MitoB, IC-03914995
Product Introduction
MitoB is a mitochondria-targeted, ratiometric mass spectrometry probe designed for the in vivo and in vitro measurement of hydrogen peroxide (H₂O₂) levels within the mitochondrial matrix of living cells and tissues. MitoB consists of a triphenylphosphonium (TPP) lipophilic cation moiety that drives its selective accumulation within the negatively charged mitochondrial matrix, conjugated to an arylboronic acid moiety that reacts specifically with H₂O₂. Upon reaction with mitochondrial H₂O₂, the arylboronic acid group is stoichiometrically converted to a phenol derivative (MitoP), with the ratio of MitoP to MitoB providing a specific and quantitative readout of average mitochondrial H₂O₂ levels over time. Unlike fluorescence-based probes that can be affected by pH, photobleaching, and non-specific oxidation, MitoB provides a definitive mass spectrometry-based measurement of mitochondrial H₂O₂, making it a uniquely powerful and specific tool for studying mitochondrial redox signaling, oxidative stress, and the role of mitochondrial H₂O₂ in physiological and pathological processes including aging, metabolic diseases, and neurodegeneration.
Product Features
1. Mitochondria-Targeted Specificity: TPP cation moiety drives selective accumulation in the mitochondrial matrix, enabling compartment-specific H₂O₂ measurement.
2. Ratiometric Mass Spectrometry Detection: Provides quantitative, stoichiometric H₂O₂ measurement via the MitoP/MitoB ratio, free from artifacts affecting optical probes.
3. In Vivo Compatible: Can be administered to live animals for measurement of mitochondrial H₂O₂ in intact tissues, enabling physiologically relevant studies.
4. Defined Reaction Chemistry: Arylboronic acid moiety reacts specifically with H₂O₂, providing direct chemical specificity superior to general ROS probes.
5. High Purity: Supplied at high purity suitable for reproducible in vitro and in vivo studies.
Specifications
Size: Available in 1 mg, 5 mg, and 10 mg
CAS Number: 1247025-84-8
Molecular Weight: 526.46
Molecular Formula: C30H38BrO2P
Purity: ¡Ý98%
Solubility: DMSO: ¡Ý20 mg/mL; Ethanol: ¡Ý10 mg/mL; limited solubility in aqueous buffers
Storage and Stability
Storage Conditions: Store at -20¡ãC, protected from light and moisture.
Shelf Life: The product is stable for 24 months from the date of manufacture when stored as directed. For reconstituted solutions, aliquot and store at -80¡ãC. Avoid repeated freeze-thaw cycles.
Protocol (For Reference Only)
Important: MitoB is a research tool for mass spectrometry-based H₂O₂ measurement. Protect from light and moisture. Prepare stock solutions freshly before use.
1. Stock Solution Preparation: Dissolve MitoB in anhydrous DMSO or ethanol to prepare a concentrated stock solution (e.g., 10-100 mM). Aliquot into single-use vials and store at -80¡ãC protected from light.
2. In Vitro Incubation with Cells: For cell culture experiments, dilute MitoB stock solution into culture medium to a final concentration of 1-10 µM. Incubate cells with MitoB at 37¡ãC for 1-6 hours to allow mitochondrial accumulation and H₂O₂-dependent conversion. Include vehicle-treated control cells and, where appropriate, cells treated with mitochondrial H₂O₂ modulators (e.g., rotenone, antimycin A, or mitochondria-targeted antioxidants).
3. In Vivo Administration: For mouse studies, administer MitoB via intraperitoneal injection at doses of 50-100 nmol/kg (or as optimized for specific experimental conditions) formulated in sterile saline. After a defined incubation period (typically 4-6 hours), euthanize animals and harvest tissues of interest. Rapidly freeze tissues in liquid nitrogen and store at -80¡ãC until processing.
4. Sample Processing for LC-MS/MS: Homogenize cell pellets or tissue samples in ice-cold extraction solvent (typically 60% acetonitrile in water containing 0.1% formic acid and an appropriate internal standard). Centrifuge at 16,000 x g for 10 minutes at 4¡ãC to remove precipitated protein. Analyze the supernatant by LC-MS/MS for both MitoB and MitoP.
5. Data Analysis: Calculate the MitoP/MitoB ratio for each sample. The ratio is proportional to the average mitochondrial H₂O₂ concentration over the incubation period. Normalize to vehicle controls and, where possible, calibrate using in situ or in vitro calibration methods for absolute quantification of H₂O₂.
Precautions
1. MitoB is a mass spectrometry probe; a triple quadrupole or high-resolution mass spectrometer with appropriate LC separation is required for analysis.
2. The MitoP/MitoB ratio reflects the average H₂O₂ level integrated over the entire incubation period; it does not provide real-time kinetic information.
3. Optimize incubation time and MitoB concentration for each experimental system; prolonged incubation or excessive doses may alter mitochondrial function.
4. For in vivo studies, tissue harvesting and processing must be performed rapidly to prevent post-mortem changes in H₂O₂ levels.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: How does MitoB differ from fluorescent mitochondrial ROS probes?
A1: Unlike fluorescent probes such as MitoSOX or DCFH-DA, MitoB provides a specific and stoichiometric reaction with H₂O₂, with the reaction product (MitoP) stable and quantified by LC-MS/MS. This avoids artifacts from pH sensitivity, photobleaching, non-specific oxidation, and auto-oxidation that commonly affect fluorescence-based probes.
Q2: What is the detection principle of MitoB?
A2: The TPP cation drives MitoB accumulation in the mitochondrial matrix. The arylboronic acid moiety reacts specifically and stoichiometrically with H₂O₂ to form MitoP. The MitoP/MitoB ratio measured by LC-MS/MS is proportional to the average mitochondrial H₂O₂ concentration.
Q3: Can MitoB be used to measure real-time H₂O₂ changes?
A3: No. MitoB provides an integrated measurement of average H₂O₂ levels over the incubation period. It does not provide real-time kinetic data. For real-time measurements, genetically encoded H₂O₂ sensors such as HyPer may be more suitable.
Q4: What mass spectrometry parameters are required for MitoB detection?
A4: MitoB and MitoP are typically analyzed by LC-MS/MS using positive electrospray ionization with multiple reaction monitoring (MRM) transitions specific for each compound. The exact transitions depend on the instrument and should be optimized in your laboratory using pure standards.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of incubation conditions, dosing, and analytical parameters is recommended for specific experimental systems.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-03914995
Product Name: MitoB
Size: 1 mg / 5 mg / 10 mg
Price:
1 mg: CNY ¥800.00 / USD $80.00 / EUR €96.00 / JPY ¥14400.00
5 mg: CNY ¥2000.00 / USD $200.00 / EUR €240.00 / JPY ¥36000.00
10 mg: CNY ¥3500.00 / USD $350.00 / EUR €420.00 / JPY ¥63000.00
2023 Version
