Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.Discovery of disease-specific biomarkers with diagnostic and prognostic utility has become an important challenge in clinical proteomics. In general, unbiased discovery experiments often result in the confident identification of thousands of proteins, hundreds of which may vary significantly between case and control samples in small discovery studies. However, because of the stochastic sampling of proteomes in discovery “omics” experiments, a large fraction of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery omics experiments do not lead to biomarkers of immediate clinical utility but rather produce candidates that must be qualified and verified in larger sample sets than were used for discovery (1).Traditional, clinical validation of biomarkers has relied primarily on immunoassays because of their specificity and sensitivity for the target analyte and high throughput capability. However, antibody reagents for a clinical grade immunoassay often only exist for a short list of candidates. The development of a reliable sandwich immunoassay for one target protein is expensive, has a long development time, and is dependent upon the generation of high quality protein antibodies. For the large majority of new, unproven candidate biomarkers, an intermediate verification technology is required that has shorter assay development time lines, lower assay cost, and effective multiplexing of dozens of candidates in low sample volumes. Ideally the approach should be capable of analyzing hundreds of samples of serum or plasma with good precision. The desired outcome of verification is a small number of highly credentialed candidates suitable for traditional preclinical and clinical validation studies.Multiple reaction monitoring (MRM)¹ coupled with stable isotope dilution (SID) MS has recently been shown to be well suited for direct quantification of proteins in plasma (2–4) and has emerged as the core technology for candidate biomarker verification. MRM assays can be highly multiplexed such that a moderate number of candidate proteins (in the range of 10–50) can be simultaneously targeted and measured in the statistically viable number of patient samples required for verification (hundreds of serum samples). However, sensitivity for unambiguous detection and quantification of proteins by MS-based assays is often constrained by sample complexity, particularly when the measurements are being made in complex fluids such as plasma.Many biomarkers of current clinical importance, such as prostate-specific antigen and the cardiac troponins, reside in the low nanogram/milliliter range in plasma and, until recently, have been inaccessible by non-antibody approaches. Our laboratory has recently shown for the first time that a combination of abundant protein depletion with limited fractionation at the peptide level prior to SID-MRM-MS provides robust limits of quantitation (LOQs) in the 1–20 ng/ml range with coefficient of variation (CV) of 10–20% at the LOQ for proteins in plasma (3).Here we demonstrate that this work flow can be extended to configure assays for a number of known markers of cardiovascular disease and, more importantly, can be deployed to measure their concentrations in clinical samples. We modeled a verification study comprising six patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of “planned” myocardial infarction (PMI), and obtained targeted, quantitative measurements for moderate to low concentrations of cardiac biomarkers in plasma. This work provides additional evidence that MS-based assays can be configured and applied to verification of new protein targets for which high quality antibody reagents are not available.     

Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine

Organophosphorus (OP) esters are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-biotin. The proteins were digested with trypsin and the labeled peptides enriched by binding to monomeric avidin. Peptides were purified by HPLC and fragmented by collision induced dissociation in a tandem ion trap mass spectrometer. Eight proteins were labeled and six were not. Tyrosine was labeled in human alpha-2-glycoprotein 1 zinc-binding protein (Tyr 138, Tyr 174 and Tyr 181), human kinesin 3C motor domain (Tyr 145), human keratin 1 (Tyr 230), bovine actin (Tyr 55 and Tyr 200), murine ATP synthase beta (Tyr 431), murine adenine nucleotide translocase 1 (Tyr 81), bovine chymotrypsinogen (Tyr 201) and porcine pepsin (Tyr 310). Only 1–3 tyrosines per protein were modified, suggesting that the reactive tyrosine was activated by nearby residues that facilitated ionization of the hydroxyl group of tyrosine. These results suggest that OP binding to tyrosine is a general phenomenon. It is concluded that organophosphorus-reactive proteins include not only enzymes in the serine hydrolase family, but also proteins that have no active site serine. The recognition of a new OP-binding motif to tyrosine suggests new directions to search for mechanisms of long-term effects of OP exposure. Another application is in the search for biomarkers of organophosphorus agent exposure. Previous searches have been limited to serine hydrolases. Now proteins such as albumin and keratin can be considered.     

