Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.     

Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides

Absolute quantification in proteomics usually involves simultaneous determination of representative proteolytic peptides and stable isotope-labeled analogs. The principal limitation to widespread implementation of this approach is the availability of standard signature peptides in accurately known amounts. We report the successful design and construction of an artificial gene encoding a concatenation of tryptic peptides (QCAT protein) from several chick (Gallus gallus) skeletal muscle proteins and features for quantification and purification.     

Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.     

Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.     

IQcat: multiplexed protein quantification by isoelectric QconCAT

Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (～2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.     

The importance of the digest: proteolysis and absolute quantification in proteomics

Virtually all mass spectrometric-based methods for quantitative proteomics are at the peptide level, whether label-mediated or label-free. Absolute quantification in particular is based on the measurement of limit peptides, defined as those peptides that cannot be further fragmented by the protease in use. Complete release of analyte and (stable isotope labelled) standard ensures that the most reliable quantification data are recovered, especially when the standard peptides are in a different primary sequence context, such as sometimes occurs in the QconCAT methodology. Moreover, in label-free methods, incomplete digestion would diminish the ion current attributable to limit peptides and lead to artifactually low quantification data. It follows that an essential requirement for peptide-based absolute quantification in proteomics is complete and consistent proteolysis to limit peptides. In this paper we describe strategies to assess completeness of proteolysis and discuss the potential for variance in digestion efficiency to compromise the ensuing quantification data. We examine the potential for kinetically favoured routes of proteolysis, particularly at the last stages of the digestion, to direct products into ‘dead-end’ mis-cleaved products.     

Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics

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.Key Words: Quantitative proteomics, mass spectrometry, absolute quantification, stable isotope labeling, label-free.     

A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme

Although significant progress has been made in protein quantification using mass spectrometry during recent years, absolute protein quantification in complex biological systems remains a challenging task in proteomics. The use of stable isotope-labeled standard peptide is the most commonly used strategy for absolute quantification, but it might not be suitable in all instances. Here we report an alternative strategy that employs a stable isotope-labeled intact protein as an internal standard to absolutely quantify the alcohol dehydrogenase (ADH) expression level in a human liver sample. In combination with a new targeted proteomics approach employing the method of multiple reaction monitoring (MRM), we precisely and quantitatively measured the absolute protein expression level of an ADH isoenzyme, ADH1C1, in human liver. Isotope-labeled protein standards are predicted to be particularly useful for measurement of highly homologous isoenzymes such as ADHs where multiple signature peptides can be examined by MRM in a single experiment.     

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.     

A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins

Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.     

CONSeQuence: prediction of reference peptides for absolute quantitative proteomics using consensus machine learning approaches

Mass spectrometric based methods for absolute quantification of proteins, such as QconCAT, rely on internal standards of stable-isotope labeled reference peptides, or "Q-peptides," to act as surrogates. Key to the success of this and related methods for absolute protein quantification (such as AQUA) is selection of the Q-peptide. Here we describe a novel method, CONSeQuence (consensus predictor for Q-peptide sequence), based on four different machine learning approaches for Q-peptide selection. CONSeQuence demonstrates improved performance over existing methods for optimal Q-peptide selection in the absence of prior experimental information, as validated using two independent test sets derived from yeast. Furthermore, we examine the physicochemical parameters associated with good peptide surrogates, and demonstrate that in addition to charge and hydrophobicity, peptide secondary structure plays a significant role in determining peptide "detectability" in liquid chromatography-electrospray ionization experiments. We relate peptide properties to protein tertiary structure, demonstrating a counterintuitive preference for buried status for frequently detected peptides. Finally, we demonstrate the improved efficacy of the general approach by applying a predictor trained on yeast data to sets of proteotypic peptides from two additional species taken from an existing peptide identification repository.     

Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition

Relative quantification methods have dominated the quantitative proteomics field. There is a need, however, to conduct absolute quantification studies to accurately model and understand the complex molecular biology that results in proteome variability among biological samples. A new method of absolute quantification of proteins is described. This method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than +/-10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested in this study. A controlled set of six exogenous proteins of varying concentrations was studied in the absence and presence of human serum. The absolute quantity of the standard proteins was determined with a relative error of less than +/-15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated protein concentrations, and this plot resulted in a linear relationship with an R(2) value of 0.9939. The analyses were applied to determine the absolute concentration of 11 common serum proteins, and these concentrations were then compared with known values available in the literature. Additionally within an unfractionated Escherichia coli lysate, a subset of identified proteins known to exist as functional complexes was studied. The calculated absolute quantities were used to accurately determine their stoichiometry.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.     

