How Are Protein Polypeptides Sequenced

The sequencing of protein peptide chains refers to the process of determining the amino acid sequence within proteins. This is accomplished by breaking proteins into smaller peptide fragments and sequencing these fragments. Currently, commonly used methods include mass spectrometry and DNA/RNA sequencing techniques.

 

Mass Spectrometry Sequencing

1. Sample Preparation

Proteins are digested, typically using enzymes such as trypsin, to generate smaller peptide fragments.

 

2. Mass Spectrometry Analysis

The peptide fragments are analyzed using a mass spectrometer. They are first ionized to form charged ions, which are subsequently separated and detected based on their mass-to-charge ratio (m/z).

 

3. Data Interpretation

By interpreting the mass spectrometry data, the mass of each peptide fragment can be determined, allowing inference of the protein's amino acid sequence.

 

DNA/RNA Sequencing Method

1. Transcription and Reverse Transcription

The gene encoding the protein is transcribed into RNA, which is then reverse-transcribed into complementary DNA (cDNA).

 

2. PCR Amplification

The cDNA is amplified by polymerase chain reaction (PCR), generating numerous copies.

 

3. Sequencing

The PCR-amplified products are sequenced using DNA sequencing techniques to determine the sequence of the gene encoding the original protein.

 

4. Translation

The DNA sequence is then translated into the corresponding amino acid sequence of the protein.

 

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

Related Services

Protein Sequencing Service