Proteomics study designs aim for inclusiveness, but extracting proteins from complex biological samples requires the use of harsh buffers that are inconsistent with the proteomics workflow. More proteins are lost in attempts to remove contaminants and interfering reagents, resulting in increased variability and decreased reproducibility of data.
Researchers at the Babraham Institute have enhanced traditional sample preparation methods in proteomics by developing a new technique that increases the inclusion of difficult-to-capture cellular proteins, thereby improving proteomics readouts. The researchers improved the recovery of less soluble proteins that contribute to the accumulation of toxic misfolded proteins in older cells. The new technique also recovered more membrane-spanning proteins.
Solvent Precipitation SP3 (SP4) improves recovery for proteomics sample preparation without magnetic beads, according to an article in the Analytical Chemistry journal. Rahul Samant, PhD, scientist in the Babraham Institute's signaling program, is the senior author of the research and Harvey Johnston, PhD, postdoctoral researcher in the Samants group.
The technological advancement builds on a single-pot, solid-phase-enhanced sample preparation technique called SP3. It involves organic solvents and magnetic beads to denature and capture protein aggregates, then washes to remove contaminants. The method risks losing proteins that do not completely attach to the magnet beads during washes. The loss and subsequent processing costs increase when samples contain higher quantities of proteins or more hydrophobic proteins.
The authors argue that a complete, reproducible extraction of protein material is critical for thorough and independent proteome investigations.
The current method excludes the need to use magnetic beads, although optional use of inert glass beads might simplify sample handling. Glass beads are available at 1/1000th the price of magnetic beads and provide a low-cost alternative to magnetic beads. The enhanced technique uses centrifugation to identify more proteins with greater consistency.
The authors removed magnetic beads altogether and instead employed acetonitrile-induced protein precipitation and centrifugation for protein capture and isolationeither bead-free or with low-cost, inert glass beads. This technique is named SP4.
In proteome characterization, the authors could obtain protein yields that were comparable or higher for 15000 microgram samples, with greater reproducibility, compared to SP3. Three other labs across eight sample types and five lysis buffers confirmed that SP4s performance was comparable or higher than SP3.
SP4 is a minimalistic protein clean-up strategy that provides cost-effective input scalability, the possibility to eliminate beads entirely, and provides important considerations for SP3 applications, all while maintaining the speed and compatibility of SP3.
SP4 appears to be a helpful addition to the sample preparation toolkit, according to Nikolai Slavov, PhD, an associate professor of bioengineering and the director of the single-cell proteomics program at Northeastern University College of Engineering. (Slavov was not involved in this research.)
The simplicity of the new technique promises to make sample preparation in proteomics simpler and more accessible. This will greatly assist proteomics-based experiments in life science laboratories with small to medium budgets. In future experiments, the authors intend to apply the new method to investigate the mechanisms underlying the aggregation and elimination of misfolded proteins in cells.