Xcalibur 3

Lipidomic analysis of kidney samples was conducted with a triple-quadrupole mass spectrometer (Thermo TSQ Quantiva) equipped with an automated nanospray ion source (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY, USA) as previously reported [41 (link)]. To prevent possible lipid aggregation, the solutions of lipid extracts were diluted in CHCl₃/MeOH/isopropyl alcohol (1:2:4, v/v/v) prior to direct infusion. All mass spectral data were acquired by different customized sequence subroutines operated under Xcalibur software (Xcalibur 3.0, Thermo Fisher Scientific Inc., San Jose, CA, USA). Data processing was performed according to the previous method [39 (link)]. All data were presented at mean ± SEM unless otherwise indicated. Kolmogorov–Smirnov test was used to check the normality of each group of variables. All p-values were greater than 0.05, indicating that the data follow the normal distribution. Statistical significance between the groups was determined by Student’s unpaired t-tests, where * p < 0.05, ** p < 0.01, and *** p < 0.001.

In this study, the XCalibur 3.0 software was used for mass spectrum analysis (Thermo Fisher, Waltham, MA, USA). Principal component analysis (PCA) was conducted to evaluate the changes in the chemical profiles of Dis1 to Dis9 using the SIMCA-P 14.0 software (Umetrics AB, Sweden). The relative quantitative data of compounds showing significant differences were obtained with different methods; among them, the nonvolatiles were relatively quantified by area normalization method and the volatiles were relatively quantified on ion peak intensities. One-way analysis of variance (ANOVA) of these quantitative results was conducted using IBM SPSS Statistics 20.0 (SPSS Institute, Chicago, IL, USA). GraphPad Prism 8.0 (GraphPad Software Inc., USA) was used to create the charts of the results.

Targeted data processing, including peak detection and comparison with the reference standard solution mix, was performed using Xcalibur™ 3.0 (Thermo Fisher Scientific). Compound intensities were normalized to the intensity of the reference internal standard (D-valine-d₈) in each sample. Univariate statistical analysis of targeted data was carried out with SPSS Statistics 26.0 (IBM). More specifically, one-way ANCOVA and Quade test with Bonferroni correction at a 5% significance level were applied for parametric and non-parametric analysis respectively. Results were adjusted for gender and age as covariates. Normality of distribution and equality of variances were assessed with Kolmogorov–Smirnov and Levine’s tests for each variable.

nESI capillaries were purchased
from Thermo. Positive ionization mode mass spectra were acquired on
a Micromass LCT ToF modified for analysis of intact protein complexes
(MS Vision, The Netherlands) equipped with an offline nanospray source.
For the LCT, the capillary voltage was 1.5 kV and the RF lens 1.5
kV. The cone voltage was set to 100 V for normal acquisition and ramped
between 50 and 300 V for collisional activation. The pressure in the
ion source was maintained at 9.0 mbar. Data were analyzed using MassLynx
V4.1 (Waters, UK). Negative ionization mode mass spectra were acquired
on an Orbitrap Fusion (Thermo Fisher Scientific, Waltham, MA) equipped
with an offline nanoelectrospray source. The instrument was operated
in intact protein mode. The capillary voltage was −1.8 kV,
the transfer tube temperature was maintained at 40 °C and the
pressure in the ion-routing multipole was 0.011 Torr. Collisional
activation was performed by increasing the HCD energy in the ion-routing
multipole. High-purity nitrogen was used as collision gas. Spectra
were recorded using the Orbitrap mass analyzer at a resolution of
60 000 with a high mass mode acquisition window of 1000–5000 m/z and a scan time of 1 ms. Data were
analyzed using Xcalibur 3.0 (Thermo Scientific, Waltham, MA).