Peakview software

To build the MS/MS spectral libraries, the peptide solutions were analyzed by a shotgun data-dependent acquisition (DDA) approach using micro-LC-MS/MS as described (17 ). The MS2 spectra (MS/MS spectra) of the identified peptides were then used to generate the spectral library for SWATH peak extraction using the add-in for PeakView Software (version 2.2, Sciex), MS/MS^ALL with SWATH Acquisition MicroApp (version 2.0, Sciex). Peptides with a confidence score above 99% (as obtained from Protein Pilot database search) were included in the spectral library. For relative quantification by SWATH-MS analysis, SWATH-MS acquisition was performed on a TripleTOF 6600 LC-MS/MS system (Sciex). Peptides from RBCs samples from all CFC and BC patients were analyzed using the data-independent acquisition method making three technical replicates per sample as previously described (17 , 18 (link)) and can seen in Supplementary Methods and Materials.

Metabolite identification was done by MetabolitePilot™ software version 2.0 (SCIEX). The MS/MS spectrum of IVM was exported as text files by the PeakView software (SCIEX) and imported to MetabolitePilot™ software (SCIEX) as a reference spectrum for creating the IVM library. Raw data files (.wiff) of metabolite sample analyses were imported to MetabolitePilot™ software and compared against the IVM‐library peak finding strategies as described in the supplementary material (Supplement Appendix S1). For the LC‐SPE‐NMR/MS system, the HPLC was operated by Hystar 3.2 (Bruker Daltonics), mass spectrum acquired by Microtof control (Bruker Daltonics), and the NMR spectrometer was operated by Topspin 3.5 (Bruker Biospin). The Complete Molecular Confidence – Structure Elucidation (CMC‐se) software version 2.6.1 (Bruker Biospin) was used for structure elucidation.

LC/MS peak lists (.peaks files) were created for all of the samples (.wiff files) using MarkerView software running under the following constraints: minimum retention time, 0.00 min; subtraction offset, 10 scans; subtraction multiplication factor, 1.3; noise threshold, 10; minimum spectral peak width, 10 ppm; and minimum RT peak width, 5 scans. Next, a peak table was created by simultaneously importing the LC/MS peak lists for all of the samples. The parameters for the second process were as follows: RT tolerance, 0.01 min; mass tolerance, 10.0 ppm; intensity threshold, 10; and maximum number of peaks, 20,000. Areas were derived using raw data, and not using the original peak findings, as suggested by the reference manual. The resulting peak table was normalized using the “total area sums” method and the data converted into common logarithms. The MS and MS/MS spectra were submitted to the Formula Finder computational tools (SCIEX) that propose probable elemental compositions within a specified mass tolerance of a given mass-to-charge ratio (m/z), using PeakView software (SCIEX). By interrogating the HMDB metabolite databases, the specific compounds giving rise to the observed m/z ions were identified, and listed in rank order based on the MS and MS/MS data. Proteomic MS/MS analyses were performed with the aid of ProteinPilot software (SCIEX).

A single 16 kDa band detected on a SDS-polyacrylamide gel after gel-filtration chromatography was subjected to liquid chromatography coupled to time-of-flight high resolution mass spectrometry (LC/Q-TOF/MS; TripleTOF 5600; SCIEX), followed by de novo sequencing. The band on the gel was excised and heated in 100 μl of 50 mM NH₄HCO₃ and 1 μl of 1 M dithiothreitol at 80°C for 10 min for reductive alkylation of the protein. Thereafter, 2 μl of 1 M iodoacetamide was added and incubated at 37°C for 15 min. After removing the liquid, 100 μl of 100% methanol and 100 μl of 10% acetic acid were added to destain the gels. The gels were contracted by shaking in 100% acetonitrile for 5 min. After the gels were dried, 20 μl of trypsin solution (0.5 mg/ml) in 50 mM NH₄HCO₃ was added and incubated overnight at 37°C. The digested proteins were analyzed by LC/Q-TOF/MS with the Analyst TF 1.7 Software (SCIEX). Chromatographic separation was carried out with a Cadenza CD-C18 column (150 × 2 mm, Imtakt, Japan). Elution buffers were 0.5% formic acid in SDW (solvent A) and acetonitrile (solvent B). Raw data were analyzed using the PeakView Software (SCIEX) and DeNovoGUI (Compomics). The single band detected on SDS-polyacrylamide gel after galactose-affinity chromatography was also analyzed in the same manner.

Briefly, a tandem mass spectrometry (MS/MS) peptide library was built from the peptides and proteins identified using data-dependent acquisition (DDA) shotgun nano LC–MS/MS analyses of the samples. The MS/MS spectra of the identified peptides were then used to generate the spectral library for SWATH peak extraction using the add-in for PeakView Software (version 2.1, Sciex, Connecticut Path Framingham, MA, USA) MS/MSALL with the SWATH Acquisition MicroApp (version 2.0, Sciex, Connecticut Path Framingham, MA, USA). Peptides with a confidence score greater than 99% determined with the Protein Pilot database search were included in the spectral library. The detailed LC and MS parameters that were used in the present study are provided in the Supplementary Materials, File S2.

Samples were quantified using the SWATH atlas human ion library with the PeakView software (Sciex). A peptide was considered as adequately integrated if it had a score higher than 1.5 or an FDR lower than 0.05. The sum of each adequately integrated peptide was computed for each protein with a max of 15 peptides per protein.