Nanoacquity uplc system with hdx technology

Peptide identification was carried out using 100 pmol of protein diluted in protiated buffer F. The sample was quenched as described above and analysed in the MS E acquisition mode in a nanoACQUITY UPLC System with HDX technology (Waters, UK) coupled to a SYNAPT G2 ESI-Q-TOF mass spectrometer over the m/z range 100-2,000 Da. The collision energy was ramped from 15 to 35 V. Other instrument parameters were as described above. Peptide identification was performed using ProteinLynx Global Server (PLGS v3.0.1, Waters, UK) using the primary sequence of SecA or derivatives as a search template. Peptides were individually assessed for accurate identification and were only considered if they had a signal to noise ratio above 10 and a PLGS score above 7.5. Further, peptides were only considered if they appeared in 3 out of 5 replicate runs for each protein. Data analysis was carried out using DynamX 3.0 (Waters, Milford MA) software to compile and process raw mass spectral data and generate centroid values to calculate relative deuteration values. All spectra were individually inspected and manually curated to ensure accurate centroid calculations. Maximum errors between replicate runs were found to be ±0.15 Da with most errors within ±0.08 Da, thus a difference of ± 0.5 Da between peptides from different states was considered significant (Houde et al., 2011) .