Using Mass Spectrometry to Determine Unknown Protein Sequences

Unknown proteins refer to those present in organisms, but their functions, structures, or affiliations have not been clearly identified. Due to the lack of corresponding data support, the identification of unknown proteins faces some challenges, and the emergence of mass spectrometry provides a powerful means for the identification of unknown proteins. De novo sequencing based on mass spectrometry is different from database-dependent searches, it does not need previous sequence information to identify the sequence of peptides or proteins, and is especially suitable for studying new or unannotated proteins.

 

Steps During the Process of De Novo Sequencing of Unknown Proteins

1. Sample Preparation

Protein samples are first broken down into shorter peptides by enzymatic digestion (usually using trypsin).

 

2. Mass Spectrometry Analysis

These peptides are then introduced into the mass spectrometer, usually through electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) technology.

 

3. Detection and Measurement of Peptide Ions

In the mass spectrometer, peptide ions are accelerated and go through the mass analyzer, being detected and separated based on their mass-to-charge ratio (m/z).

 

4. Sequence Inference

By analyzing the peptide's mass spectrometry fragmentation map, especially the fragments generated by collision-induced dissociation (CID) or electron transfer dissociation (ETD), the amino acid sequence of the peptide can be inferred. This requires the interpretation of the fragment ion's patterns and how they correspond to possible amino acid sequences.

 

The challenge of De novo sequencing in identifying unknown proteins lies in its high demand for the quality of mass spectrometry data, including the strength of the signal, the complexity of the fragmentation map, and the accuracy of the measurement. In addition, the sequence inference process is computationally intensive, requiring specialized software and algorithms to process data and identify possible sequences.