Protein Identification by SDS-PAGE and LC-MS/MS
  • OVERVIEW

Protein Identification by SDS-PAGE and LC-MS/MS


The identification of protein glue spots and strips is currently the most widely used proteomics research method. Two-dimensional gel electrophoresis (2-DE) and polyacrylamide gel electrophoresis (SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) to separate the protein mixture, collect the target protein glue spots and strips, and use them for mass spectrometry analysis to obtain protein information after in-gel enzymatic digestion.

Technical Features

The sample is simple and easy to detect.

Targeted and easy to analyze.

High accuracy, high specificity, and short lead time.

Scope of applications

Pharmacokinetics

LC-MS is widely used in the field of bioanalysis and is specially involved in pharmacokinetic studies of pharmaceuticals. Pharmacokinetic studies are needed to determine how quickly a drug will be cleared from the body organs and the hepatic blood flow. MS analyzers are useful in these studies because of their shorter analysis time, and higher sensitivity and specificity compared to UV detectors commonly attached to HPLC systems.

Proteomics/metabolomics

LC-MS is used in proteomics as a method to detect and identify the components of a complex mixture. The bottom-up proteomics LC-MS approach generally involves protease digestion and denaturation using trypsin as a protease, urea to denature the tertiary structure, and iodoacetamide to modify the cysteine residues. After digestion, LC-MS is used for peptide mass fingerprinting, or LC-MS/MS (tandem MS) is used to derive the sequences of individual peptides. LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometry.

Drug development

LC-MS is frequently used in drug development because it allows quick molecular weight confirmation and structure identification. These features speed up the process of generating, testing, and validating a discovery starting from a vast array of products with potential application. LC-MS applications for drug development are highly automated methods used for peptide mapping, glycoprotein mapping, lipodomics, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.

Workflow of Our Service

- Securing initial sample (for example, by cell lysis, immunoprecipitation, FPLC, anything that may be necessary for the task at hand).

- Separation by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE).

- Staining with MS-compatible stain.

- Band excision.

- Extensive de-staining, if no MS-compatible stain was employed.

- In-gel reduction and alkylation.

- In-gel enzymatic digestion with sequencing-grade trypsin.

- Digest concentration.

- LC-MS/MS data analysis


Sample Requirements

Sample content: 50-1000fmol (depending on the test item).

Glue samples: test staining or silver staining (without using glutaraldehyde as a fixative).

Storage method: It is recommended to use low temperature for liquids, and deionized water for glues to prevent drying.

Sample Information Sheet: The source, content, status and other basic information of the sample should be filled in in detail.


Deliverables

An experimental report, including the experimental process, analysis process and data results.

Protein Identification by SDS-PAGE and LC-MS/MS


The identification of protein glue spots and strips is currently the most widely used proteomics research method. Two-dimensional gel electrophoresis (2-DE) and polyacrylamide gel electrophoresis (SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) to separate the protein mixture, collect the target protein glue spots and strips, and use them for mass spectrometry analysis to obtain protein information after in-gel enzymatic digestion.

Technical Features

The sample is simple and easy to detect.

Targeted and easy to analyze.

High accuracy, high specificity, and short lead time.

Scope of applications

Pharmacokinetics

LC-MS is widely used in the field of bioanalysis and is specially involved in pharmacokinetic studies of pharmaceuticals. Pharmacokinetic studies are needed to determine how quickly a drug will be cleared from the body organs and the hepatic blood flow. MS analyzers are useful in these studies because of their shorter analysis time, and higher sensitivity and specificity compared to UV detectors commonly attached to HPLC systems.

Proteomics/metabolomics

LC-MS is used in proteomics as a method to detect and identify the components of a complex mixture. The bottom-up proteomics LC-MS approach generally involves protease digestion and denaturation using trypsin as a protease, urea to denature the tertiary structure, and iodoacetamide to modify the cysteine residues. After digestion, LC-MS is used for peptide mass fingerprinting, or LC-MS/MS (tandem MS) is used to derive the sequences of individual peptides. LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometry.

Drug development

LC-MS is frequently used in drug development because it allows quick molecular weight confirmation and structure identification. These features speed up the process of generating, testing, and validating a discovery starting from a vast array of products with potential application. LC-MS applications for drug development are highly automated methods used for peptide mapping, glycoprotein mapping, lipodomics, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.

Workflow of Our Service

- Securing initial sample (for example, by cell lysis, immunoprecipitation, FPLC, anything that may be necessary for the task at hand).

- Separation by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE).

- Staining with MS-compatible stain.

- Band excision.

- Extensive de-staining, if no MS-compatible stain was employed.

- In-gel reduction and alkylation.

- In-gel enzymatic digestion with sequencing-grade trypsin.

- Digest concentration.

- LC-MS/MS data analysis


Sample Requirements

Sample content: 50-1000fmol (depending on the test item).

Glue samples: test staining or silver staining (without using glutaraldehyde as a fixative).

Storage method: It is recommended to use low temperature for liquids, and deionized water for glues to prevent drying.

Sample Information Sheet: The source, content, status and other basic information of the sample should be filled in in detail.


Deliverables

An experimental report, including the experimental process, analysis process and data results.

Request Quote
First Name *
Last Name *
Email Address *
Instiution *
Prodcuts/Services *
Questions & Comments *