High-abundance proteins

The quantitation of small-sized therapeutic proteins or peptides in plasma can be conducted with EC—MS/MS detection of the intact proteins. Electrospray ionization (ESI) is the most widely used ionization technique for quantitative EC—MS/MS analysis of proteins or peptides. There are some occasions where atmospheric pressure chemical ionization (APCI) was used to circumvent matrix effects (Volosov et al., 2001). ESI typically generates multiply-charged protein or peptide ions, depending upon the number of basic charges in the polypeptide backbone. For small proteins, high abundance peaks of multiply... 

The use of CIEF in combination with FTICR has been demonstrated in an analysis of the E. coli proteome (Jensen et al., 1999). For these experiments, E. coli was grown in a medium depleted of rare isotopes in order to increase the mass measurement accuracy. The high abundance isotopes are present at approximately 98.89% 12C, 99.63% 14N and 99.985% H. For peptides, the presence of rare isotopes does not significantly change the spectra but with undigested proteins, mass accuracy can be limited by the broadened distribution of ions of any given protein due to the incorporation... 

The ratio of peaks for peptides derived from 42 high abundance yeast proteins was examined for the wild type versus cln2 mutant strains (Oda et al., 1999). Only two of the proteins, a peroxisomal membrane protein and S-adenosylmethionine synthase 2, exhibited significant differences in expression between the strains. The biological significance of this observation is not yet known but the study does indicate that changes of >20% in expression levels can be detected using the technique (Oda et al., 1999). 

SR-BI is highly abundant in POS (Tserentsoodol et al., 2006a). Therefore it may be speculated that it plays a role in further uptake of carotenoid-enriched (lipo)proteins and their transport from the outer segment and then into deeper layers of the retina. 

The proteins of high abundance are often identified by multiple peptides and high statistical confidence. On the contrary, many medium and low abundant proteins are typically identified by only a single peptide. This is a direct result of the semirandom nature of the DDA algorithm (Liu et al., 2004), as discussed above. In addition, these peptide hits often cannot be confirmed by subsequent DDA experiments conducted... 

Figure 12.5 illustrates a typical problem of analysis of minor components present in a matrix of highly abundant ones. Despite the availability of large LC-MS peak capacity (Table 12.3), the number of peptides detected in a semm/plasma digest does not exceed several hundreds (Kapp et al., 2005). These peptides typically match 30-50 high abundant proteins. We believe that the maj ority of remaining proteins/peptides in the sample are present at concentrations well below the LOD of MS instrument. 

Zolotaijova, N., Martosella, J., Nicol, G., Bailey, J., Boyes, B.E., Barrett, W.C. (2005). Differences among techniques for high-abundant protein depletion. Proteomics 5, 3304-3313. 

Many precolumns and trap cartridges for sample clean-up are commercially available. In our experience, a 2 to 3 cm short column with twice the analytical column inner diameter and packed with the same particles performs satisfactorily. An antibody affinity column for selective removal of highly abundant proteins from human serum samples provides better sensitivity for the discovery of low abundance protein markers that may represent revolutionary therapeutic diagnosis and monitoring. 

The Agilent multiple affinity removal system utilizes the specificity of antibody-antigen recognition for 14 highly abundant proteins from human serum samples. The affinity column achieves reproducible and specific depletion from human serum and plasma to eliminate 94% of interfering proteins. It allows identification of proteins down to nanograms per milliliter level as reported by Agilent. 

Quantitative studies comparing the relative abundances of proteins in different cellular states may be performed with MS. Methods such as 2D-GE have been utilized extensively with great success and differentially represent spots excised and then subjected to MS/MS for final identification of the differentially expressed proteins. 2D-GE requires approximately 50 pg of starting material and is limited by its bias toward high abundance proteins and propensity to detect proteins with extreme pi values. Furthermore, proteins at both extremes of molecular weight and those associated with membrane fractions are not well represented by 2D-GE.10... 

In contrast to the Bantu, consumption of high-meat diets by the North American Eskimos has been accompanied by severe osteoporosis. Mazess and Mather ( 0 measured bone densities of both male and female Eskimos of all ages. As early as the fourth decade of life, Eskimo women had bones with less than 857> of the density of age, and sex-matched white women living in the United States. Markedly larger differences of bone occurred in later decades this was true of Eskimos of both sexes. The Eskimo diet, very high in protein, is abundantly supplied with fish, reindeer, moose, caribou, and other meats. 

On the basis of these limitations, in-cell NMR experiments are most useful for the investigation of the folding state of proteins, the binding of small molecules that are highly abundant or are added externally or whenever a catalytic relationship between the interaction partner and the protein of... 

Thioneins are apoproteins that are exceptionally sulfur-rich (composed of greater 30 mol% cysteine). These proteins are found in high abundance in liver and kidney cytoplasm where they form metallothioneins (the holo-protein forms) upon complexation with metal ions. Thi-onein synthesis is induced by the presence of metals, especially zinc, copper, mercury, and cadmium. 

However, analysis of protein mixtures derived from cells, tissues, and body fluids by 2D PAGE by no means represents a comprehensive picture of the proteins in the mixture. Proteins with extreme isoelectric points, large proteins, small proteins, and hydrophobic proteins are commonly not amenable to 2D PAGE and hence can be easily missed. Furthermore, low abundant proteins are often not detected in 2D gels when proteins of high abundance are present. This limitation is particularly relevant when analyzing serum or other body fluids, where protein amounts vary by ten orders of magnitude (Anderson and Anderson 1998). 

Therefore, it appears that the amino acid spectrum in shell matrix proteins and perio-straca of Haliotis is largely a result of the mixing of three different peptide fractions, each one characterized by two amino acids. The six amino acids involved in these three fractions account for about 70 to 80 per cent of the total. The principal difference between shell matrix proteins und periostraca proteins is the high abundance of alanine and serine in the calcified tissue which suggests that the fraction containing these two amino acids is critical in biomineralization processes. 

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