PTH (7-84) N15 Human

What is PTH (7-84) N15 Human and how does it differ from full-length PTH?

PTH (7-84) N15 Human is a recombinant protein fragment of human parathyroid hormone containing amino acids 7-84 of the native sequence, labeled with the stable nitrogen isotope 15N. This fragment differs from full-length PTH (1-84) in several critical ways. While full-length PTH primarily stimulates bone resorption and calcium release, PTH (7-84) acts as an antagonist for the PTH1R receptor and may interact with a distinct carboxy-terminus interacting PTH receptor . The fragment is produced as a single, non-glycosylated polypeptide chain containing 78 amino acids with a molecular mass of approximately 8.9 kDa .

The biological function contrast is significant: PTH (7-84) is secreted by the parathyroid glands when extracellular calcium levels are high, specifically to offset the effects of full-length PTH, creating a physiological counterbalance in calcium homeostasis regulation . The N15 stable isotope labeling provides a valuable tracking mechanism for distinguishing the exogenous protein from endogenous PTH in experimental systems .

How should PTH (7-84) N15 Human be stored and reconstituted for optimal stability?

The proper storage and reconstitution of PTH (7-84) N15 Human is critical for maintaining its structural integrity and biological activity. The lyophilized protein should be stored desiccated at temperatures below -18°C for long-term stability . While the lyophilized form remains stable at room temperature for approximately three weeks, refrigerated or frozen storage is strongly recommended to prevent degradation .

For reconstitution, the following protocol is recommended:

• Reconstitute the lyophilized powder in sterile water at a concentration not less than 100 μg/ml .

• After reconstitution, store the solution at +4°C if used within 2-7 days .

• For longer storage periods, maintain the reconstituted solution below -18°C .

• For extended storage, add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to prevent adsorption to container surfaces and improve stability .

• Avoid repeated freeze-thaw cycles as they can significantly degrade protein integrity .

The reconstituted solution can be further diluted in appropriate buffers for specific experimental applications. Each laboratory should validate storage conditions for their specific experimental systems .

What are the primary research applications of PTH (7-84) N15 Human?

PTH (7-84) N15 Human serves multiple critical research applications in endocrinology, bone metabolism, and calcium homeostasis investigations:

• Receptor Antagonism Studies: As an antagonist for PTH1R, it enables investigation of receptor signaling mechanisms and downstream pathway modulation .

• Isotope Dilution Assays: The 15N labeling facilitates quantitative analysis through isotope dilution mass spectrometry for precise measurement of endogenous PTH fragments in biological samples .

• Metabolic Tracking: Researchers can follow the metabolic fate of the fragment through biological systems, distinguishing it from endogenous PTH .

• Bone-Kidney Axis Research: The fragment helps elucidate the complex interplay between bone metabolism and renal calcium handling mechanisms .

• Analytical Standard: It serves as a reference standard for method development in proteomics and hormone analysis .

The high purity (>97% as determined by RP-HPLC and SDS-PAGE analyses) makes it particularly valuable for mechanistic studies requiring precise quantification and minimal interfering factors .

How can PTH (7-84) N15 Human be used to study calcium homeostasis mechanisms?

PTH (7-84) N15 Human provides unique research opportunities for dissecting calcium homeostasis mechanisms through several experimental approaches:

• Receptor Competition Assays: By antagonizing PTH1R, PTH (7-84) can be used in competition assays with full-length PTH to determine receptor binding kinetics and downstream signaling modulation in calcium regulation .

• Calcium Flux Measurements: The fragment can be applied in cellular models to measure how PTH receptor antagonism affects calcium transport across cell membranes, particularly in osteoblasts, osteoclasts, and renal tubular cells .

• Ex Vivo Bone Studies: Using bone organ cultures, researchers can investigate how varying ratios of PTH (1-84) to PTH (7-84) influence calcium release from the bone mineral matrix .

• Renal Calcium Handling: The 15N labeling allows tracking of how the fragment modulates renal calcium excretion and reabsorption processes in kidney perfusion models .

Experimental designs typically incorporate dose-response relationships to determine physiologically relevant concentrations, with calcium levels measured through fluorometric assays, radioisotope tracing, or ion-selective electrode techniques. The antagonistic effects can be quantified by measuring cAMP production, as the biological activity has been calculated to correspond to a specific activity of 9,000 Units/mg using the UMR106 cell/cAMP method .

What are the recommended detection methods for N15-labeled PTH fragments in biological samples?

