Unknown Proteins Sequencing Service

The amino acid sequence of proteins forms the basis for determining protein structure and function. Protein sequencing, which determines the types and arrangement of amino acids composing a protein, is a crucial aspect of proteomic studies. Common methods include mass spectrometry (such as tandem mass spectrometry) and the Edman degradation method. Unknown proteins typically refer to those proteins that have not been thoroughly studied and characterized. These proteins may be encoded by newly discovered genes or may be gene products from known organisms whose structure, function, and roles have not been fully understood. Sequencing unknown proteins contributes to revealing the complete picture of an organism's genome and proteome, which is crucial for studying protein structure and function. It also promotes advancements in biotechnology, drug development, and disease diagnosis.

The MtoZ Biolabs utilizes the latest Thermo Obitrap Exploris 240 mass spectrometer for sequencing unknown protein samples. It employs six commonly used proteases (Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) to cleave the target proteins and conduct mass spectrometric analysis. While obtaining fragmented peptide segments, the protein sequence is determined 100% by piecing together these peptide segments.

Analysis Workflow

1. Sample Preparation

Extraction of proteins and removal of impurities through purification processes.

2. Protein Fractionation/Isolation

Proteins are fractionated or isolated, typically using one-dimensional or two-dimensional gel electrophoresis (1D/2D-PAGE) and liquid chromatography (such as HPLC).

3. Protease Digestion

Proteins are digested into smaller peptide segments through multiple enzyme digestions (such as Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) to facilitate subsequent mass spectrometric analysis.

4. Peptide Separation

Peptide segments obtained from protease digestion are further separated using liquid chromatography (such as RP-HPLC).

5. Mass Spectrometric Analysis

Peptide segments are analyzed using a mass spectrometer to obtain information about their mass, charge states, etc.

6. Peptide Sequencing

Peptide segments are further sequenced using tandem mass spectrometry (MS/MS).

7. Data Processing and Analysis

Specialized mass spectrometry software such as Mascot, Sequest, etc., are used to process and analyze mass spectrometry data in combination with known protein databases.

Applications

1. Discovery of New Proteins

Sequencing and functional annotation of unknown proteins aid in discovering new biological catalysts, signal transduction proteins, transcription factors, and other molecular functions.

2. Protein Function Studies

Protein sequencing provides data support for functional annotation and protein interaction network studies.

3. Discovery of Disease Biomarkers

Comparative analysis of proteomes between pathological and normal tissues helps in identifying potential disease biomarkers, providing clues for disease diagnosis and treatment.

4. Drug Development

Identification of target proteins for drug action and discovery of new drug targets accelerate the development and optimization of new drugs.

Sample Submission Requirements

We accept the following types of samples:

1. Purified Proteins

Please provide protein samples with purity greater than 85%. The recommended sample volume is 50-200 μg, and the protein concentration should be between 0.1-1 mg/mL.

2. Protein Extracts

Including but not limited to protein extracts from cells, tissues, or organisms. The recommended sample volume is 200-500 μg, and the protein concentration should be between 1-5 mg/mL.

3. Protein Gel Bands/Spots

Protein gel bands separated by 1D-SDS PAGE, protein spots from 2D PAGE, IP, Co-IP samples, and GST fusion protein pull-downs, etc. For gel bands/spots, please ensure a protein content of at least 10 ng, while for immunoprecipitation samples, please provide at least 20 μg of protein.

Deliverables

In the technical report, MtoZ Biolabs will provide you with detailed technical information, including: