of information for proteomics research, enzymology, and protein chemistry in general. The techniques require only miniscule amounts of sample, so they can be readily applied to the small amounts of protein that can be extracted from a two-dimensional electrophoretic gel. The accurately measured molecular mass of a protein is one of the critical parameters in its identification. Once the mass of a protein is accurately known, mass spectrometry is a convenient and accurate method for detecting changes in mass due to the presence of bound cofactors, bound metal ions, covalent modifications, and so on. 

Swart, R., Mitulovic, G., Smoluch, M. and Chervet, J.P. (2003) Two dimensional nano LC/MS for the analysis of complex samples in proteomics. European Journal of Pharmaceutical Sciences 19, S10. 

Mouledous L, Hunt S, Harcourt R, Harry J, Williams KL, Gutstein HB. Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples. Proteomics 2003 3(5) 610-615. 

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