Detection of N15-labeled PTH fragments in biological samples requires specialized analytical approaches that capitalize on the isotopic labeling. The following methods are particularly effective:

• Mass Spectrometry: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers the highest specificity and sensitivity for detecting and quantifying N15-labeled peptides. The mass shift created by 15N substitution throughout the peptide enables precise discrimination between endogenous and exogenous PTH fragments .

• Immunoassays with MS Confirmation: Specialized immunoassays using antibodies specific to the C-terminal region of PTH can be used for initial enrichment, followed by mass spectrometric analysis to confirm isotope incorporation .

• NMR Spectroscopy: For structural studies, nuclear magnetic resonance spectroscopy can detect 15N-labeled proteins with high specificity, providing information on protein conformation and interaction dynamics .

• Isotope Ratio MS: For quantitative studies tracking metabolic incorporation, isotope ratio mass spectrometry can measure small changes in 15N/14N ratios in biological matrices .

Sample preparation typically involves immunoaffinity purification or solid-phase extraction prior to instrumental analysis. This multi-step approach enables detection of labeled fragments at concentrations as low as 1-10 ng/mL in complex biological matrices such as plasma or tissue extracts .

How should researchers validate the purity and activity of PTH (7-84) N15 Human before experimental use?

Comprehensive validation of PTH (7-84) N15 Human before experimental use is essential for ensuring reliable research outcomes. A multi-faceted approach to quality control should include:

• Purity Assessment:

  • Reverse-phase HPLC analysis to confirm >97% purity

  • SDS-PAGE under reducing and non-reducing conditions to verify molecular weight and absence of aggregates

  • Mass spectrometry to confirm complete 15N incorporation and correct molecular weight (~8.9 kDa)

• Functional Validation:

  • cAMP antagonism assay using UMR106 cells to confirm biological activity (expected specific activity of approximately 9,000 Units/mg)

  • PTH1R receptor binding competition assay against labeled PTH (1-84)

  • Calcium mobilization inhibition assays in responsive cell lines

• Physical Characterization:

  • Visual inspection to confirm appearance as a sterile filtered white lyophilized powder

  • Solubility testing in recommended reconstitution buffer

  • Stability assessment at different temperatures following reconstitution

Each validation step should be documented with appropriate acceptance criteria based on certificate of analysis specifications. Researchers should perform at minimum the purity and functional validation before initiating critical experiments to ensure consistency across studies .

How can PTH (7-84) N15 be used in mass spectrometry-based quantitative proteomics studies?

PTH (7-84) N15 represents a powerful tool for quantitative proteomics studies investigating parathyroid hormone metabolism and signaling. Advanced mass spectrometry applications include:

• Absolute Quantification (AQUA): The 15N-labeled fragment serves as an ideal internal standard for absolute quantification of endogenous PTH fragments in biological samples. When added at known concentrations, it allows precise determination of endogenous fragment concentrations through isotope dilution principles .

• Multiple Reaction Monitoring (MRM): Targeted proteomics approaches using MRM can track specific peptide fragments generated from both the labeled standard and endogenous PTH. This enables highly selective quantification even in complex matrices .

• PTH Fragment Profiling: The standard enables comprehensive profiling of the various PTH fragments present in circulation under different physiological and pathological conditions, providing insights into PTH metabolism .

• Turnover Studies: By monitoring the appearance and disappearance of labeled fragments over time, researchers can determine the biological half-life and clearance mechanisms of different PTH fragments .

• Post-translational Modification Analysis: When used as a reference, the N15-labeled fragment helps identify and quantify post-translational modifications on endogenous PTH fragments, which may affect biological activity .

Experimental workflows typically involve sample enrichment through immunoaffinity purification, enzymatic digestion optimization, and careful selection of signature peptides that retain the isotope label for reliable quantification .

What are the methodological considerations when designing isotope dilution experiments using PTH (7-84) N15?

Designing robust isotope dilution experiments with PTH (7-84) N15 requires careful attention to several critical methodological considerations:

• Standard Curve Development:

  • Prepare a calibration curve using varying concentrations of N15-labeled PTH (7-84) in a matrix matching the experimental samples

  • Include matrix-matched blank samples to account for background interference

  • Validate linearity across the anticipated concentration range (typically 1-1000 ng/mL)

• Sample Preparation Optimization:

  • Add the labeled standard early in the sample preparation workflow to account for procedural losses

  • Validate recovery rates across different sample types (plasma, urine, tissue extracts)

  • Minimize proteolytic degradation by including appropriate protease inhibitors

• Mass Spectrometric Method Development:

  • Select signature peptides that retain sufficient 15N atoms to provide clear mass discrimination

  • Optimize collision energies for each selected transition

  • Monitor at least 3-5 transitions per peptide for confirmation of identity

  • Account for potential overlap with naturally occurring isotope patterns

• Validation Parameters:

  • Determine limits of detection and quantification

  • Assess inter-day and intra-day precision (target CV <15%)

  • Evaluate accuracy through spike recovery experiments

  • Test for potential matrix effects in different biological samples

• Data Analysis Considerations:

  • Calculate concentration using the ratio of unlabeled to labeled peak areas

  • Apply appropriate correction factors for incomplete isotope incorporation if necessary

  • Validate results using orthogonal techniques when possible

The high purity (>97%) of commercial PTH (7-84) N15 preparations supports precise quantification, but each laboratory should verify performance in their specific experimental system .

