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Mass spectrometry-based approaches toward absolute quantitative proteomics.

Kito K, Ito T - Curr. Genomics (2008)

Bottom Line: More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides.On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides.Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8561, Japan.

ABSTRACT
Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.

No MeSH data available.


Different types of stable isotope standard are spiked at different steps of the sample preparation procedure. Intact protein standard can be spiked as soon as proteins are extracted from cells, tissues or bloods, even if subsequent fractionation steps (e.g., SDS-PAGE, gel filtration) are included in the procedure. While synthetic peptide standard is spiked before or after digestion with protease, peptide-concatenated standard has to be spiked prior to digestion to allow co-proteolysis of target and standard. Note that synthetic peptide and peptide concatenated standard have to be spiked after protein fractionation steps.
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Figure 1: Different types of stable isotope standard are spiked at different steps of the sample preparation procedure. Intact protein standard can be spiked as soon as proteins are extracted from cells, tissues or bloods, even if subsequent fractionation steps (e.g., SDS-PAGE, gel filtration) are included in the procedure. While synthetic peptide standard is spiked before or after digestion with protease, peptide-concatenated standard has to be spiked prior to digestion to allow co-proteolysis of target and standard. Note that synthetic peptide and peptide concatenated standard have to be spiked after protein fractionation steps.

Mentions: In MS-based absolute quantification, a known amount of isotope-labeled authentic standard is mixed with the analyte, and the mixture is introduced into mass spectrometer. The absolute amount of the analyte is calculated from the ratio of ion intensity between the analyte and its standard. Accordingly, known amounts of stable isotope-labeled synthetic peptides, proteins, or peptide concatemers have been used as a standard for absolute or stoichiometric quantification of proteins. Different types of standard are added to the samples at the different stages of the procedure, and have distinct pros and cons (Fig. 1 and Table 1). Accordingly, the most suitable standard should be selected, depending on the purpose of the experiment, or on whether it intends to quantify a small number of targets including their post-translational modifications, obtain highly accurate data for a single unique protein, or measure absolute or stoichiometric abundance of many proteins.


Mass spectrometry-based approaches toward absolute quantitative proteomics.

Kito K, Ito T - Curr. Genomics (2008)

Different types of stable isotope standard are spiked at different steps of the sample preparation procedure. Intact protein standard can be spiked as soon as proteins are extracted from cells, tissues or bloods, even if subsequent fractionation steps (e.g., SDS-PAGE, gel filtration) are included in the procedure. While synthetic peptide standard is spiked before or after digestion with protease, peptide-concatenated standard has to be spiked prior to digestion to allow co-proteolysis of target and standard. Note that synthetic peptide and peptide concatenated standard have to be spiked after protein fractionation steps.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682933&req=5

Figure 1: Different types of stable isotope standard are spiked at different steps of the sample preparation procedure. Intact protein standard can be spiked as soon as proteins are extracted from cells, tissues or bloods, even if subsequent fractionation steps (e.g., SDS-PAGE, gel filtration) are included in the procedure. While synthetic peptide standard is spiked before or after digestion with protease, peptide-concatenated standard has to be spiked prior to digestion to allow co-proteolysis of target and standard. Note that synthetic peptide and peptide concatenated standard have to be spiked after protein fractionation steps.
Mentions: In MS-based absolute quantification, a known amount of isotope-labeled authentic standard is mixed with the analyte, and the mixture is introduced into mass spectrometer. The absolute amount of the analyte is calculated from the ratio of ion intensity between the analyte and its standard. Accordingly, known amounts of stable isotope-labeled synthetic peptides, proteins, or peptide concatemers have been used as a standard for absolute or stoichiometric quantification of proteins. Different types of standard are added to the samples at the different stages of the procedure, and have distinct pros and cons (Fig. 1 and Table 1). Accordingly, the most suitable standard should be selected, depending on the purpose of the experiment, or on whether it intends to quantify a small number of targets including their post-translational modifications, obtain highly accurate data for a single unique protein, or measure absolute or stoichiometric abundance of many proteins.

Bottom Line: More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides.On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides.Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8561, Japan.

ABSTRACT
Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.

No MeSH data available.