Extremely High Frequency Electromagnetic Radiation Enforces Bacterial Effects of Inhibitors and Antibiotics

The coherent electromagnetic radiation (EMR) of the frequency of 51.8 and 53 GHz with low intensity (the power flux density of 0.06 mW/cm(2)) affected the growth of Escherichia coli K12(lambda) under fermentation conditions: the lowering of the growth specific rate was considerably (approximately 2-fold) increased with exposure duration of 30-60 min; a significant decrease in the number of viable cells was also shown. Moreover, the enforced effects of the N,N'-dicyclohexylcarbodiimide (DCCD), inhibitor of H(+)-transporting F(0)F(1)-ATPase, on energy-dependent H(+) efflux by whole cells and of antibiotics like tetracycline and chloramphenicol on the following bacterial growth and survival were also determined after radiation. In addition, the lowering in DCCD-inhibited ATPase activity of membrane vesicles from exposed cells was defined. The results confirmed the input of membranous changes in bacterial action of low intensity extremely high frequency EMR, when the F(0)F(1)-ATPase is probably playing a key role. The radiation of bacteria might lead to changed metabolic pathways and to antibiotic resistance. It may also give bacteria with a specific role in biosphere.     

Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.     

Changes in the activity of enzymes,participating in glycogen metabolism of alloxan diabetic rats

Summary Alloxan diabetes induced in white rats by intraperitoneal injection of Aloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished.AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities.The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.     

Synthesis of a Double Transmembrane Domain Fragment of Ste2p by Native Chemical Ligation

Native chemical ligation (NCL) approaches have been applied extensively to soluble proteins. Fewer successes have been achieved with membrane peptides. In this report, the synthesis and semisynthesis by NCL of peptides corresponding to 1.7 transmembrane domains of the α-factor receptor from Saccharomyces cerevisiae is described. Synthesis was achieved when the ligation point was approximately in the middle of the loop joining the two transmembrane regions. In contrast, little to no ligation was observed when the ligation point was at the putative membrane interface of the sixth transmembrane domain (TM6) and the third extracellular loop (EL3). Ligations of a chemically synthesized 22-residue thioester with a synthetic 29-residue N-Cys peptide and a biosynthetic 73-residue N-Cys peptide were successfully achieved in both trifluoroethanol/guanidinium hydrochloride (TFE/GnHCl) and sodium dodecyl sulfate (SDS) media when mercaptoethanesulfonic acid (MESNA) was used as a catalyst. The resulting 51-residue and 95-residue ligation products were purified by reversed phase HPLC and recovered on a mg scale. Both peptides were >95% pure as determined by HPLC and had the expected molecular weight as judged by mass spectrometry. Segmental labeling of the 95-residue fragment, in which the N-Cys portion was [¹⁵N] labeled, resulted in a peptide that gave an NMR spectrum which was comparable to that of the unligated 73-residue peptide alone. R B Merrifield personified the finest qualities of a human being. He was an outstanding individual who influenced the way research is conducted by tens of thousands of scientists. At the same time he was a warm, humble, sincere man who was extremely kind and generous. I (FN) personally saw his generosity during a seminar he invited me to give at Rockefeller University. He was already a Nobel laureate but he treated me as a colleague and the encouragement he offered concerning my research program was very important for my future in academia. It is an honor to be among the participants in a volume honoring his contributions to peptide science.     

Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop

Interleukin-1 stimulation leads to the recruitment of MyD88, interleukin-1 receptor-associated kinase 1 (IRAK-1) and interleukin-1 receptor-associated kinase 4 (IRAK-4) to the IL-1 receptor. The formation of the IL-1 receptor complex triggers a series of IRAK-1 autophosphorylations, which result in activation. IRAK-4 is upstream of IRAK-1 and may act as IRAK-1 kinase to transmit the signal. To date, there is no upstream kinase reported for IRAK-4; the activation mechanism of IRAK-4 remains poorly understood. Here, for the first time, we report three autophosphorylation sites that are responsible for IRAK-4 kinase activity. LC-MS/MS analysis has identified phosphorylations at T342, T345, and S346, which reside within the activation loop. Site-directed mutants at these positions exhibit significant reductions in the catalytic activity of IRAK-4 (T342A: 57%; T345A: 66%; S346A: 50%). The absence of phosphorylation in kinase-dead IRAK-4 indicates that phosphorylations in the activation loop result from autophosphorylation rather than from phosphorylation by an upstream kinase. Finally, we demonstrate that autophosphorylation is an intramolecular event as wild-type IRAK-4 failed to transphosphorylate kinase-inactive IRAK-4. The present data indicate that the kinase activity of IRAK-4 is dependent on the autophosphorylations at T342, T345, and S346 in the activation loop.     

Mass spectral characterization of organophosphate-labeled,tyrosine-containing peptides: Characteristic mass fragments and a new binding motif for organophosphates

We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8–8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.     

A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease

We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.