How does PTH (7-84) antagonize the effects of full-length PTH at the receptor level?

The antagonistic mechanism of PTH (7-84) against full-length PTH involves sophisticated receptor interactions that can be experimentally elucidated:

• Competitive Binding: PTH (7-84) competitively binds to the PTH1 receptor (PTH1R) but lacks the N-terminal residues (amino acids 1-6) that are critical for receptor activation. This allows binding without initiating the full signaling cascade triggered by intact PTH .

• Receptor Conformational Differences: Research indicates that binding of PTH (7-84) induces a different conformational change in PTH1R compared to full-length PTH, resulting in altered G-protein coupling. This can be studied using FRET-based conformational sensors in receptor-expressing cell lines .

• Second Messenger Inhibition: While full-length PTH stimulates cAMP production and PKA activation, PTH (7-84) fails to activate this pathway and may even reduce baseline cAMP levels in some cellular contexts. The specific activity has been measured using the UMR106 cell/cAMP method, corresponding to approximately 9,000 Units/mg .

• Alternative Receptor Interaction: Evidence suggests PTH (7-84) may interact with a distinct carboxy-terminus interacting PTH receptor, separate from the classical PTH1R. This presents an additional mechanism for its antagonistic effects that can be investigated through receptor binding studies .

• Downstream Signaling Modulation: The fragment can modulate MAPK pathway activation differently than full-length PTH, potentially activating inhibitory pathways that counteract PTH-induced effects on calcium mobilization and bone resorption .

Experimental approaches to study these mechanisms include receptor binding assays, signaling pathway activation measurement, receptor mutagenesis studies, and functional outcomes in cellular models of bone or kidney function .

What are common issues encountered when working with PTH (7-84) N15 and how can they be resolved?

Researchers working with PTH (7-84) N15 frequently encounter several technical challenges that can be systematically addressed:

• Solubility Issues:

  • Problem: Incomplete dissolution after reconstitution

  • Solution: Ensure reconstitution at concentrations not less than 100 μg/ml in sterile water before diluting to working concentrations. Gentle vortexing or rotation rather than vigorous shaking helps maintain protein integrity .

• Activity Loss During Storage:

  • Problem: Diminished antagonistic activity in functional assays

  • Solution: Add carrier protein (0.1% HSA or BSA) for long-term storage, strictly avoid freeze-thaw cycles, and store reconstituted protein at -18°C or lower for extended periods .

• Adsorption to Labware:

  • Problem: Significant loss of peptide concentration due to binding to plastic surfaces

  • Solution: Use low-binding microcentrifuge tubes and pipette tips, pre-coat vessels with carrier protein, and include 0.01-0.05% Tween-20 in working solutions .

• Inconsistent Mass Spectrometry Results:

  • Problem: Variable detection of N15-labeled peptides

  • Solution: Optimize collision energies for selected transitions, use at least three transitions per peptide, and ensure complete tryptic digestion by extending incubation times .

• Unexpected Fragmentation:

  • Problem: Detection of additional PTH fragments not corresponding to PTH (7-84)

  • Solution: Include protease inhibitor cocktails in experimental buffers, minimize sample handling time, and keep samples cold during processing .

• Matrix Interference in Complex Samples:

  • Problem: Suppressed signal in biological matrices

  • Solution: Implement effective sample clean-up procedures such as solid-phase extraction or immunoaffinity purification prior to analysis .

By implementing these targeted solutions, researchers can significantly improve experimental reliability and reproducibility when working with PTH (7-84) N15 in diverse research applications .

How can researchers ensure accurate quantification of PTH (7-84) N15 in biological samples?

