An analytical method using lc-ms/ms proteomics to characterize proteins translated from mrna

A method for assessing translation efficacy of one or more mRNAs, the method comprising:

• (a) translating the one or more mRNAs, wherein the one or more mRNAs are present in a cell lysate (cell-free translation) or within a cell (cell-based translation) thereby obtaining a translation reaction product comprising a protein or proteins translated from the one or more mRNAs;
• (b) digesting the translation reaction product with a first proteolytic enzyme, thereby obtaining a first enzymatic digest;
• (c) subjecting the first enzymatic digest to liquid chromatography-tandem mass spectrometry (LC-MS/MS), thereby obtaining a first peptide profile;
• and
• (d) analyzing the first peptide profile by detecting peptides derived from the one or more proteins;
• and
• (e) optionally further analyzing the first peptide profile, thereby determining amino acid sequence or sequences of the one or more proteins;
• and comparing the amino acid sequence or sequences thus obtained to an amino acid sequence or sequences expected from translation of the one or more mRNAs, thereby verifying, nucleotide sequence or sequences of the one or more mRNAs;
• thereby assessing the translation efficacy of the one or more mRNAs.