Workflow of Unknown Proteins Sequencing

Proteins are essential molecules in living organisms, fulfilling roles in structure, catalysis, signaling, and more. To fully understand the functions of proteins and their roles in biological processes, protein sequence analysis is a crucial step.

 

Sample Preparation

1. Sample Collection and Processing

Protein samples can be derived from various sources, including tissue samples, cell cultures, and blood. Before sequencing, samples need to undergo several processing steps such as cell lysis, protein extraction, and purification. These steps aim to obtain high-purity and high-concentration protein samples to ensure accuracy in subsequent analyses.

 

2. Protein Separation

After protein extraction, the proteins in the sample usually need to be separated. Common methods include gel electrophoresis (SDS-PAGE) and liquid chromatography (LC). These methods can separate complex protein mixtures into individual proteins for subsequent mass spectrometry analysis.

 

Protein Digestion

1. Enzymatic Digestion

To facilitate mass spectrometry analysis, proteins typically need to be cleaved into smaller peptides. This process is usually achieved through enzymatic digestion, with trypsin being the most commonly used enzyme. Trypsin specifically recognizes and cleaves lysine and arginine residues in proteins, breaking down long-chain proteins into shorter peptides.

 

2. Sample Purification

After enzymatic digestion, the resulting peptides need to be purified. Reverse-phase high-performance liquid chromatography (RP-HPLC) is commonly used for this purpose. This method effectively removes salts, buffers, and other interfering substances, yielding purified peptide samples.

 

Mass Spectrometry Analysis

1. Choice of Mass Spectrometer

Mass spectrometers are the core tools for protein sequencing. Common mass spectrometers include electrospray ionization mass spectrometers (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOF-MS). Choosing the appropriate mass spectrometer can enhance the sensitivity and accuracy of peptide detection.

 

2. Data Acquisition

During mass spectrometry analysis, peptide samples are ionized and accelerated through the mass spectrometer, separated, and detected based on their mass-to-charge ratio (m/z). The mass spectrometer generates mass spectra, showing the m/z and relative abundance of each peptide. These data are fundamental to protein sequence analysis.

 

Data Analysis

1. Peptide Matching

Once mass spectra are generated, the detected peptides need to be matched with known protein sequences through database searching or de novo sequencing methods. Database searching relies on known protein databases like UniProt, while de novo sequencing deduces peptide sequences directly from mass spectrometry data.

 

2. Protein Assembly

The sequence fragments obtained through peptide matching usually require further assembly to reconstruct the entire protein sequence. This process may involve combining various algorithms and bioinformatics tools to ensure the accuracy and completeness of the sequence assembly.

 

Result Validation

1. Sequence Validation

To verify the accuracy of the sequencing results, further experimental validation is often necessary. For example, the synthesized peptides can be compared to the original sample through mass spectrometry to confirm their consistency. Additionally, functional experiments can be conducted to verify the biological activity of the protein.

 

2. Database Updating

Upon confirmation of the sequencing results, new protein sequences should be added to public databases for reference by other researchers. This not only helps the scientific community share resources but also provides essential data for future protein research.

 

Unknown protein sequencing is a complex and multi-step process that requires precise operations and strict controls at each step. Through sample preparation, protein digestion, mass spectrometry analysis, data analysis, and result validation, high-quality protein sequence information can be obtained, providing valuable fundamental data for biological research. MtoZ Biolabs provides integrate unknown proteins sequencing service.