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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 strategy using peptide-concatenated standard, tryptic peptides to be quantified are selected from target proteins and concatenated to generate an artificial protein, which serves as a stable isotope-labeled standard. Colored fragments denote the selected tryptic peptides. Target and standard peptides that form an isotope pair are indicated by similar colors (blue and light blue, red and pink, and green and light green). Peptide-concatenation not only guarantees equimolar spiking of every standard peptide but also eliminates the need to prepare many standard molecules. Two types of peptide-concatenated standard, namely QconCAT [66] and PCS [67], have been reported. While each peptide included in QconCAT is the tryptic peptide per se, that in PCS carries its natural flanking sequences at both sides to faithfully recapitulate the efficiency of proteolytic cleavage in the target protein. Even a peptide excised less efficiently from the target protein (colored in pink) can be precisely quantified with PCS, because its standard (colored in red) is also excised from the PCS at a similar efficiency.
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Figure 2: In strategy using peptide-concatenated standard, tryptic peptides to be quantified are selected from target proteins and concatenated to generate an artificial protein, which serves as a stable isotope-labeled standard. Colored fragments denote the selected tryptic peptides. Target and standard peptides that form an isotope pair are indicated by similar colors (blue and light blue, red and pink, and green and light green). Peptide-concatenation not only guarantees equimolar spiking of every standard peptide but also eliminates the need to prepare many standard molecules. Two types of peptide-concatenated standard, namely QconCAT [66] and PCS [67], have been reported. While each peptide included in QconCAT is the tryptic peptide per se, that in PCS carries its natural flanking sequences at both sides to faithfully recapitulate the efficiency of proteolytic cleavage in the target protein. Even a peptide excised less efficiently from the target protein (colored in pink) can be precisely quantified with PCS, because its standard (colored in red) is also excised from the PCS at a similar efficiency.

Mentions: To overcome these bottlenecks, two groups independently conceived a strategy that uses a peptide concatemer as a standard, namely QconCAT [66] and PCS for peptide-concatenated standard [67]. In both strategies, tryptic peptides used for quantification are concatenated into a single artificial protein. This protein was metabolically labeled with stable isotope in E. coli, purified, and mixed with a protein sample to obtain absolute or stoichiometric quantities of multiple proteins (Fig. 2). This peptide concatenation strategy eliminates the daunting task for the preparation of many standard peptides/proteins. Furthermore, since all isotope-labeled peptides are contained in a single protein, they are always added to the sample at exactly the same molarity. The QconCAT approach provided absolute quantitative data of more than 10 proteins in chick skeletal muscle of different developmental stages [66, 68]. It was also used for quantification of plasma proteins [69]; absolute amounts of 13 proteins in human plasma of 20–10,000 fmol/μl concentrations were successfully measured in an MRM mode.


Mass spectrometry-based approaches toward absolute quantitative proteomics.

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

In strategy using peptide-concatenated standard, tryptic peptides to be quantified are selected from target proteins and concatenated to generate an artificial protein, which serves as a stable isotope-labeled standard. Colored fragments denote the selected tryptic peptides. Target and standard peptides that form an isotope pair are indicated by similar colors (blue and light blue, red and pink, and green and light green). Peptide-concatenation not only guarantees equimolar spiking of every standard peptide but also eliminates the need to prepare many standard molecules. Two types of peptide-concatenated standard, namely QconCAT [66] and PCS [67], have been reported. While each peptide included in QconCAT is the tryptic peptide per se, that in PCS carries its natural flanking sequences at both sides to faithfully recapitulate the efficiency of proteolytic cleavage in the target protein. Even a peptide excised less efficiently from the target protein (colored in pink) can be precisely quantified with PCS, because its standard (colored in red) is also excised from the PCS at a similar efficiency.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: In strategy using peptide-concatenated standard, tryptic peptides to be quantified are selected from target proteins and concatenated to generate an artificial protein, which serves as a stable isotope-labeled standard. Colored fragments denote the selected tryptic peptides. Target and standard peptides that form an isotope pair are indicated by similar colors (blue and light blue, red and pink, and green and light green). Peptide-concatenation not only guarantees equimolar spiking of every standard peptide but also eliminates the need to prepare many standard molecules. Two types of peptide-concatenated standard, namely QconCAT [66] and PCS [67], have been reported. While each peptide included in QconCAT is the tryptic peptide per se, that in PCS carries its natural flanking sequences at both sides to faithfully recapitulate the efficiency of proteolytic cleavage in the target protein. Even a peptide excised less efficiently from the target protein (colored in pink) can be precisely quantified with PCS, because its standard (colored in red) is also excised from the PCS at a similar efficiency.
Mentions: To overcome these bottlenecks, two groups independently conceived a strategy that uses a peptide concatemer as a standard, namely QconCAT [66] and PCS for peptide-concatenated standard [67]. In both strategies, tryptic peptides used for quantification are concatenated into a single artificial protein. This protein was metabolically labeled with stable isotope in E. coli, purified, and mixed with a protein sample to obtain absolute or stoichiometric quantities of multiple proteins (Fig. 2). This peptide concatenation strategy eliminates the daunting task for the preparation of many standard peptides/proteins. Furthermore, since all isotope-labeled peptides are contained in a single protein, they are always added to the sample at exactly the same molarity. The QconCAT approach provided absolute quantitative data of more than 10 proteins in chick skeletal muscle of different developmental stages [66, 68]. It was also used for quantification of plasma proteins [69]; absolute amounts of 13 proteins in human plasma of 20–10,000 fmol/μl concentrations were successfully measured in an MRM mode.

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.