Dataset Information

Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

ABSTRACT: Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible.

SUBMITTER: Xian F

PROVIDER: S-EPMC4974354 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

Xian Feng F Zi Jin J Wang Quanhui Q Lou Xiaomin X Sun Haidan H Lin Liang L Hou Guixue G Rao Weiqiao W Yin Changcheng C Wu Lin L Li Shuwei S Liu Siqi S

Molecular & cellular proteomics : MCP 20160527 8

Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptide ...[more]

PMID: 27234506

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Project description:Absolute quantification of peptides by mass spectrometry requires a reference, frequently using heavy isotope-coded peptides as internal standards. These peptides have traditionally been generated by chemical stepwise synthesis. Recently a new way to supply such peptides was described in which nucleotide sequences coding for the respective peptides are concatenated into a synthetic gene (QconCAT). These QconCATs are then expressed to produce a polypeptide consisting of concatenated peptides, purified, quantified by various methods, and then digested to yield the final internal standard peptides. Although both of these methods for peptide production are routinely used for absolute quantifications, there is currently no information regarding the accuracy of the quantifications made in each case. In this study, we used sets of synthetic and biological peptides in parallel to evaluate the accuracy of either method. We also addressed some technical issues regarding the preparation and proper utilization of such standard peptides. Twenty-five peptides derived from the Caenorhabditis elegans proteome were selected for this study. Twenty-four were successfully chemically synthesized. Five QconCAT genes were designed, each a concatenation of the same 25 peptides but each in separate, different randomized order, and expressed via in vitro translation reactions that contained heavy isotope-labeled lysine and arginine. Three of the five QconCATs were successfully produced. Different digestion conditions, including various detergents and incubation conditions, were tested to find those optimal for the generation of a reproducible and accurate reference sample mixture. All three QconCAT polypeptides were then digested using the optimized conditions and then mixed in a 1:1 ratio with their synthetic counterparts. Multireaction monitoring mass spectrometry was then used for quantification. Results showed that the digestion protocol had a significant impact on equimolarity of final peptides, confirming the need for optimization. Under optimal conditions, however, most QconCAT peptides were produced at an equimolar ratio. A few QconCAT-derived peptides were largely overestimated due to problems with solubilization or stability of the synthetic peptides. Although the order in which the peptide sequences appeared in the QconCAT sequence proved to affect the success rate of in vitro translation, it did not significantly affect the final peptide yields. Overall neither the chemical synthesis nor the recombinant genetic approach proved to be superior as a method for the production of reference peptides for absolute quantification.

Dataset Information

Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

Publications

Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

Similar Datasets

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