Proteomics, mass spectrometry quantification

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

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. 

Kuhn E,Wu J, Karl J, et ah Quantification of C-reactive protein in the semm of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics (2004) 4 1-12. 

FIGURE 1 Example of a gel-free-oriented proteomics nano-LC/MS-MS workflow in which bacterial culture proteins digested to tryptic peptides are separated via LC and peptides subsequently analyzed by mass spectrometry. In the process, the spectrometer rapidly cycles every few seconds and examines a size window in which peptide-derived MSI ions are analyzed to define MS/MS (MS2) spectra. The MS/MS (MS2) spectrum generated for each peptide then enters a bioinformatic pipeline for sequence identification, statistical validation, and quantification. 

Keshishian H, Addona T, Burgess M, Mani DR, Shi X, Kuhn E et al (2009) Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 8 2339-2349... 

In the proteomic analysis of the brain, two-dimensional gels for protein separation, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for protein identification, have mainly been employed. This classical proteomics approach allows for the quantification of changes in protein levels and modifications. Simultaneously, it is a robust, well-established method that finds wide application in the study of biological systems. In this article, we provide a description of the protocols of the proteomic analysis used in our laboratory and a summary of the major findings from our group and other neuroproteomics groups. 

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. 

One of the main problems to overcome in order to realise quantitative proteomics is that mass spectrometry is not strictly a quantitative technique with respect to molecular abundance. One approach to overcome this problem is to perform comparative experiments in parallel where one sample is labelled with an isotope (such as 0 or H) and the other is unlabelled. Mass differences between labelled and unlabelled samples may be observed in a mass spectrometer and the relative ratios quantified (Figure 9.21). Obviously this method of quantification relies on equivalent mass expression by both samples in spite of the isotopic labelling of the one and not the other. In other words, in spite of the labelling, both samples should behave identically chemically. The simplest way to effect labelling is to use isotope-coded affinity tags (ICATs). According to this technique, two identical populations of proteins... 

Maass, S. et al (2011) Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem., 83 (7), 2677-2684. 

Background Untreatable metastasis, rather than the primary tumor, is the cause of mortality in breast cancer. Myeloid-derived suppressor cells (MDSCs) are hema-topoetic cells that home specifically to the tumors and have a major role in tumor invasion and metastasis and the development of resistance to chemotherapy. MDSCs proliferate in response to tumors and accumulate in the spleen, from which they can be isolated using their Grl and CDllb surface markers. The objective was to use label-free mass spectrometry and shotgun proteomics to characterize MDSCs that associate with two mouse cell lines derived from the same tumor, one from the primary tumor (67NR) and the other from cells that have already metastasized to various organs (4T1). Spectral counting, for quantification, and protein network analysis were used to search for MDSC biomarkers characteristic to metastasis. 

The field of proteome analysis is challenging because of the complexity of any given proteome, the broad dynamic range of protein abundance, the existence of mnltiple protein forms dne to PTMs, proteolysis and splice variants, and the dif-flcnlty of quantification of absolute protein content. Some of these challenges can be met by the use of complementary approaches to proteomics. In general, four basic steps are involved in proteome analysis (1) sample preparation, (2) separation and purification, (3) mass spectrometry analysis, and (4) database search. 

Barnidge DR, Goodmanson MK, Klee GG, Muddiman DC. Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-MS/MS using protein cleavage and isotope dilution mass spectrometry. J Proteome Res 2004 3(3) 644-652. 

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

Hamdan M and Righetti PG (2002) Modern strategies for protein quantification in proteome analysis advantages and limitations. Mass Spectrometry Reviews 21 287-302. 

Hand in hand with identification and quantification of proteins remains the need to determine full three-dimensional protein structures. Even though thousands of proteins are encoded in the genome, only a relative handful of them have been studied in depth in terms of detailed structure and function. Full structure determination will therefore continue to be central to the field of proteomics. X-ray crystallography, NMR, and mass spectrometry are key tools that will be applied ever more fervently as the quest intensifies to elucidate as many structures in the proteome as possible. 

Langenfeld E, Zanger UM, Jung K, Meyer HE, Marcus K (2009) Mass spectrometry-based absolute quantification of microsomal cytochrome P450 2D6 in human liver. Proteomics 9 2313 2323... 

Seibert C, Davidson BR, Fuller BJ, Patterson LH, Griffiths WJ, Wang Y (2009) Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry. J Proteome Res 8 1672-1681... 

Wang, M., You, J., Bemis, K.G., Tegeler, T.J., Brown, D.P. (2008) Label-free mass spectrometry-based protein quantification technologies in proteomic analysis. Briefings in Functional Genomics and Proteomics, 7, 329-339. 

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