Quantification in proteomics through stable isotope coding: a review

This review focuses on techniques for quantification and identification in proteomics by stable isotope coding. Methods are examined for analyzing expression, post-translational modifications, protein:protein interactions, single amino acid polymorphism, and absolute quantification. The bulk of the quantification literature in proteomics focuses on expression analysis, where a wide variety of methods targeting different features of proteins are described. Methods for the analysis of post-translational modification (PTM) focus primarily on phosphorylation and glycosylation, where quantification is achieved in two ways, either by substitution or tagging of the PTM with an isotopically coded derivatizing agent in a single process or by coding and selecting PTM modified peptides in separate operations. Absolute quantification has been achieved by age-old internal standard methods, in which an isotopically labeled isoform of an analyte is synthesized and added to a mixture at a known concentration. One of the surprises is that isotope coding can be a valuable aid in the examination of intermolecular association of proteins through stimulus:response studies. Preliminary efforts to recognize single amino acid polymorphism are also described. The review ends with the conclusion that (1) isotope ratio analysis of protein concentration between samples does not necessarily relate directly to protein expression and rate of PTM and (2) that multiple new methods must be developed and applied simultaneously to make existing stable isotope quantification methods more meaningful. Although stable isotope coding is a powerful, wonderful new technique, multiple analytical issues must be solved for the technique to reach its full potential as a tool to study biological systems.     

Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards

An important challenge for proteomics is to be able to compare absolute protein levels across biological samples. Here we introduce an approach based on the use of culture-derived isotope tags (CDITs) for quantitative tissue proteome analysis. We cultured Neuro2A cells in a stable isotope-enriched medium and mixed them with mouse brain samples to serve as internal standards. Using CDITs, we identified and quantified a total of 1,000 proteins, 97-98% of which were expressed in both mouse whole brain and Neuro2A cells. CDITs also allow comprehensive and absolute protein quantification. Synthetic unlabeled peptides were used to quantify the corresponding proteins labeled with stable isotopes in Neuro2A cells, and the results were used to obtain the absolute amounts of 103 proteins in mouse whole brain. The expression levels correlated well with those in Neuro2A cells. Thus, the use of CDITs allows both relative and absolute quantitative proteome studies.     

Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.     

Amine-reactive isobaric tagging reagents: requirements for absolute quantification of proteins and peptides

Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented.     

Absolute quantification strategies in proteomics based on mass spectrometry

The strong need for quantitative information in proteomics has fueled the development of mass spectrometry-based analytical methods that are able to determine protein abundances. This article reviews mass spectrometry experiments aimed at providing an absolute quantification of proteins. The experiments make use of the isotope-dilution concept by spiking a known amount of synthetic, isotope-labeled reference peptide into the analyte sample. Quantification is achieved by comparing the mass spectrometry signal intensities of the reference with an endogenous peptide that is generated upon proteolytic cleavage of the target protein. In an analogous manner, the level of post-translational modification at a distinct residue within a target protein can be determined. Among the strengths of absolute quantification are low detection limits reaching subfemtomole levels, a high dynamic range spanning approximately five orders of magnitude, low requirements for sample clean-up, and a fast and straightforward method development. Recent studies have demonstrated the compatibility of absolute quantification with various mass spectrometry readout techniques and sample purification steps such as 1D gel electrophoresis, size-exclusion chromatography, isoelectric peptide focusing, strong cation exchange and reversed phase or affinity chromatography. Under ideal conditions, quantification errors and coefficients of variation below 5% have been reported. However, the fact that at the start of the experiment the analyte is a protein and the internal standard is a peptide, severe quantification errors may result due to the selection of unsuitable reference peptides and/or imperfect protein proteolysis. Within the ensemble of mass spectrometry-based quantification methods, absolute quantification is the method of choice in cases where absolute numbers, many repetitive experiments or precise levels of post-translational modifications are required for a few, preselected species of interest. Consequently, prominent application areas include biomarker quantification, the study of post-translational modifications such as phosphorylation or ubiquitination and the comparison of concentrations of interacting proteins.