Acquity m class μlc system

Final choice of HDIT101 surrogate
peptides and further workflow optimizations
were performed on a Waters Xevo TQ-S mass spectrometer equipped with
a Waters Acquity M-class μLC system and an IonKey/MS separation
device. The final chromatographic conditions such as mobile-phase
composition, analytical column, and initial conditions were set as
described in the previous part and in Table S1. ESI parameters are detailed in Table S1, as well as MRM acquisition parameters, which were optimized on
synthetic peptides using MassLynx V4.1 IntelliStart optimization procedures
(Waters) with argon as collision gas for collision-induced dissociation.
Optimized collision energies were set for each transition as described
in Table S1, and dwell time was set to
52 ms for the MS method with six MRM transitions to record and to
80 ms for the MS method with four MRM transitions to record. MRM data
were subsequently transferred to TargetLynx V 4.1 software (Waters)
to compute peak intensity, peak area, response, calibration parameters,
calculated concentrations, and residual standard deviations.

The surrogate peptide discovery analyses were performed
on a Waters Acquity M-class μLC system coupled to a Waters Xevo
G2-XS Q-TOF mass spectrometer featured with an IonKey/MS separation
device (Waters). The solid-phase-extracted (SPE) desalted peptide
digests were injected (two analyses for each sample with injection
volumes of 2 and 5 μL) onto a trapping C18 column (Waters Acquity
UPLC M-Class Trap Symmetry, 300 μm × 50 mm, 100 Å,
5 μm). Flow was then inverted toward the analytical C18 column
(Waters Peptide CSH C18 iKey, 150 μm × 50 mm, 130 Å,
1.7 μm). μLC and electrospray ionization (ESI)–MS/MS
parameters used for the peptide discovery experiments are detailed
in Supporting Information part I (Table
S1). Data were acquired in data-independent acquisition mode MS^e, for which all incoming precursor ions are fragmented in
the collision cell and all fragment ions are subsequently detected.
Acquired MS^e data were interpreted using Biopharmalynx
software (Waters) to search for tryptic peptides from the HDIT101
sequence. From these results, sequence coverage of light and heavy
chains and global sequence coverage were calculated using the data-processing
parameters given in Table S1.