Absolute quantitation of peptides and proteins by coulometric mass spectrometry after derivatization (original) (raw)
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Absolute Quantitation of Oxidizable Peptides by Coulometric Mass Spectrometry
Journal of The American Society for Mass Spectrometry
Quantitation methods for peptides using mass spectrometry have advanced rapidly. These methods rely on using standard and/or isotope-labeled peptides, which might be difficult or expensive to synthesize. To tackle this challenge, we present a new approach for absolute quantitation without the use of standards or calibration curves based on coulometry combined with mass spectrometry (MS). In this approach, which we call coulometric mass spectrometry (CMS), the mass spectrum of a target peptide containing one or more tyrosine residues is recorded before and after undergoing electrochemical oxidation. We record the total integrated oxidation current from the electrochemical measurement, which according to the Faraday's Law of coulometry, provides the number of moles of oxidized peptide. The ion intensity ratio of the target peptide before and after oxidation provides an excellent estimate of the fraction of the peptide that has been oxidized, from which the total amount of peptide is calculated. The striking strength of CMS is that it needs no standard peptide, but CMS does require the peptide to contain a known number of oxidizable groups. To illustrate the power of this method, we analyzed various tyrosinecontaining peptides such as GGYR, DRVY, oxytocin, [Arg 8 ]-vasotocin and angiotensinogen 1-14 with a quantification error ranging from − 7.5 to + 2.4%. This approach is also applicable to quantifying phosphopeptides and could be useful in proteomics research.
Absolute Quantitation of Proteins by Coulometric Mass Spectrometry
Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotopelabeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins β-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from Escherichia coli. In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3−3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.
Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry
Journal of Chromatography B, 2008
With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC-MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded.
Biological Mass Spectrometry, 1987
Treatment of peptides containing methionine and/or tryptophan with dimethylsuifoxide/hydrochloric acidlacetic acid resulted in oxidation of these amino acids respectively to methionine sulfoxide and oxyindolalanine. This reaction was monitored by fast atom bombardment mass spectrometry using a dithiothreitoVdithioerythrito1 liquid matrix. Under these conditions, only methionine and tryptophan were oxidized. Comparison of mass spectra of a sample before and after oxidation should provide a rapid screening procedure for determination of these residues in peptides.
Molecular & Cellular Proteomics, 2011
Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and -galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power
Journal of Proteome Research, 2004
A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, R1 antichymotrypsin, interleukin-6, and tumor necrosis factor-R) from a pool of 10 203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.