Comparative Evaluation of Current Peptide Production Platforms Used in Absolute Quantification in Proteomics

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. 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. Systems biology depends intimately on the ability to make quantitative measurements of the transcriptome, proteome, and metabolome. Quantitative proteomics has grown in the last 6–7 years to become a major source of information for systems biology studies where iterative measurements of reproducible sets of proteins from differentially perturbed biological states are essential. Quantification in proteomics is typically performed by a means of relative measurement (1Aebersold R. 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Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3721) Google Scholar). Label-free quantification has also been established as a reliable method for relative quantification in recent years (5Rinner O. Mueller L.N. Hubalek M. Mueller M. Gstaiger M. Aebersold R. An integrated mass spectrometric and computational framework for the analysis of protein interaction networks.Nat. Biotechnol. 2007; 25: 345-352Crossref PubMed Scopus (146) Google Scholar, 6Wang G. Wu W.W. Zeng W. Chou C. Shen R. Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: reproducibility, linearity, and application with complex proteomes.J. Proteome Res. 2006; 5: 1214-1223Crossref PubMed Scopus (229) Google Scholar, 7Askenazi, M. (July 12, 2006) Iterative Base Peak Framing of Mass Spectrometry Data. World Patent WO/2006/130368Google Scholar, 8Kipping M. Pollack L. Langridge J. Label free quantitative proteomics: high resolution electrospray LC-MS for functional proteome analysis.BIOspektrum. 2005; 11: 780-781Google Scholar). Despite many attractive features of these technologies, relative quantification methods suffer from serious limitations that constitute a major bottleneck in large scale proteomics (9Puetz S. Reinders J. Reinders Y. Sickmann A. Mass spectrometry-based peptide quantification: applications and limitations.Expert Rev. Proteomics. 2005; 2: 381-392Crossref PubMed Scopus (42) Google Scholar). For example, for all relative quantification methods, the -fold changes of proteins in the sample of interest (disease, treated, etc.) are measured versus a normal reference sample. Any error in the normal or the normal sample to serious in quantitative measurements for tagging a of be with the reference in a Although the reference sample be in and used as a reference to many and a In quantification methods, the conditions need to be at all to the and the used for proteomics and in a and a major Absolute on the not have of the limitations S. W. and absolute quantitative proteomics: proteins in 2006; PubMed Scopus Google it of from conditions for different and In absolute quantification is in the it information for standard for absolute quantification of proteins and peptides is this method is and requires of that is in the analysis and of the protein or peptide sequence M. and analysis of Chromatogr. A. PubMed Scopus Google Scholar). In the quantified to be to the of mass spectrometry-based new for large scale absolute quantification of proteins have These methods on peptides as internal standards. peptides of interest are in a heavy and to the sample of interest in to mass analysis of a synthetic peptide for absolute protein quantification using Proteome Res. 2004; 3: PubMed Scopus Google Scholar). for the light and heavy peptides are and absolute of the light peptides, the biological be from the ratio of the respective heavy isotope-coded peptides be either by chemical synthesis or biological method has limitations of chemical synthesis is synthesis is also and peptides need to be In some peptides are or due to These and issues in absolute measurements In and J. Multiplexed absolute quantification for proteomics using concatenated peptides by QconCAT 2006; PubMed Scopus Google Scholar, J. R. and S. J. (July 12, 2006) for Absolute Quantification of and World Patent Scholar, Multiplexed absolute quantification in proteomics using proteins of concatenated 2005; 2: PubMed Scopus Google Scholar, J. Absolute quantitative analysis of protein using Cell. Proteomics. 2007; Full Text Full Text PDF PubMed Scopus Google a new technology to produce reference peptides by In this a synthetic gene is produced and expressed in in a heavy lysine and in the of a protein in which peptides of interest are concatenated J. 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