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.
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IJS, KILJ, NUK, PNG, UL, UM
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.
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•Acetone precipitation requires salt to recover low molecular weight peptides.•The cation trend follows a reverse Hofmeister series; Zn2+ is optimal for recovery.•The method applies to peptides as small as 1 kDa with high recovery (>90%).•Mass spectrometry reveals precipitation is unaffected by amino acid sequence.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•For most proteins, ionic species are required for precipitation in organic solvent.•The amount of salt correlates with the protein and organic solvent concentrations.•Ionic species are also required ...to precipitate complex proteome mixtures.•A model of ion pairing is proposed to explain this synergistic precipitation.
Solvent precipitation is commonly used to purify protein samples, as seen with the removal of sodium dodecyl sulfate through acetone precipitation. However, in its current practice, protein loss is believed to be an inevitable consequence of acetone precipitation. We herein provide an in depth characterization of protein recovery through acetone precipitation. In 80% acetone, the precipitation efficiency for six of 10 protein standards was poor (ca. ≤15%). Poor recovery was also observed for proteome extracts, including bacterial and mammalian cells. As shown in this work, increasing the ionic strength of the solution dramatically improves the precipitation efficiency of individual proteins, and proteome mixtures (ca. 80–100% yield). This is obtained by including 1–30mM NaCl, together with acetone (50–80%) which maximizes protein precipitation efficiency. The amount of salt required to restore the recovery correlates with the amount of protein in the sample, as well as the intrinsic protein charge, and the dielectric strength of the solution. This synergistic approach to protein precipitation in acetone with salt is consistent with a model of ion pairing in organic solvent, and establishes an improved method to recover proteins and proteome mixtures in high yield.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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, 20oC) 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 –20oC.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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. fractionation, purification) that are amenable to MS analysis of intact proteins. Chromatographic strategies are improving but pose higher risk of sample loss. Gel-based separations (e.g. GELFrEE) may alleviate this concern but at the expense of requiring sodium dodecyl sulfate (SDS). While this surfactant maintains protein solubility during fractionation, the advantage is short-lived, as the detergent must ultimately be depleted to avoid MS signal suppression. To do so requires overcoming strong interactions between SDS and protein. Adding to the challenge, one must now consider upholding the solubility of purified protein(s) in the absence of SDS. This review explores uses of SDS in TDP workflows, addressing front-end strategies that reduce matrix interferences while maximizing recovery of intact proteins in MS-compatible formats.
The benefits of employing SDS in a TPD workflow can easily outweigh the disadvantages. Several SDS depletion strategies are available, though not all are equally amenable to TDP. This review provides a comprehensive and critical accounting of SDS in TDP, demonstrating methods that are suited to MS analysis of intact proteins.
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•SDS is the most effective additive to enhance and maintain protein solubility.•Depletion strategies must overcome the interactions between SDS and protein.•Front-end strategies amenable to top-down MS maximize protein recover and purity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
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.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.
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IJS, KILJ, NUK, PNG, UL, UM
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Reliable size-based protein separation is an invaluable biological technique. Unfortunately, size separation in solution is underutilized, owing perhaps to the poor resolution of conventional ...techniques. Here, we report an enhanced multiplexed GELFrEE (gel-eluted liquid fraction entrapment electrophoresis) device which incorporates eight independent separation channels, operating with high repeatability. This enables simultaneous size separation of independent proteome samples, each into 16 well resolved liquid fractions, covering 10−150 kDa in 1.5 h. A novel strategy to increase sample loads while maintaining electrophoretic resolution is presented by distributing the sample among the eight channels with subsequent pooling of collected fractions. Liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis of the S. cerevisiae proteome following GELFrEE separation and sodium dodecyl sulfate (SDS) removal demonstrates the resolution and high correlation achieved between molecular weight and fraction number for the identified proteins. This device is highly orthogonal to solution isoelectric focusing, enabling our disclosure of a fully multiplexed high-throughput two-dimensional liquid electrophoretic (2D LE) platform that separates analogously to 2D polyacrylamide gel electrophoresis (PAGE). With 2D LE, a total of 128 well-resolved liquid fractions are obtained from 1 mg of S. cerevisiae proteins covering ranges 3.8 < pI < 7.8 and 10 kDa < MW < 150 kDa in an unprecedented 3.25 h total separation time.
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IJS, KILJ, NUK, PNG, UL, UM
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.
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IJS, KILJ, NUK, PNG, UL, UM
