Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection.

Enhanced Electrophoretic Depletion of Sodium Dodecyl Sulfate with Methanol for Membrane Proteome Analysis by Mass Spectrometry.

Membrane proteins are underrepresented during proteome characterizations, primarily owing to their lower solubility. Sodium dodecyl sulfate (SDS) is favored to enhance protein solubility but interferes with downstream analysis by mass spectrometry. Here, we present an improved workflow for SDS depletion using transmembrane electrophoresis (TME) while retaining a higher recovery of membrane proteins.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS.

Tips And Tricks for Proteome Sample Preparation.

Kovalchuk, S. I., Ziganshin, R.

Maximizing Cumulative Trypsin Activity with Calcium at Elevated Temperature for Enhanced Bottom-Up Proteome Analysis.

Bottom-up proteomics relies on efficient trypsin digestion ahead of MS analysis. Prior studies have suggested digestion at elevated temperature to accelerate proteolysis, showing an increase in the number of MS-identified peptides. However, improved sequence coverage may be a consequence of partial digestion, as higher temperatures destabilize and degrade the enzyme, causing enhanced activity to be short-lived.

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.

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 ZnSO, 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.

Automated Electrokinetic Platform for High-Throughput Sodium Dodecyl Sulfate Depletion Ahead of Proteome Analysis by Mass Spectrometry.

Sodium dodecyl sulfate (SDS) provides numerous benefits for proteome sample preparation. However, the surfactant can be detrimental to downstream mass spectrometry analysis. Although strategies are available to deplete SDS from proteins, each is plagued by unique deficiencies that challenge their utility for high-throughput proteomics.

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.

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.

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry.

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (., 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.

Developing front-end devices for improved sample preparation in MS-based proteome analysis.

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.

A robust strategy for proteomic identification of biomarkers of invasive phenotype complexed with extracellular heat shock proteins.

As an extension of their orchestration of intracellular pathways, secretion of extracellular heat shock proteins (HSPs) is an emerging paradigm of homeostasis imperative to multicellular organization. Extracellular HSP is axiomatic to the survival of cells during tumorigenesis; proportional representation of specific HSP family members is indicative of invasive potential and prognosis. Further significance has been added by the knowledge that all cancer-derived exosomes have surface-exposed HSPs that reflect the membrane topology of cells that secrete them.

Membrane-Based SDS Depletion Ahead of Peptide and Protein Analysis by Mass Spectrometry.

SDS interferes with both bottom-up and top-down MS analysis, requiring removal prior to detection. Filter-aided sample preparation (FASP) is favored for bottom-up proteomics (BUP) while acetone precipitation is popular for top-down proteomics (TDP). We recently demonstrated acetone precipitation in a membrane filter cartridge.

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating.

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE).

Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment.

The complexity of human tissue fluid precludes timely identification of cancer biomarkers by immunoassay or mass spectrometry. An increasingly attractive strategy is to primarily enrich extracellular vesicles (EVs) released from cancer cells in an accelerated manner compared to normal cells. The Vn96 peptide was herein employed to recover a subset of EVs released into the media from cellular models of breast cancer.

The benefits (and misfortunes) of SDS in top-down proteomics.

Unlabelled: Top-down proteomics (TDP) has great potential for high throughput proteoform characterization. With significant advances in mass spectrometry (MS) instrumentation permitting tandem MS of large intact proteins, a limitation to the widespread adoption of TDP still resides on front-end sample preparation protocols (e.g.

Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone.

Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis.

Membrane proteins are underrepresented in proteome analysis platforms because of their hydrophobic character, contributing to decreased solubility. Sodium dodecyl sulfate is a favored denaturant in proteomic workflows, facilitating cell lysis and protein dissolution; however, SDS impedes MS detection and therefore must be removed prior to analysis. Although strategies exist for SDS removal, they provide low recovery, purity, or reproducibility.

Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O-formylation) for peptides incubated in formic acid.

Comparison of sodium dodecyl sulfate depletion techniques for proteome analysis by mass spectrometry.

In proteomics, sodium dodecyl sulfate (SDS) is favored for protein solubilization and mass-based separation (e.g. GELFrEE or SDS PAGE).

Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins.

A two-stage spin cartridge for integrated protein precipitation, digestion and SDS removal in a comparative bottom-up proteomics workflow.

Unlabelled: Protein precipitation with organic solvent is an effective means of depleting contaminants such as sodium dodecyl sulfate (SDS), while maintaining high analyte recovery. Here, we report the use of a disposable two-stage spin cartridge to facilitate isolation of the precipitated protein, with subsequent enzyme digestion and peptide cleanup in the cartridge. An upper filtration cartridge retains over 95% of the protein (10 μg BSA), with 99.

Reprint of "GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)".

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures.