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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.

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.

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL-1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.

Chemical analysis has long relied on instrumentation, from the simplest (eg, burets) to the more sophisticated (eg, mass spectrometers) to facilitate precision measurements. Regardless of their complexity, the development of a new instrumental device can be a valued approach to address problems in science. In this perspective, we outline the process of novel device design, from early phase conception to the manufacturing and testing of the tool or gadget. Focus is placed on the development of improved front-end devices to facilitate protein sample manipulations ahead of mass spectrometry, which therefore augment the proteomics workflow. Highlighted are some of the many training secrets, choices, and challenges that are inherent to the often iterative process of device design. In hopes of inspiring others to pursue instrument design to address relevant research questions, we present a summary list of points to consider prior to innovating their own devices.