A New Method for Sample Preparation to Accelerate Mass Spectrometric Analysis

Mass spectrometry is a widely used protein analysis method, but its sample preparation process is complex and time-consuming. Researchers at Ewon University in Japan have recently developed a new method to significantly shorten the preparation time by anion exchange solid phase extraction centrifugal columns.

The findings were recently published in Chemical Communications. The benefit of the new method is that it does not require highly specialized operating techniques to perform mass spectrometry analysis of trace amounts of cell and tissue samples with good reproducibility.

In recent years, with the popularity of proteomics research, researchers have used mass spectrometry to analyze protein components in biological samples. When detecting proteins, it is often necessary to extract protein components from cells and digest them with proteases to peptides suitable for mass spectrometry analysis. Digestion of proteins in this manner often takes 20 hours or more.

In addition, sodium dodecyl sulfate (SDS) is a surfactant commonly used in protein extraction processes. It interferes with mass spectrometry and protein digestion and must therefore be completely removed before analysis. However, removal of the SDS is at the cost of sample loss.

To overcome these challenges, researchers at Ewon University have developed a novel sample preparation method, AnExSP. It uses a microliter-sized spin column and an anion-exchange solid-phase extraction membrane installed in a pipette tip called a StageTip as a tool for protein digestion.

The researchers stated that by enriching the protein components on the surface of the diaphragm, they successfully completed the enzymatic digestion process within 60 minutes. At the same time, they established optimal conditions to elute digested peptides from the diaphragm alone, while retaining the SDS contained in the sample on the diaphragm, thereby minimizing sample loss.

They combined AnExSP with data-independent acquisition (DIA) of the Orbitrap mass spectrometer to successfully detect approximately 7,000 different protein components from 1 μg of cultured cell protein extracts. " Using small volumes of StageTip to establish sample pretreatment conditions, AnExSP pretreatment can be used in single-cell proteomics research in the future, and the field has received increasing attention in recent years," they said.

In addition, the AnExSP method can also be used for protein pretreatment in SDS-PAGE electrophoresis experiments. Compared with traditional pretreatment methods, AnExSP performs excellently, especially in detecting long-chain digested peptides. The researchers successfully achieved the purification of the target protein complex as well as the efficient detection of long-chain cross-linked peptides, which are difficult to detect with conventional methods.