Accurate quantification of PTH (7-84) N15 in biological samples requires attention to multiple factors throughout the analytical workflow:

• Reference Standards and Calibration:

  • Use properly characterized PTH (7-84) N15 reference material with verified purity (>97% by RP-HPLC and SDS-PAGE)

  • Prepare calibration curves in matched matrix to account for matrix effects

  • Include quality control samples at low, medium, and high concentrations within the calibration range

• Sample Preparation Optimization:

  • Validate extraction recovery across the anticipated concentration range

  • Evaluate and minimize protein binding to labware through appropriate buffer formulation

  • Implement internal standardization to correct for procedural losses

  • Process samples at 4°C whenever possible to minimize degradation

• Instrument Parameters and Method Validation:

  • Determine limits of detection and quantification specific to the analytical platform

  • Validate linearity, accuracy (80-120% recovery), and precision (CV <15%)

  • Assess potential ion suppression or enhancement effects in mass spectrometry applications

  • Evaluate freeze-thaw stability of analyte in the specific biological matrix

• Data Analysis Considerations:

  • Apply appropriate regression models for calibration curves (linear, quadratic, or weighted options)

  • Implement blank subtraction procedures for endogenous background correction

  • Utilize bracketing calibrators for long analytical sequences

  • Apply correction factors for isotopic abundance if necessary

• Confirmation Approaches:

  • Consider orthogonal detection methods to verify quantitative results

  • Monitor multiple transitions or fragments for mass spectrometric confirmation

  • Include positive and negative biological controls to verify biological plausibility of results

By implementing this comprehensive approach to quantification, researchers can achieve reliable measurements of PTH (7-84) N15 with minimal bias and acceptable precision for research applications .

What emerging applications are being developed for PTH (7-84) N15 in metabolic bone disease research?

PTH (7-84) N15 is enabling several innovative research directions in metabolic bone disease investigation:

• PTH Fragment Ratio Diagnostics: Emerging research suggests that the ratio of PTH (1-84) to PTH (7-84) may serve as a more sensitive indicator of bone turnover status than either fragment alone. N15-labeled standards enable precise quantification of these ratios in clinical samples .

• Renal Osteodystrophy Mechanisms: The antagonistic effects of PTH (7-84) on bone and kidney function are particularly relevant in chronic kidney disease. Isotope-labeled fragments allow tracking of altered PTH metabolism in renal failure models .

• Aging and PTH Resistance: Age-related PTH resistance may involve changes in the balance between full-length and truncated PTH fragments. N15-labeled standards facilitate investigation of age-dependent changes in PTH fragment profiles .

• Pharmacodynamic Biomarkers: In clinical studies of osteoporosis treatments, especially those targeting the PTH pathway, N15-labeled standards enable precise quantification of endogenous PTH fragments as pharmacodynamic biomarkers .

• Circadian Rhythm Effects: Recent evidence suggests circadian variations in PTH fragment ratios. Isotope-labeled standards support time-course studies investigating these temporal patterns and their relationship to bone turnover markers .

• Multi-Omics Integration: Combining quantitative proteomics of PTH fragments with genomics and metabolomics data offers new opportunities to develop integrated models of calcium homeostasis regulation in health and disease .

These emerging applications highlight the continuing importance of high-quality, isotope-labeled PTH fragments as analytical tools in bone and mineral metabolism research .

How might PTH (7-84) N15 be used to investigate the cross-talk between bone and kidney function?

PTH (7-84) N15 represents a sophisticated tool for investigating the complex cross-talk between bone and kidney in mineral homeostasis:

• Dual-Tissue Signaling Studies: By using N15-labeled PTH (7-84) in co-culture systems containing both osteoblastic/osteocytic cells and renal tubular cells, researchers can track the fragment's trafficking between tissues and its differential effects on each cell type .

• Competing Receptor Populations: The N15 label enables quantitative assessment of how PTH (7-84) distributes between PTH receptors in bone versus kidney tissues, potentially explaining tissue-specific responses to circulating PTH fragments .

• FGF23-PTH Axis Investigation: Emerging evidence suggests PTH fragments modulate FGF23 production by bone, which in turn affects renal phosphate handling. Isotope-labeled PTH (7-84) allows precise tracking of this bone-kidney signaling axis .

• Chronic Kidney Disease Models: In CKD models, where PTH metabolism is significantly altered, N15-labeled fragments help distinguish between retention of circulating fragments and altered production patterns .

• Calcium-Sensing Receptor Interactions: PTH (7-84) may interact differently with calcium-sensing receptors in parathyroid, bone, and kidney tissues. The N15 label facilitates quantitative binding studies across these tissue types .

Methodologically, these investigations often employ:

• Ex vivo organ culture systems

• Microfluidic co-culture platforms

• Tissue-specific receptor expression models

• Isotope ratio imaging mass spectrometry for tissue localization

• Functional readouts including calcium flux, phosphate transport, and gene expression profiles