Mechanism of Nano LC-MS/MS in Protein Sequence Analysis

Analyzing protein sequences is crucial for understanding their functions, structures, and interactions. Mass spectrometry (MS) technology, especially nano liquid chromatography-tandem mass spectrometry (Nano LC-MS/MS), has become a primary tool in proteomics research.

 

Basic Principles of Nano LC-MS/MS

Nano LC-MS/MS combines the advantages of nano liquid chromatography (nano LC) and tandem mass spectrometry (MS/MS) for efficient separation and precise analysis of proteins and peptides. Nano LC achieves high-resolution separation with nanometer-scale columns, while MS/MS provides high-sensitivity detection through two-stage mass analysis.

 

1. Nano Liquid Chromatography (Nano LC)

Nano LC employs columns with an inner diameter of less than 100 micrometers, using minimal sample and solvent amounts to achieve high-resolution and high-sensitivity separation. The workflow of Nano LC mainly includes sample preparation, sample loading, gradient elution, and detection.

 

2. Tandem Mass Spectrometry (MS/MS)

Tandem mass spectrometry enables in-depth analysis of complex samples through two-stage mass analysis. The first mass spectrometry (MS1) detects and separates ionized proteins or peptides, while the second mass spectrometry (MS2) fragments and analyzes selected precursor ions to obtain structural information of specific ions.

 

Application of Nano LC-MS/MS in Protein Sequence Analysis

1. Protein Digestion

Proteins in the sample are first digested, usually using trypsin, into smaller peptides. These digested peptides are easier to separate by Nano LC and detect by MS/MS.

 

2. Peptide Separation

The peptide mixture is separated by Nano LC. Due to the high surface area and low flow rate of nano-scale columns, Nano LC achieves efficient separation and concentration of peptides, significantly enhancing analytical sensitivity and resolution.

 

3. Peptide Ionization

The separated peptides are ionized as they enter the mass spectrometer, typically using electrospray ionization (ESI). The ionized peptides generate charged ions in the mass spectrometer, entering the mass analysis stage.

 

4. Mass Spectrometry Analysis

In mass spectrometry analysis, MS1 first detects and separates all ionized peptides based on their mass-to-charge ratio (m/z), selecting target precursor ions. The selected precursor ions enter the collision cell for fragmentation, producing a series of product ions. MS2 then detects these product ions, obtaining the fragmentation spectrum of the peptides.

 

5. Data Analysis and Sequence Elucidation

The obtained fragmentation spectra are analyzed using specialized software. By comparing with known protein databases, the amino acid sequences of the peptides can be deduced, further inferring the sequences of the original proteins.

 

Nano LC-MS/MS technology, through efficient peptide separation and high-sensitivity mass spectrometry detection, has significantly advanced proteomics research. Its widespread application in protein sequence analysis has greatly enhanced our understanding of protein structures and functions, promoting progress in biological and medical fields. MtoZ Biolabs provides integrate protein sequencing service.