Dataset Information

Rapid trypsin digestion at elevated temperature with calcium for enhanced bottom-up proteome analysis

ABSTRACT: Trypsin is frequently employed to cleave proteins ahead of mass spectrometry characterization. Traditionally, enzyme digestion involves overnight incubation of proteins at 37 °C, which is not only time consuming but can still yield in poor digestion efficiency. While raising the temperature should theoretically accelerate the digestion, this also destabilizes the enzyme and enhances trypsin degradation. We therefore questioned whether elevated temperature digestion is beneficial for improving tryptic digestion. Here we quantify protein digestion kinetics at elevated temperatures for calcium-stabilized trypsin and enforce the critical importance of calcium ions to preserve the enzyme. We quantitatively demonstrate that 1 hour at 47 °C provides a superior digest when compared to conventional (overnight, 37 °C) processing of the proteome. The impact of our enhanced digestion protocol is shown through bottom-up mass spectrometry analysis of a complex proteome mixture.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bacteria Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Jessica Nickerson

LAB HEAD: Alan Austin Doucette

PROVIDER: PXD035682 | Pride | 2023-03-11

REPOSITORIES: Pride

ACCESS DATA

Publications

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

Nickerson Jessica L JL Doucette Alan A AA

Biology 20221001 10

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 acti ...[more]

PMID: 36290348

Similar Datasets

Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

Dataset Information

Rapid trypsin digestion at elevated temperature with calcium for enhanced bottom-up proteome analysis

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure