Application of Proteonano™ SP3 Proteome Extraction Kit

SP3 Technology for Proteomics

In the proteomics workflow, the process of extracting proteins from biological samples and enzymatically digesting proteins into peptides is very important, as it is the major determinant of the sensitivity and accuracy of proteomics analysis. Conventional pre-treatment methods, such as liquid-phase digestion, in-gel digestion, and filter membrane-assisted sample processing, often require complex transfer steps and large contact areas between the samples and the container walls. These issues may lead to sample loss and hinder the realization of deep proteomics analysis.

With the rapid development of materials science, a single-pot, solid-phase-enhanced sample preparation (SP3) based on paramagnetic beads has been developed, with excellent performance. The SP3 method involves capturing proteins using the carboxyl groups on the surface of magnetic beads under the drive of a high-concentration organic solvent. Subsequently, the proteins bound to the SP3 magnetic beads are digested with trypsin in an aqueous solution. The SP3 magnetic beads are compatible with surfactants, and the proteins or peptides after digestion can be eluted under aqueous conditions. The SP3 technology uses a hydrophilic interaction process to replace or eliminate the substances typically used for cell or tissue lysis, protein dissolution, and enzymatic digestion (such as detergents, salt precipitants, salts, buffers, acids, and solvents) prior to downstream proteomic analysis.

Proteonano™ SP3 Proteome Extraction Kit

Amerigo Scientific provides the Proteonano™ SP3 proteome extraction kit that uses multivalent, multi-affinity, and superparamagnetic nanoprobes to enrich low-abundance proteins in complex samples. Our kit based on the SP3 technology can enable swift, reliable, and efficient processing of proteins for proteomic research. This Proteonano™ kit can be compatible with many automated systems, such as the Opentrons OT-2, KingFisher Flex, and KingFisher Apex systems.

Experimental Procedures

Comparison of SP3 Sample Preparation Kits

The components, performance, application, and compatibility of the Proteonano™ SP3 proteome extraction kit are compared with those of three similar products as shown in the table below.

Protein Identification of Plant and Animal Tissue Samples

In the application of protein identification in plant and animal tissue samples, using the Proteonano™ SP3 proteome extraction kit, nanogram-level protein loading can be achieved for the deep coverage of proteomics. The mass spectrometry data was collected using the next-generation Orbitrap Astral high-resolution mass spectrometer. Following automatic protein capture and tryptic protein digestion, 300 ng of peptides were aspirated by the automatic sampler and combined with the analytical column (5 μm*150 mm, C18, 2 μm, 100 Å) for separation. An 8-minute analysis gradient was established using two mobile phases (mobile phase A: 0.1% formic acid, mobile phase B: 0.1% formic acid, 80% ACN). The flow rate of the liquid phase was set to 1.8 μL/min, and mass spectrometry results were collected in DIA mode.

After treating frozen Arabidopsis (At.) samples with the Proteonano™SP3 proteome extraction kit, 9,414 proteins were detected in the samples through mass spectrometry analysis. In the similarly treated frozen tobacco samples, 9916 proteins were detected. Compared with the other commercially available SP3 kit, the samples treated with the Proteonano™ SP3 kit can be identified with an additional 100 to 400 proteins. Processing fresh corn samples with the Proteonano™ SP3 protein extraction kit allowed for the detection of 11934 proteins, while 12521 proteins were detected in treated fresh soybean samples. Compared with the filtration-assisted sample preparation (FASP) method, the samples treated with the Proteonano™ SP3 kit can be identified with 800 to 1000 more soybean proteins.

Figure A. The number of proteins identified in plant tissue samples

Frozen rabbit intestinal tissue samples were treated with the Proteonano™ SP3 proteome extraction kit, and over 8300 proteins could be detected, which was similar to the results obtained using the other commercially available SP3 kit. The results were highly reproducible in parallel processed samples.

Figure B. The number of proteins identified in the animal tissue samples