2.4. Analytical Instrumentation and Data Acquisition

The resulting spiked digests were analysed by nanoLC-HDMS^e using an M-Class nano-Acquity system (Waters UK, Wilmslow, Cheshire, UK) with a trap valve manager, coupled to a Synapt G2-Si mass spectrometer (Waters UK, Wilmslow, Cheshire, UK).

The sample (1.0 μL) was loaded onto the trapping column (Waters UK, Wilmslow, Cheshire, UK; NanoEase™ M/Z Symmetry C18 100Å, 5 um, 180 um × 20 mm trap column), using partial loop injection with a sample loading time of 3 min at a flow rate of 0.3 μL/min.

The sample was resolved on an analytical column (nanoEase™ M/Z Peptide BEH C18 130Å, 1.7 μm, 75 μm × 100 mm column) using a gradient of 99% A (Optima^® Water with 0.1% formic acid) 1% B (Optima^® ACN with 0.1% formic acid) (Sigma-Aldrich, Dorset, UK) to 60% A, 40% B over 90 min at a flow rate of 0.3 μL/min and then to 15% A, 85% B over 90 min. At 95 min, the conditions returned to the initial state and held for 15 min in preparation for the next injection.

The mass spectrometry (MS) data was acquired in HDMS^e mode, e.g., ion mobility was enabled and all ions within a specified m/z range were observed and fragmented. In essence, four signals are available from an HDMS^e acquisition: firstly, a low energy MS, secondly, a high energy MS/MS, the third signal contains the lockmass data for mass axis correction and the fourth relates to the ion mobility aspect of HDMS^e.

A mass range of 50 to 2000 Da was selected in resolution mode (ToF W mode) using a positive polarity continuum acquisition. The scan time was set at 0.5 s and a ramp transfer collision energy of 15–45 eV was used for the high energy signal. The time-of-flight analyser was calibrated before running batches against the fragment ions of glufibrinopeptide and throughout the analytical run at 6 min intervals using the Waters ZSpray™ NanoLockSpray™ source with leucine enkephaline.