Bead-based Protein Digestion Protocol

Digestion was performed on beads using a digestion buffer containing 50 mM NH₄HCO₃. The beads were then treated with 1 mM DTT at 25 °C for 30 min, followed by addition of 5 mM iodoacetamide (IAA) at 25 °C for 30 min in the dark. Lysyl endopeptidase (Wako) was added to the mixture at a 1:50 (w/w) enzyme to protein ratio and digestion proceeded at 25 °C overnight. Samples were further digested overnight with 1:50 (w/w) trypsin (Promega) at 25 °C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.

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de Amorim J.L., Leung S.W., Haji-Seyed-Javadi R., Hou Y., Yu D.S., Ghalei H., Khoshnevis S., Yao B, & Corbett A.H. (2024). The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops). The Journal of Biological Chemistry, 300(2), 105646.

Publication 2024

Lysyl endopeptidase trypsin Sep-Pak C18 column

Corresponding Organization : Emory and Henry College

Other organizations : Emory University

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Variable analysis

independent variables

Digestion buffer containing 50 mM NH4HCO3
Treatment with 1 mM DTT at 25 °C for 30 min
Addition of 5 mM iodoacetamide (IAA) at 25 °C for 30 min in the dark
Digestion with Lysyl endopeptidase (Wako) at a 1:50 (w/w) enzyme to protein ratio
Digestion with trypsin (Promega) at a 1:50 (w/w) enzyme to protein ratio

dependent variables

Resulting peptides

control variables

Temperature (25 °C)
Incubation time (30 min, overnight)

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