Abstract
A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.
Highlights
A protein of a biological sample is usually quantified by immunological techniques based on antibodies
PHENYX Z-score correlation remained unchanged (R2 ϭ 0.995) after applying a more stringent acceptance criteria in the form of a p value threshold of Յ10Ϫ8. This result indicated that true probabilistic Z-scores, as calculated by PHENYX, are as good to represent protein abundance as spectrum sampling (SpS) and correlated better than MASCOT ion or PeptideProphet probability scores when only first rank identification was used as the sole acceptance criteria for identification
Our results indicate that serum amyloid A4 is constitutively associated with high density lipoproteins (HDLs) where it is maintained at a constant ratio relative to apoA-I as long as HDL cholesterol (HDL-C) is at physiologically normal levels of Ͼ1 mM (Fig. 6, lower left panel)
Summary
A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Integrated peak areas can be influenced by different effects including ion suppression, by limited ion trapping capacity of mass spectrometers, or by the parameters applied to create extracted ion chromatograms, e.g. m/z tolerance, background subtraction, etc Fundamental to all these approaches are correct peptide identifications. Absolute protein quantitation in complex protein mixtures can be determined by the staining intensity of a protein spot on 2DE relative to the total intensity on the gel This approach relies on complete separation of all proteins without any loss, or equal losses between repetitions, in the first dimensional gel and an equal staining efficiency for all proteins. The authors found that PAI values correlated better with logarithmic protein concentrations for unknown reasons
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