Recent advances in top-down proteome sample processing ahead of MS analysis.

Top-down proteomics is emerging as a preferred approach to investigate biological systems, with objectives ranging from the detailed assessment of a single protein therapeutic, to the complete characterization of every possible protein including their modifications, which define the human proteoform. Given the controlling influence of protein modifications on their biological function, understanding how gene products manifest or respond to disease is most precisely achieved by characterization at the intact protein level. Top-down mass spectrometry (MS) analysis of proteins entails unique challenges associated with processing whole proteins while maintaining their integrity throughout the processes of extraction, enrichment, purification, and fractionation. Recent advances in each of these critical front-end preparation processes, including minimalistic workflows, have greatly expanded the capacity of MS for top-down proteome analysis. Acknowledging the many contributions in MS technology and sample processing, the present review aims to highlight the diverse strategies that have forged a pathway for top-down proteomics. We comprehensively discuss the evolution of front-end workflows that today facilitate optimal characterization of proteoform-driven biology, including a brief description of the clinical applications that have motivated these impactful contributions.

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry.

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion.

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow-including the digestion step-can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Mass spectrometry profiling of low molecular weight proteins and peptides isolated by acetone precipitation.

Solvent-based protein precipitation provides exceptional recovery, particularly when the ionic strength of the solution is controlled. While precipitation is ideally suited for intact protein purification ahead of mass-spectrometry, low molecular weight (LMW) proteins and peptides are considered less susceptible to aggregation in organic solvent. As the combination of salt and organic solvent (i.e. acetone) has yet to be exploited to precipitate LMW proteins, we herein determine the low mass limit for solvent-based protein precipitation. We establish optimized conditions for high recovery precipitation of LMW proteins and peptides. Our results demonstrate a strong dependence on the type of salt to recover LMW components from complex mixtures. Inclusion of 100 mM ZnSO4 with 97% acetone provides near quantitative recovery of all peptides down to 2 kDa, and continues to exceed 90% yield for peptides at a molecular weight of 1 kDa. A detailed characterization of the precipitated peptides resulting from trypsin and pepsin digestion of complex systems is provided by bottom-up mass spectrometry.