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. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

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. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

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.

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. An optimal approach would rapidly and reproducibly achieve less than 10 ppm residual SDS while simultaneously maximizing analyte recovery. Here, we describe improvements to a simple electrokinetic device termed transmembrane electrophoresis, which we previously reported for automated, rapid SDS depletion of proteome samples. Voltage-driven transport of SDS across a molecular weight cutoff membrane is enhanced at higher electric fields, which is herein achieved by integrating an active cooling mechanism to mitigate the impacts of Joule heating. We report 99.9% reduction of SDS (final concentration < 5 ppm) in 5 min. The device is employed in a detergent-based proteomic workflow for analysis of an enriched yeast membrane proteome extract, demonstrating quantitative protein recovery (>98%) and increasing the number of identifications by liquid chromatography-tandem mass spectrometry.

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 (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.

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. Alternatively, our automated electrophoretic device, termed transmembrane electrophoresis (TME), depletes SDS for both TDP and BUP studies. Here TME is compared to these two alternative methods of SDS depletion in both BUP and TDP workflows. To do so, a modified FASP method is described applicable to the SDS purification and recovery of intact proteins, suitable for LC/MS. All three methods reliably deplete >99.8% SDS. TME provide higher sample yields (average 90%) than FASP (55%) or acetone precipitation (57%), translating into higher total protein identifications (973 vs 877 FASP or 890 acetone) and higher spectral matches (2.5 times) per protein. In a top down workflow, each SDS-depletion method yields high-quality MS spectra for intact proteins. These results show each of these membrane-based strategies is capable of depleting SDS with high sample recovery and high spectra quality for both BUP and TDP studies.

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. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

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. However as shown here, exposure of histone H4 to 80% formic (1 h, 20(o) C) induces N-formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N-formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below -20(o) C.

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. Here we present a simple automated device, termed transmembrane electrophoresis (TME), incorporating the principles of membrane filtration, but with an applied electric current to ensure near-complete (99.9%) removal of the surfactant, including protein-bound SDS. Intact proteins are recovered in solution phase in high yield (90-100%) within 1 h of operation. The strategy is applied to protein standards and proteome mixtures, including an enriched membrane fraction from E. coli, resulting in quality MS spectra free of SDS adducts. The TME platform is applicable to both bottom-up MS/MS as well as LC-ESI-MS analysis of intact proteins. SDS-depleted fractions reveal a similar number of protein identifications (285) compared wit a non-SDS control (280), being highly correlated in terms of protein spectral counts. This fully automated approach to SDS removal presents a viable tool for proteome sample processing ahead of MS analysis. Data are available via ProteomeXchange, identifier PXD003941.

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. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at -20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins.

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. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

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. Though higher levels of organic solvent lower proteome solubility, we found that the inclusion of 40% methanol provided optimal solubility of membrane proteins, with 86% recovery relative to extraction with SDS. Incorporating 40% methanol during the electrophoretic depletion of SDS by TME also maximized membrane protein recovery. We further report that methanol accelerates the rate of detergent removal, allowing TME to deplete SDS below 100 ppm in under 3 min. This is attributed to a three-fold elevation in the critical micelle concentration (CMC) of SDS in the presence of methanol, combined with a reduction in the SDS to protein binding ratio in methanol (0.3 g SDS/g protein). MS analysis of membrane proteins isolated from the methanol-assisted workflow revealed enhanced proteome detection, particularly for proteins whose pI contributed a minimal net charge and therefore possessed reduced solubility in a purely aqueous solvent. This protocol presents a robust approach for the preparation of membrane proteins by maximizing their solubility in MS-compatible solvents, offering a tool to advance membrane proteome characterization.

Dual LC-MS platform for high-throughput proteome analysis.

We describe a dual-column interface for parallel chromatography to improve throughput during LC-MS experimentation. The system employs a high-voltage switch to operate two capillary column/nanospray emitters fixed at the MS orifice. Sequentially loading one column while operating the second nearly doubles the LC-MS duty cycle. Given the innate run-to-run variation of a nanospray LC-MS (12% RSD peak area; 2% retention time), the intercolumn variability of the platform showed no meaningful difference for proteome analysis, with equal numbers of proteins and peptides identified per column. Applied to GeLC analysis of an E. coli extract, throughput was increased using one of three methods: doubling the number of replicates, increasing the LC gradient length, or sectioning the gel into twice as many fractions. Each method increased the total number of identifications as well as detection throughput (number of peptides/proteins identified per hour). The greatest improvement was achieved by doubling the number of gel slices (10 vs 5). Analysis on the dual column platform provided a 26% increase in peptides identified per hour (24% proteins). This translates into ~50% more total proteins and peptides identified in the experiment using the dual LC-MS platform.

Critical assessment of the spectroscopic activity assay for monitoring trypsin activity in organic-aqueous solvent.

Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous-organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems. However, as reported here, the changing solvent polarity induces bias in the absorbance measurement, lending upward of 35% error in the apparent trypsin activity as the acetonitrile is raised to 70%. Furthermore, time-dependent spectroscopic and mass spectrometric measurements reveal a progressive deactivation of trypsin over a 5- to 10-min period in as little as 30% acetonitrile.

