Critical assessment of accelerating trypsination methods

Optimizing the Identification of Citrullinated Peptides by Mass Spectrometry: Utilizing the Inability of Trypsin to Cleave after Citrullinated Amino Acids

“…To ensure a complete tryptic digestion, we employed a significantly higher trypsin concentration in the performed tryptic digestions (1:10) combined with 12 h incubation at 37°C [25]. However, cleavage after citrulline could not be detected for any of the investigated peptides, whereas the arginine and lysine cleavage sites were fully cleaved.…”

A Critical Review of Trypsin Digestion for LC-MS Based Proteomics

“…Information about the enzyme to protein ratio needed for digestion of a protein sample is crucial to ensure an enzyme amount sufficient to perform the digestion, but not too high resulting in autolysis products from the trypsin used. Recent experiments indicate that a sufficient ratio of enzyme to substrate (E+S) is 1+20 (Hustoft et al 2011). For targeted proteomics it may be beneficial to perform a pilot study on the necessary digestion time for the type of sample to be analyzed, to obtain an optimal digestion efficiency of the sample.…”

“…Thus dealing with these long digestion times, an overnight digestion is often more convenient, starting with the post digestion sample preparation steps the following day. Proteins may act differently in different environments and less effective digestions have been observed when model proteins were digested in a mixture as compared to being digested separately (Hustoft et al 2011). One reason for these observations could be increased competition for the trypsin cleavage sites, when more proteins are digested together.…”

“…The area of the intact protein peak decreased with increasing digestion time, -indicating better trypsination efficiency. The size of the intact protein peak could hence in some cases be used to compare the efficiency of digestion methods (Hustoft et al 2011). When exploring the potential of microwave oven accelerated digestion of a mixture of proteins, different temperatures were examined both for the microwave oven and the Thermoshaker control samples.…”

“…Such ZipTips can be used for purification of, for instance peptides, proteins and nucleic acids. Purifying tryptic peptides with the C18 ZipTip results in high recovery, but noteworthy is that the capacity of the C18 ZipTips is limited; however, up to 10 µg digested protein could be loaded without losses (Hustoft et al 2011). Another possible disadvantage of the C18 ZipTip procedure is the loss of small hydrophilic peptides which may be lost due to washing with an aqueous mobile phase containing 0.1 % trifluoroacetic acid (TFA) (Hustoft et al 2011).…”