Profinder b 08

We used Agilent MassHunter B.07 software for initial data processing and compound peak identification. In brief, compound peaks are extracted by matching MS signals with theoretical monoisotopic masses of glycans to within 10 ppm mass tolerance. The assignments are further confirmed by MS2 fragmentation when available. From the exported data, we then create a new library containing the chemical formulae and retention times of identified glycoconjugates for targeted isotopologue extraction. Agilent Profinder B.08 software was used to extract quantitative isotopologue data for each identified glycan through its Batch Isotopologue Analysis function. The program averages the MS signals across a chosen peak in an extracted ion chromatogram to form the MS spectra from which isotopologue signals are extracted. It also corrects for the natural distribution of ¹³C and returns the relative abundance of each isotopologue. Mass tolerance was set at ± (10 ppm + 2 mDa) to screen out background noise or signals from other co-eluting compounds. For each glycan, the percent abundance of each isotopologue signal from M + 0 to M + n, where n is the total number of carbons in the glycan, can then be exported for further analysis.

Samples were separated by aqueous normal phase chromatography using a Cogent Diamond Hydride column. Samples were then analyzed using an Agilent Technologies 6230 high resolution, accurate mass TOF LC/MS. Detected ions were characterized by chromatographic retention time and ion mass. Data analysis was performed using Profinder B08 and Qualitative analysis (Agilent Technologies). Molecules were characterized by mass, time, and abundance (as reported by ion intensity) [16 (link)].
Experiments were repeated to ensure reproducibility using unfrozen urine aliquots from the discovery cohort (technical replicates). Repeat experiments were performed using 4 independent randomized subsets of cases and controls (n = 5–7 cases and 5–7 controls per experiment). Molecule abundance remained significantly different in repeat experiments and ROC curve analysis of repeated sample sets confirmed an area under the ROC curve of 87–100 for each molecule when comparing molecules within each experiment independently. Validation cohort samples were randomized and blinded prior to sample prep and HPLC analysis. Longitudinal samples were randomized prior to HPLC analysis. Sample prep and HPLC analysis of longitudinal samples was repeated three times to ensure reproducibility. Creatinine normalization did not significantly alter molecule ion counts, significance calculations or ROC curves.

Data analysis was done with MassHunter Qualitative Analysis B.07 and Profinder B.08 (Agilent, CA). GSL compounds were identified with the find by molecular feature function, and filtered through an in-house library of GSL compositions. The glycan and lipid composition of the GSLs was further confirmed through tandem MS fragmentation spectra. A library that includes the chemical formulas and retention times of identified GSLs was composed from the data exported from MassHunter. This library was then used in Profinder to perform batch targeted feature extraction and to quantitate the peak areas of the identified GSL compounds. The program included both proton and ammonium adducts in its integration of compound signals. A mass tolerance of 10 ppm was applied for signal extraction. Peak smoothing using a Gaussian function was applied prior to integration.