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. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

Tips And Tricks for Proteome Sample Preparation.

ARTICLES DISCUSSED: Kovalchuk, S. I., Ziganshin, R., Shelukhina, I. Simple in-house ultra-high performance capillary column manufacturing with the FlashPack approach. Journal of Visualized Experiments. (178), e62522 (2021). Sirois, I., Isabelle, M., Duquette, J. D., Saab, F., Caron, E. Immunopeptidomics: Isolation of mouse and human MHC Class I- and II-associated peptides for mass spectrometry analysis. Journal of Visualized Experiments. (176), e63052 (2021). Han, Y., Thomas, C. T., Wennersten, S. A., Lau, E., Lam, M. P. Y. Shotgun proteomics sample processing automated by an open-source lab robot. Journal of Visualized Experiments. (176), e63092 (2021). Nickerson, J. L. et al. Organic solvent-based protein precipitation for robust proteome purification ahead of mass spectrometry. Journal of Visualized Experiments. (180), e63503 (2022). Li, D., Liang, J., Zhang, Y., Zhang, G. An integrated workflow of identification and quantification on FDR control-based untargeted metabolome. Journal of Visualized Experiments. doi: 10.3791/63625-v (2022). Petelski, A. A., Nikolai Slavov, N., Specht, N. Single-cell proteomics preparation for mass spectrometry analysis using freeze-heat lysis and an isobaric carrier. Journal of Visualized Experiments. doi: 10.3791/63802 (2022).

Perfluorooctanoic acid and ammonium perfluorooctanoate: volatile surfactants for proteome analysis?

RATIONALE: Fluorinated surfactants are being explored as mass spectrometry (MS)-friendly alternatives to sodium dodecyl sulfate (SDS) for proteome analysis. Previous work demonstrates perfluorooctanoic acid (PFOA) to be compatible with electrospray ionization (ESI)-MS. The high volatility of PFOA provides an intrinsic approach to potentially eliminate the surfactant during ESI, or alternatively through solvent evaporation prior to MS. The ammonium salt of PFOA, ammonium perfluorooctanoate (APFO), is likely favored for proteome experiments; the MS and liquid chromatography (LC)/MS tolerance of APFO has not been established for proteome applications. METHODS: Standard proteins and peptides, as well as a yeast proteome mixture, were individually spiked with surfactants (APFO, PFOA, SDS), and subjected to direct infusion ESI-MS, LC/MS/MS and LC/UV. The level of fluorinated surfactant remaining after solvent evaporation under varying conditions (time, pH, salt and protein content) was quantified and compared to the threshold tolerance level of the surfactant in an MS experiment (determined herein). RESULTS: Whereas PFOA is found ineffective at assisting protein solubilization, APFO is as effective as SDS for resolubilization of acetone-precipitated yeast proteins (~100% recovery). Unfortunately, the LC and MS threshold tolerance of APFO is only minimally greater than SDS (~2-fold higher concentration to cause 50% suppression). Nonetheless, the benefits of APFO in a proteome experiment are realized following a one-step evaporation protocol for removal of the surfactant in acidified solvent. CONCLUSIONS: APFO is considered a favoured alternative to SDS for proteome solubilization. Strictly speaking, APFO is not an 'MS-friendly' surfactant for proteome characterization; the detergent not only suppresses ESI signals at high concentration, but also perturbs reversed phase separation. However, the simplicity of APFO removal ahead of LC/MS justifies its use over the conventional SDS.

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. Here, we use a spectroscopic (BAEE) assay to quantify calcium-stabilized trypsin activity over the complete time course of a digestion. At 47 °C, the addition of calcium contributes a 25-fold enhancement in trypsin stability. Higher temperatures show a net decrease in cumulative trypsin activity. Through bottom-up MS analysis of a yeast proteome extract, we demonstrate that a 1 h digestion at 47 °C with 10 mM Ca2+ provides a 29% increase in the total number of peptide identifications. Simultaneously, the quantitative proportion of peptides with 1 or more missed cleavage sites was diminished in the 47 °C digestion, supporting enhanced digestion efficiency with the 1 h protocol. Trypsin specificity also improves, as seen by a drop in the quantitative abundance of semi-tryptic peptides. Our enhanced digestion protocol improves throughput for bottom-up sample preparation and validates the approach as a robust, low-cost alternative to maximized protein digestion efficiency.

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.

Intact proteome fractionation strategies compatible with mass spectrometry.

Proteome fractionation refers to separation at the level of intact proteins. Proteome fractionation may precede sample digestion and subsequent peptide-level separation and detection (i.e., bottom-up mass spectrometry [MS]). For top-down MS, proteome fractionation acts as a stand-alone separation platform, since intact proteins are directly analyzed by the mass spectrometer. Regardless of the MS identification strategy, separation of intact proteins has clear benefits as a result of decreasing sample complexity. However, this stage of the workflow also creates considerable challenges, which are generally absent from the counterpart peptide separation experiment. For example, maintaining protein solubility is a key concern before, during and after separation. To this end, surfactants such as sodium dodecyl sulfate may be employed during fractionation, so long as they are eliminated prior to MS. In this article, current strategies for proteome fractionation in a MS-compatible format are reviewed, illustrating the challenges and outlooks on this important aspect of proteomics.