Researchers from Ehime University and the Kazusa DNA Research Institute have developed a sample preparation method for cellular proteomics and protein structural analysis that only requires trace amounts of cells and is orders of magnitude faster than current techniques.
Mass spectrometry is a popular method for identifying protein components of biological samples, but comes with challenges. Protein components have to be extracted from cells and digested with proteases to peptide sizes that are easy for the machine to analyze. This digestion can take 20-plus hours. Also, sodium dodecyl sulfate (SDS), a surfactant often used in protein extraction processes, interferes with mass spectrometry and protein digestion and must be thoroughly removed prior to analysis, resulting to sample loss.
The new method, dubbed AnExSP for Anion-Exchange Disc-Assisted Sequential Sample Preparation by the Ehime/Kazusa team, leverages a microliter-sized spin column fitted with anion-exchange solid-phase extraction discs in a StageTip pipette tip as a tool for protein digestion. “We succeeded in completing the enzymatic digestion process in a minimum of 60 minutes by enriching the protein components on the disc surface,” the authors say. “Furthermore, we established optimal conditions for eluting only the digested peptides from the discs while keeping the SDS contained in the sample on the discs, and achieved sample pretreatment with simple operation and minimal loss.”
Using data-independent acquisition (DIA) analysis by Orbitrap mass spectrometry, they also succeeded in detecting approximately 7,000 different protein components in one microgram of human cultured cell protein extract. “By establishing sample pretreatment conditions using the reduced-size StageTip in the future, AnExSP pretreatment can be used for single-cell proteomics research, which is of growing interest in recent years,” the team adds.
AnExSP can also be used for pretreatment of proteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which is commonly used in protein experiments. Compared to conventional pretreatment methods, AnExSP demonstrated superior performance, especially in the detection of long-chain digested peptides.
The work, entitled Bottom-up/cross-linking mass spectrometry via simplified sample processing on anion-exchange solid-phase extraction spin column, was published in Chemical Communications. Because of its far-reaching versatility, AnExSP is expected to be applied to highly sensitive structural analysis methods for trace protein complexes in biological samples.