The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed –1 ribosomal frameshifting

Liao, Pei-Yu; Choi, Yong Seok; Dinman, Jonathan D.; Lee, Kelvin H.

A representative flow chart of the mass spectrometry analysis is shown in Supplementary Data (Supplementary Figure S1 ). Trypsin-digested frameshift protein samples resulted in target peptides spanning the recoding sites with a single amino acid difference between FS z and FS m . These peptides were analysed by nano-flow liquid chromatography tandem mass spectrometry using multiple reaction monitoring (nLC-MRM/MS). The digested sample was vacuum dried, reconstituted with 30 ml of 0.1% formic acid (FA), and a portion of each reconstituted sample was injected into Dionex 3000 nLC system (Sunnyvale, CA, USA). First, the sample was loaded onto an Acclaim PepMap 100 C18 trap column (300 mm  5 mm, 5 mm) and on-line desalting was carried out with water (0.1% FA) at a flow rate of 30 ml/min for 5 min. Then, peptides trapped in the trap column were GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGA AGG CCA GGG AAT TTT CTT GGA TAA AG MB2 GCU CCT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGA AGG CCA GGG AAT TTT CTT GGA TAA AG MB2UCC GCC CCT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGA AGG CCA GGG AAT TTT CTT GGA TAA AG MB2CCC TTC CCT TTA AAC CAG AAC GCC TCC AGG CCT TGC AAC ACT TGG TCC GGA AGG CCC TGG AGG CAG GCC TAA (3)]. Samples were analysed in triplicates (except duplicates of CCC and TLV).