Nanoelute nanoflow ultrahigh pressure lc system

The streptavidin beads with captured biotin-labeled proteins were resuspended in fresh 50 mM NH₄HCO₃, then reduced using 10 mM dithiotreitol (DTT), and alkylated using 15 mM iodoacetamide. Protein samples were digested using 1 µg trypsin (Promega, Wisconsin, USA) and incubated overnight at 37°C. Following digestion, the samples were acidified and desalted using homemade C18 stage tips. Peptides were eluted from the stage tips using 50% acetonitrile (ACN)/0.1% formic acid (FA), dried to completion by speed vacuum, and resuspended in 0.1% FA. LC-MS/MS analysis was performed using a nanoElute nanoflow ultrahigh pressure LC system (Bruker Daltonics, Bremen, Germany) coupled to a timsTOF fleX mass spectrometer (Bruker Daltonics) with CaptiveSpray nanoelectrospray ion source (Bruker Daltonics). The peptides were separated using a 60-min gradient at a flow rate of 300 nL/min. The timsTOF fleX was operated in PASEF mode, and the data-dependent acquisition was performed using 10 PASEF MS/MS scans per cycle with a near 100% duty cycle.

Samples were acidified with
0.5% trifluoroacetic acid. The desalting and concentration step was
performed with ZipTip C18 (Millipore), the digested samples were speed-vacuum-dried,
and the total protein content was analyzed by a bicinchoninic acid
assay. LC-TIMS-MS/MS was carried out using a nanoElute nanoflow ultrahigh-pressure
LC system (Bruker Daltonics, Bremen, Germany) coupled to a timsTOF
Pro 2 mass spectrometer equipped with a CaptiveSpray nanoelectrospray
ion source (Bruker Daltonics). Details of the methodology can be found
in Montserrat-de la Paz et al.,^{16 (link)} although
in this case, the reference library is acquired from UniProt_proteome_Salvia-ssp_Feb22.