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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.


In the strategy based on intensities of individual peptides, average of intensities of the three most intense ions is used to generate reliable absolute quantitative data. In the strategy based on identification frequency, spectral count (i.e., number of identified MS/MS spectra) is the most useful indicator to estimate protein abundance. Since larger proteins tend to provide more observable peptides than smaller ones, each spectral count is divided by observable peptide number for normalization to ensure accuracy of quantification. For instance, while proteins A and C present at the same abundance have different spectral counts (i.e., 6 and 3, respectively), they share the same normalized spectral count (i.e., 1.5).
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Figure 3: In the strategy based on intensities of individual peptides, average of intensities of the three most intense ions is used to generate reliable absolute quantitative data. In the strategy based on identification frequency, spectral count (i.e., number of identified MS/MS spectra) is the most useful indicator to estimate protein abundance. Since larger proteins tend to provide more observable peptides than smaller ones, each spectral count is divided by observable peptide number for normalization to ensure accuracy of quantification. For instance, while proteins A and C present at the same abundance have different spectral counts (i.e., 6 and 3, respectively), they share the same normalized spectral count (i.e., 1.5).

Mentions: Stable isotope-labeling strategies described above have enabled quantitative MS-based proteomics. However, they inevitably require additional steps for isotope labeling and/or preparation of the standards. By contrast, the so-called label-free (standard-free) method is simple and requires no additional experimental steps; it just exploits peak intensity of peptide ion or identification frequency for a particular protein to obtain quantitative data (Fig. 3 and Table 1). While absolute quantification with stable-isotope labeling can quantify only the proteins with corresponding isotope standards, label-free strategies can, in principle, quantify any protein from which a peptide is unambiguously identified. This implies that the label-free methods are amenable to a large scale analysis. On the other hand, they provide less accurate quantitative values than those by label-based ones, due to run-to-run variations and a stochastic nature of the measurement.


Mass spectrometry-based approaches toward absolute quantitative proteomics.

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

In the strategy based on intensities of individual peptides, average of intensities of the three most intense ions is used to generate reliable absolute quantitative data. In the strategy based on identification frequency, spectral count (i.e., number of identified MS/MS spectra) is the most useful indicator to estimate protein abundance. Since larger proteins tend to provide more observable peptides than smaller ones, each spectral count is divided by observable peptide number for normalization to ensure accuracy of quantification. For instance, while proteins A and C present at the same abundance have different spectral counts (i.e., 6 and 3, respectively), they share the same normalized spectral count (i.e., 1.5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: In the strategy based on intensities of individual peptides, average of intensities of the three most intense ions is used to generate reliable absolute quantitative data. In the strategy based on identification frequency, spectral count (i.e., number of identified MS/MS spectra) is the most useful indicator to estimate protein abundance. Since larger proteins tend to provide more observable peptides than smaller ones, each spectral count is divided by observable peptide number for normalization to ensure accuracy of quantification. For instance, while proteins A and C present at the same abundance have different spectral counts (i.e., 6 and 3, respectively), they share the same normalized spectral count (i.e., 1.5).
Mentions: Stable isotope-labeling strategies described above have enabled quantitative MS-based proteomics. However, they inevitably require additional steps for isotope labeling and/or preparation of the standards. By contrast, the so-called label-free (standard-free) method is simple and requires no additional experimental steps; it just exploits peak intensity of peptide ion or identification frequency for a particular protein to obtain quantitative data (Fig. 3 and Table 1). While absolute quantification with stable-isotope labeling can quantify only the proteins with corresponding isotope standards, label-free strategies can, in principle, quantify any protein from which a peptide is unambiguously identified. This implies that the label-free methods are amenable to a large scale analysis. On the other hand, they provide less accurate quantitative values than those by label-based ones, due to run-to-run variations and a stochastic nature of the measurement.

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.