Nanolc ultra hplc system

10 μg of recombinant, full-length STAT3 (SignalChem) was incubated in the presence or absence of 500 μM of S3I-201 in a 100 mM ammonium bicarbonate buffer for 1 h at 37°C. The incubation was then quenched by the addition of dithiothreitol (DTT) to a final concentration of 0.5 mM and incubation at 55°C for 30 min. Free sulfhydryl groups were alkylated with 2-iodoacetamide (Sigma) at a final concentration of 1.5 mM for 45 min at room temperature. The samples were subjected to trypsin (Promega) digestion (0.5 μg of trypsin per reaction), lyophilized, re-suspended in 0.1% formic acid, and desalted using ZipTip C18 (EMD Millipore) prior to analysis. Samples were analyzed by LC-MS/MS on a nanoLC ultra HPLC system (Eksigent) coupled to a Thermo Scientific Orbitrap-Elite.
Tryptic STAT3 peptides were resolved over an CH₃CN gradient at a flow rate of 300 nl/min on a 15 cm long, 75-micrometer diameter C18 CL column with a particle size of 3 microns and 120 Angstrom pores (Eksigent). The aqueous and organic mobile phases consisted of 0.1% formic acid in water or CH₃CN respectively. Organic mobile phase composition was varied over each hour long LC-MS/MS analysis as follows: 2% from 0 to 1 min, 10% to 35% from 1 to 35 min, 80% from 35.5 to 44.5 min, 2% from 45 to 60 min.

Nano-LCMS using a home-packed 0.75 μm x 10cm C18 emitter tip (Reprosil-Pur 120 C18-AQ, 3 μm). A Nano LC-Ultra HPLC system (Eksigent) was coupled to an LTQ Orbitrap Elite (ThermoFisher) and samples were analyzed in data-dependent acquisition mode. A 60000 resolution MS scan was followed by 10 CID MS/MS ion trap scans on multiply charged precursor ions with a dynamic exclusion of 20 s. The LC gradient was delivered at 200 nl/minute and consisted of a ramp of 2–35% acetonitrile (0.1% formic acid) over 90 min, 35–80% acetonitrile (0.1% formic acid) over 5 min, 80% acetonitrile (0.1% formic acid) for 5 min, and then 2% acetonitrile for 20 min.

Samples were reconstituted in 2% ACN, 0.1% FA (v/v) prior to analysis using a Triple TOF 6600 mass spectrometer (Sciex, UK) delivered into the instrument using an Eksigent NanoLC Ultra HPLC system. Samples were injected onto a nanoACQUITY UPLC Symmetry C18 Trap Column (P/N Waters, MA, USA) and washed for 10 min at 2 µL/min with 0.1% FA. A gradient from 1.6% ACN/0.1% FA to 95% ACN/0.1% FA was applied over 95 min at a flow rate of 300 nL/min through a Peptide BEH C18 nanoACQUITY Column (Waters, MA, USA). MS was operated as described in previous methods (Meng et al., 2016 (link)).