Origin 2015

Data were analyzed with Clampfit and ORIGIN 2015 (OriginLab) and are
presented as means ± SEM as noted in the text and figure legends. Number
(n) of hair cells recorded is indicated for each mean value
given in the text and figures legends. Number of mice used is indicated in the
figures legends. One-way analysis of variance (ANOVA) or Student’s t test
was used to compare biophysical properties of sensory transduction currents
using the statistical function in ORIGIN 2015 (OriginLab). Statistical
significance is indicated as p values.

PvDBP Region II and monoclonal antibodies were purified as described [29 (link)] and buffer exchanged into PBS. Stock antigen and antibodies solutions in PBS buffer were exchanged into 200 mM ammonium acetate solution (pH = 6.5–7). Before each experiment, the antigen and antibody were mixed and diluted to 5 μM and 6 μM, respectively. The sample solution was incubated at 25 ºC for 30 min before MS analysis.
Samples were analyzed using a quadrupole ion-mobility time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Protein and complex ions were generated in a nESI source in the positive-ion mode. The source parameters were optimized to provide gentle ESI conditions. The traveling-wave IM separator was operated at a N₂ pressure of ~1.5 mbar; the IMS wave velocity was 650 m/s; and the IMS wave height 40 V. The ToF-MS was operated over a m/z range of 100–15,000. For a CIU experiment, the trap-collision voltage applied to the ions in the traveling-wave-based ion trap situated prior to the IM separator was increased from 10 to 200 V in 10 V increments, and ion-mobility mass spectra were acquired at each increment.
Mass spectra were processed with Masslynx V4.1 software (Waters). Arrival time distributions at each collision voltage were extracted in the Drift Scope (Waters) and plotted as a 2D contour plot using Origin 2015 (OriginLab, Northampton, MA).

Curve fitting and statistical analysis were performed using GraphPad Prism v7.05 (GraphPad Software, San Diego, CA). Data were tested with a D’Agostino and Pearson normality test. Since at least one experimental group in each condition significantly deviated from a Gaussian distribution, we chose non-parametric tests for statistical analysis. We selected a Mann-Whitney test for unpaired comparisons between two groups; whereas a Kruskal-Wallis test with a Dunn’s post hoc test was used for multiple comparisons. For comparing paired data in more than two conditions, we selected a Friedman test with a Dunn’s post hoc test for multiple comparisons. No power analysis was conducted a priori. Graphics were constructed using Origin 2015 (OriginLab, Northampton, MA). Descriptive statistics are specified in the figure legends.