An efficient in‐gel digestion method on small amounts of protein sample from large intact gel pieces

Abstract

In‐gel digestion is an essential method, but there has been concern that a large ratio of gel volume to protein volume will reduce digestion efficiency and make it difficult to recover peptides after digestion. Few methods for handling large gel pieces have been established. Therefore, we optimized the in‐gel digestion method to handle large intact gel pieces containing small amounts of proteins. As a model for large intact gel pieces containing small amounts of protein, gel pieces of 7 × 10 mm in size obtained by short‐range sodium dodecyl‐sulfate polyacrylamide gel electrophoresis of 150 ng of total protein extracted from Arabidopsis cells were used. The use of a positive pressure manifold and 96‐well filter enabled rapid and uniform liquid exchange and gel drying, even when handling multiple gel pieces, and reduced the labor required for a liquid exchange. This facilitated the study of conditions using multiple gels and enabled a detailed study of the enzyme solution permeation and elution methods. By using the optimized method, we were able to establish a method for recovering small amounts of protein from large intact gel pieces without compromising reproducibility or recovery rate.

Isocitrate dehydrogenase 1 upregulation in urinary extracellular vesicles from proximal tubules of type 2 diabetic rats

“…Protein bands were excised from the SDS gel and subjected to in‐gel digestion according to a previously published protocol 21–23 . The gels were fixed, washed with water and 1:1 acetonitrile/50 mM ammonium bicarbonate, and then dehydrated in acetonitrile.…”

“…Protein bands were excised from the SDS gel and subjected to in-gel digestion according to a previously published protocol. [21][22][23] The gels were fixed, washed with water and 1:1 acetonitrile/50 mM ammonium bicarbonate, and then dehydrated in acetonitrile. Subsequently, 50 mM tris (2-carboxyethyl) phosphine (TCEP) was added, and the gels were incubated for 10 min at 60°C before they were alkylated with 100 mM iodoacetamide for 1 h at room temperature in the dark.…”

Lipid droplets in Arabidopsis thaliana leaves contain myosin-binding proteins and enzymes associated with furan-containing fatty acid biosynthesis

“…Each lane was divided into six pieces. In-gel digestion was performed according to a method described previously (Kano et al, 2023). The digested peptides were analyzed by nano-flow reverse-phase LC followed by tandem MS using a Q-Exactive hybrid mass spectrometer (Thermo Fisher Scientific).…”

LASP1, CERS6, and Actin Form a Ternary Complex That Promotes Cancer Cell Migration

“…Supernatants were collected and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After in-gel digestion was performed [21], peptides were analyzed by liquid chromatography-tandem mass spectrometry (for details, see Supplementary Methods).…”