Xcalibur qual browser

Heart powder was serially extracted in 80% methanol, evaporated under N₂, and resuspended in 50% methanol. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was essentially as previously described (Nadtochiy et al., 2015 (link)). Metabolites were resolved by HPLC (Shimadzu Prominence 20 System) and identified by single-reaction monitoring on a triple-quadrupole mass spectrometer (Thermo Quantum TSQ) (see the Supplemental Experimental Procedures for details). Data were analyzed using MzRock (https://code.google.com/archive/p/mzrock/). Selected metabolites were also analyzed using XCalibur Qual Browser (Thermo Scientific). Data were normalized to protein content.

Tryptic peptide of the splice junction in AML1/ETO splice product was initially validated by manual inspection of chromatograms and MS1 spectra with theoretical m/z values of peptide candidates; m/z = 519.517 (z = 4) and m/z = 692.353 (z = 3)
Validated MS1 ions were further analyzed by “MS2 run only” mode to identify the splice junction peptide with the desired b- and y- ions. The LC-MS/MS experiment was performed with an Easy-nLC 1200 UPLC system employing a 45 cm × 75 μm (inner diameter) nano-capillary column packed with 1.9 μm C18-AQ resin (Dr. Maisch, Germany) mated to a metal emitter in-line with an Orbitrap Fusion Lumos (Thermo Scientific, USA). The column temperature was 45 °C, and a one-hour gradient method was run at a flow rate of 300 nL/min. The mass spectrometer was operated to scan MS2 from MS1 of the inclusion list with a 60,000 resolution (positive mode, profile mode, AGC target of 4 × 10⁵, maximum IT of 118 ms) in the Orbitrap, followed by HCD fragmentation in the ion trap with 30% collision energy. The isolation window for precursor ions was set to 0.4 m/z in the quadrupole.
Raw files were processed with Thermo XCalibur Qual Browser, and MS2 peaks were manually annotated.

Data were processed using Xcalibur QualBrowser (Thermo Fisher Scientific), ProSight Lite 1.4 (http://prosightlite.northwestern.edu/), ProSight PD 4.0 (Thermo Fisher Scientific), and TDValidator 1.0 (Proteinaceous) (68 (link), 69 ). For ProSight PD searches, a custom RAS proteoform sequence and PTM database was created with Protein Annotator (http://proteinannotator.kelleher.northwestern.edu/) and was deposited on MassIVE. RAS isoform sequences were downloaded from UniProt, and mutations, SNPs, and PTMs were manually annotated. Raw files were run through ProSight PD with subsequent manual validation by ProSight Light or TDValidator (70 (link)). Observed masses, error, and p-scores of completely characterized RAS proteoforms are reported in Table S5. The top two most abundant RAS proteoforms as determined by protein ion relative ratios and fragment ion relative ratios for Figure 4 were quantified by the method described in the study by Pesavento et al. (71 (link)). Fragment ion relative ratios were taken from 3 m/z isolation windows for the canonical forms of KRAS WT and mutant (Table S6). Incompletely characterized RAS proteoforms are reported in Table S7. Raw files and the custom ProSight PD database are deposited on MassIVE (MSV000088748). Proteoform record numbers can be searched in a database provided by the Consortium for Top–Down Proteomics.