Workflow of Protein Full-Length Sequencing

Full-length protein sequencing is a sophisticated and vital technique that plays a crucial role in proteomics research. By determining the precise amino acid sequence of proteins, researchers can gain a deeper understanding of protein structure and function, thereby advancing fields such as disease research and drug development.

 

Sample Preparation

Sample preparation is the first step in full-length protein sequencing and is fundamental to the success of subsequent steps. Samples can be obtained from cells, tissues, or bodily fluids. The general steps include:

 

1. Sample Lysis

Disrupting cell membranes through physical or chemical methods to release proteins.

 

2. Protein Extraction

Separating proteins and removing other impurities using buffer solutions and centrifugation techniques.

 

3. Protein Quantification

Measuring protein concentration using BCA or Bradford methods to ensure the sample quantity meets experimental requirements.

 

Protein Separation and Purification

Accurate sequencing depends on effective protein separation and purification, typically employing electrophoresis or chromatography techniques.

 

1. SDS-PAGE

Separating proteins by polyacrylamide gel electrophoresis, which grades them based on molecular weight.

 

2. Chromatography

Further purifying target proteins using methods such as ion-exchange chromatography and affinity chromatography.

 

Mass Spectrometry Analysis

Mass spectrometry analysis is the core step in full-length protein sequencing, providing high-precision mass spectra of proteins.

 

1. Protein Digestion

Using specific enzymes like trypsin to break proteins into peptide fragments.

 

2. Mass Spectrometry Detection

Introducing peptide fragments into mass spectrometers, commonly using techniques such as MALDI-TOF and LC-MS/MS.

 

3. Data Acquisition

Generating mass spectrometry data through ionization, time-of-flight, and detection processes.

 

Data Processing and Analysis

The processing and analysis of mass spectrometry data are crucial for ensuring the accuracy and reliability of protein sequencing.

 

1. Data Processing

Using specialized software for baseline correction, noise removal, and peak identification.

 

2. Sequence Analysis

Matching fragment ion peaks in mass spectra to known sequences through database searches and algorithms to determine amino acid sequences.

 

3. Sequence Assembly

Integrating sequence information from multiple peptide fragments to reconstruct the full-length protein sequence.

 

Result Validation

The final step to ensure sequencing accuracy is result validation, which typically involves experimental validation and bioinformatics analysis.

 

1. Experimental Validation

Synthesizing peptide fragments of the determined sequence for verification through mass spectrometry or Western Blot analysis.

 

2. Bioinformatics Analysis

Using protein databases and tools to analyze the function, structure, and interactions of the sequenced proteins.

 

MtoZ Biolabs provides integrate protein full-length sequencing service.