The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.     

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of G_i2 (a heterotrimeric G-protein α-subunit) is described in detail.     

Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation status of cyclin-dependent kinases

Multisite phosphorylation is an important mechanism for achieving intricate regulation of protein function. Here we extended the absolute quantification of abundance (AQUA) methodology and validated its applicability to quantitatively study multisite phosphorylation. As a test case, we chose the conserved inhibitory site of the cyclin-dependent kinases (CDKs), Cdk1, Cdk2, and Cdk3, which are important regulators of cell cycle transitions and apoptosis. Inhibitory phosphorylation at Thr(14) and Tyr(15) of the CDKs is modulated by complex regulatory mechanisms involving multiple kinases and phosphatases. Yet the resulting quantitative dynamics among the four possible phosphorylated and non-phosphorylated versions of CDKs (T14p-Y15p, T14p-Y15, T14-Y15p, and T14-Y15) has not been investigated to date. Hence we used the heavy isotope-labeled tryptic peptides spanning the inhibitory site as internal standards and quantified all four versions by LC-selected reaction monitoring. Quantification of the phosphorylation status of the inhibitory site in the cell extracts provided novel quantitative insights. 1) The transition to mitotic phase was dominated by the conversion of "T14p-Y15p" to the "T14-Y15" form, whereas the two monophosphorylated forms were considerably lower in abundance. 2) The amount of all four forms decreased during the progression of apoptosis but with differing kinetics. Analysis of immunoprecipitated Cdk1 and Cdk2 revealed that the inhibitory site phosphorylation state of both kinases at different stages of the cell cycle followed the same trend. Quantitative immunoblotting using antibodies to Cdk1 and Cdk2 and to the T14-Y15p form suggested that quantification by AQUA was reliable and accurate. These results highlight the utility of internal standard peptides to achieve accurate quantification of multisite phosphorylation status.     

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.     

Direct quantitation of MHC-bound peptide epitopes by selected reaction monitoring

We describe a cell-free approach that employs selected reaction monitoring (SRM) in tandem mass spectrometry to identify and quantitate T-cell epitopes. This approach utilises multiple epitope-specific SRM transitions to identify known T-cell epitopes and an absolute quantitation (AQUA) peptide strategy to afford AQUA. The advantage of a mass spectrometry-based approach over more traditional cell-based assays resides in the robustness and transferability of an SRM approach between laboratories and the ability of this strategy to detect multiple peptides simultaneously without the requirement of epitope-specific reagents such as T-cell lines. Thus, the SRM strategy for epitope quantitation will find application in studies of antigen density, the link between epitope abundance and immunogenicity, the dynamic range of epitope presentation and the abundance of T-cell epitopes in disease.     

Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers

A major bottleneck for validation of new clinical diagnostics is the development of highly sensitive and specific assays for quantifying proteins. We previously described a method, stable isotope standards with capture by antipeptide antibodies, wherein a specific tryptic peptide is selected as a stoichiometric representative of the protein from which it is cleaved, is enriched from biological samples using immobilized antibodies, and is quantitated using mass spectrometry against a spiked internal standard to yield a measure of protein concentration. In this study, we optimized a magnetic-bead-based platform amenable to high-throughput peptide capture and demonstrated that antibody capture followed by mass spectrometry can achieve ion signal enhancements on the order of 10(3), with precision (CVs <10%) and accuracy (relative error approximately 20%) sufficient for quantifying biomarkers in the physiologically relevant ng/mL range. These methods are generally applicable to any protein or biological fluid of interest and hold great potential for providing a desperately needed bridging technology between biomarker discovery and clinical application.     

A novel strategy for the targeted analysis of protein and peptide metabolites

In many biological applications such as epitope discovery or drug metabolism studies, the detection of naturally processed exogenous proteins (e.g. vaccines or peptide therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related or even identical compounds. We describe a method that incorporates stable isotope labelling of the protein of interest, allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low-molecular-weight fragment ions produced during MS/MS that distinguish isotopically labelled peptides from related endogenous compounds. All isotopically labelled peptides can be selected using this method. The technique makes no assumptions about the processed or post-translational state of the peptide, and hence can selectively screen out modified peptides that would otherwise be missed by single reaction monitoring approaches. This method does not replace single reaction monitoring or regular precursor scanning techniques; instead, it is a method that can be used when the assumptions required for the former two techniques cannot be predicted. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α-synuclein in serum and the brain.     

Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards

A general method for the quantification of proteins in human serum was developed using mass spectrometry (MS) and stable isotope-labeled synthetic peptides as internal standards. Using this approach, C-reactive protein (CRP), a diagnostic marker of rheumatoid arthritis (RA), was detected in serum samples taken from patients with either erosive or nonerosive RA and compared to healthy individuals. Small volumes of serum samples were enriched for low-abundance proteins through the selective removal of human serum albumin (HSA), immunoglobulin G (IgG), and haptoglobin. After depletion of abundant proteins, the complexity of the protein mixture was further simplified using size exclusion chromatography (SEC) to fractionate denatured proteins into discrete molecular weight ranges. Fractions of interest containing CRP, M(r) = 25 000, were pooled, digested with trypsin, and then fixed quantities of the synthetic peptides were added to the mixture. The mixture of tryptic peptides was subsequently analyzed by nanoflow chromatography-tandem MS (nanoLC-MS/MS) using multiple-reaction monitoring (MRM) on a triple quadrupole mass spectrometer (TQ-MS). The ratio of transition ions derived from the endogenous and isotope-labeled peptides provided a quantitative measure of CRP in the original samples as assessed by independent measurement of CRP in the same patient samples using an immunoassay. The use of isotope-labeled synthetic peptides and MRM is a powerful analytical method for the prescreening of candidate protein biomarkers in human serum prior to antibody and immunoassay development.     

Mass spectrometry-based detection and quantification of plasma glycoproteins using selective reaction monitoring

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.     

Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100?000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14?000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.     

The selected reaction monitoring/multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases

Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.     

Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

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, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) 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.     

Several inhibitors of the Plk1 Polo-Box Domain turn out to be non-specific protein alkylators

For almost a decade, there has been much interest in the development of chemical inhibitors of Polo-like kinase 1 (Plk1) protein interactions. Plk1 is a master regulator of the cell division cycle that controls numerous substrates. It is a promising target for cancer drug development. Inhibitors of the kinase domain of Plk1 had some success in clinical trials. However, they are not perfectly selective. In principle, Plk1 can also be inhibited by interfering with its protein interaction domain, the Polo-Box Domain (PBD). Selective chemical inhibitors of the PBD would constitute tools to probe for PBD-dependent functions of Plk1 and could be advantageous in cancer therapy. The discovery of Poloxin and thymoquinone as PBD inhibitors indicated that small, cell-permeable chemical inhibitors could be identified. Other efforts followed, including ours, reporting additional molecules capable of blocking the PBD. It is now clear that, unfortunately, most of these compounds are non-specific protein alkylators (defined here as groups covalently added via a carbon) that have little or no potential for the development of real Plk1 PBD-specific drugs. This situation should be minded by biologists potentially interested in using these compounds to study Plk1. Further efforts are needed to develop selective, cell-permeable PBD inhibitors.     

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 1?N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.     

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.     

Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.     

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.     

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.     

Critical assessment of proteome‐wide label‐free absolute abundance estimation strategies

There is a great interest in reliable ways to obtain absolute protein abundances at a proteome‐wide scale. To this end, label‐free LC‐MS/MS quantification methods have been proposed where all identified proteins are assigned an estimated abundance. Several variants of this quantification approach have been presented, based on either the number of spectral counts per protein or MS1 peak intensities. Equipped with several datasets representing real biological environments, containing a high number of accurately quantified reference proteins, we evaluate five popular low‐cost and easily implemented quantification methods (Absolute Protein Expression, Exponentially Modified Protein Abundance Index, Intensity‐Based Absolute Quantification Index, Top3, and MeanInt). Our results demonstrate considerably improved abundance estimates upon implementing accurately quantified reference proteins; that is, using spiked in stable isotope labeled standard peptides or a standard protein mix, to generate a properly calibrated quantification model. We show that only the Top3 method is directly proportional to protein abundance over the full quantification range and is the preferred method in the absence of reference protein measurements. Additionally, we demonstrate that spectral count based quantification methods are associated with higher errors than MS1 peak intensity based methods. Furthermore, we investigate the impact of miscleaved, modified, and shared peptides as well as protein size and the number of employed reference proteins on quantification accuracy.