In eukaryotic cells, phosphorylation is widely involved in various biological processes such as signal transduction, cell cycle regulation, transcriptional control, and stress response. The reversible phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues alters protein conformation and function, serving as an indispensable part of cellular regulatory systems. However, in complex biological samples, phosphorylated peptides are typically of low abundance, present in small proportions, and exhibit poor ionization efficiency, making them easily masked by high-abundance non-phosphorylated peptides.
Effective enrichment of phosphorylated peptides prior to mass spectrometry analysis is essential for accurate phosphorylation research. To address this need, MtoZ Biolabs offers a phosphopeptide enrichment kit which providing an efficient, stable, and user-friendly sample pretreatment solution. This kit assists researchers in enhancing mass spectrometry detection sensitivity and gaining deeper insights into the roles of phosphorylation in both physiological and pathological processes.
Product Overview
The MtoZ Biolabs' phosphopeptide enrichment kit is designed based on titanium dioxide (TiO₂) material. Under acidic conditions, TiO₂ carries a positive charge and specifically binds to the negatively charged phosphate groups present on phosphorylated peptide fragments in the sample. After non-phosphorylated peptides are removed by a washing buffer, a basic elution buffer is used to release the phosphorylated peptides bound to TiO₂, thereby achieving specific enrichment of phosphopeptides. Subsequently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is employed to identify the phosphorylation sites on the peptides.
This product is compatible with various digested sample sources and is suitable for the analysis of protein samples from mammalian cells, tissues, microorganisms, or plants, making it an ideal tool for phosphoproteomics research.
Product Details
|
Product Details |
Size |
Storage Conditions |
|
Loading buffer |
30 mL×1 |
4℃ |
|
Wash buffer 1 |
10 mL×1 |
4℃ |
|
Wash buffer 2 |
10 mL×1 |
4℃ |
|
Wash buffer 3 |
5 mL×1 |
4℃ |
|
Elution buffer 1 |
1 mL×1 |
RT |
|
Elution buffer 2 |
1 mL×1 |
RT |
|
TiO2 Material |
20 mg×1 |
RT |
|
Tip |
12 pcs |
RT |
Protocol
MtoZ Biolabs provides a complete operating manual and matched buffer system. It is recommended to follow the steps below to achieve optimal enrichment performance:
1. Sample Preparation
(1) Desalt the digested protein sample using a C18 column or equivalent method, then lyophilize.
(2) A starting amount of >200 µg of digested protein is recommended. Reconstitute in Loading Buffer, ensuring complete dissolution of peptides with a concentration >2 µg/µL.
2. Phosphopeptide Binding
(1) Wash the TiO₂ material three times.
(2) Incubate the reconstituted peptides with different ratios of TiO₂ material, each incubation lasting 30 minutes.
3. Tip Pre-treatment
(1) Pre-equilibrate the Tip three times using 200 µL of Loading Buffer (centrifuge at 1000 ×g for 1 minute each time).
4. Washing Impurities
(1) Transfer the incubated TiO₂ material onto the Tip.
(2) Sequentially wash with 3 × 200 µL of Wash Buffer 1/2 (centrifuge at 1000 ×g for 1 minute each), to remove unbound and nonspecific peptides.
(3) Perform a final wash with 200 µL of Wash Buffer 3.
5. Eluting Phosphopeptides
(1) Use a new collection tube, add 60 µL of Elution Buffer 1 and let stand for 3 minutes, then centrifuge to collect. Repeat with 60 µL of Elution Buffer 2 for a total of 120 µL.
(2) It is recommended to immediately adjust the eluent to pH < 3 to prevent phosphate hydrolysis under high pH. Concentrate the eluate to dryness using a vacuum concentrator.
6. Downstream Analysis
Reconstitute the dried product in 20 µL of 0.1% formic acid and use directly for LC-MS/MS analysis or peptide concentration measurement.
Features and Benefits
The MtoZ Biolabs phosphopeptide enrichment kit offers the following advantages in phosphoproteomics research:
1. High Selectivity Binding
Titanium dioxide (TiO₂) material specifically binds to phosphate groups, significantly enhancing target peptide enrichment efficiency while reducing background interference.
2. Fast and Efficient
The standard workflow can be completed within 2 hours, making it ideal for mid- to low-throughput laboratories to quickly process samples.
3. Simple Operation
The material + Tip design avoids tedious procedures and is compatible with standard benchtop centrifugation equipment without the need for specialized instruments.
4. Ready-to-Use Buffers
All buffers in phosphopeptide enrichment kit are pre-formulated to ensure reproducibility and stability, eliminating the need for manual buffer preparation.
5. High Compatibility
The enriched products can be directly applied to various downstream analytical platforms, including DDA, DIA, PRM, and other mass spectrometry techniques.
Applications
The MtoZ Biolabs phosphopeptide enrichment kit is applicable to a wide range of fields including basic research and translational medicine, and is especially suitable for the following applications:
1. Phosphorylation Site Identification
Used to localize phosphorylation modifications on Ser/Thr/Tyr residues in proteins.
2. Signal Pathway Studies
Combined with mass spectrometry to reveal phosphorylation changes between kinases and substrates during signal transduction processes.
3. Drug Mechanism Evaluation
Investigates drug intervention effects by comparing phosphorylation profiles before and after treatment.
4. Target Screening and Validation
Assists in identifying disease-related phosphorylation events to support target discovery and validation.
5. Multi-Sample Omics Analysis
Enables quantitative phosphorylation analysis under multiple conditions using TMT/iTRAQ labeling.
Phosphorylation is a critical post-translational modification that regulates protein function and cellular behavior. Efficient enrichment of phosphorylated peptides is fundamental to studying phosphorylation-related mechanisms. The MtoZ Biolabs' phosphopeptide enrichment kit provides researchers with a high-performance and cost-effective sample preparation solution based on its scientifically designed principles, optimized protocol, and broad compatibility.
Whether you are investigating signaling pathways, screening kinase substrates, exploring disease mechanisms, or conducting high-throughput phosphoproteomic quantification, MtoZ Biolabs offers dependable support for your research. Visit official website for more details or contact us for technical assistance and customized recommendations.
FAQs
Q1: Can the Eluted Phosphopeptides Be Stored for a Long Time?
A1: Long-term storage is not recommended. The elution buffer has a high pH, which may lead to hydrolysis of phosphate groups. It is advised to immediately adjust the pH and dry the sample.
Q2: Does the Sample pH Affect the Enrichment Efficiency?
A2: Yes. It is recommended that the pH of the redissolved peptides be below 3 to enhance the binding efficiency between metal ions and phosphate groups.
Q3: Can the Tip Be Reused?
A3: To ensure data accuracy and prevent cross-contamination, this product is designed for single use only.
Q4: Will Residual EDTA or Reducing Agents in the Sample Affect the Process?
A4: Yes, they will affect enrichment. EDTA can chelate metal ions and reduce binding efficiency. These components should be thoroughly removed before digestion.
Q5: Can the Elution Buffer Be Used Directly for Mass Spectrometry Analysis?
A5: It is recommended to dry the eluate and reconstitute with 0.1% formic acid to improve compatibility with mass spectrometry and enhance ionization efficiency.
