Biopharma finder software

To identify the proteolysis sites, LC-MS intact mass analysis was conducted for control samples and the reaction mixtures of disulfide HMGB1 cleaved by neutrophil elastase in the presence or absence of the 20 bp DNA duplex under the conditions described above. The reaction was stopped by freezing and kept at −20 °C until ready for use. Immediately after thawing the reaction mixtures, samples were diluted 1:1 with a solution of 50% acetonitrile and 0.2% formic acid and analyzed by LC-MS to determine the intact mass profile of each sample. LC-MS intact mass analysis was accomplished using a desalting size-exclusion chromatography column (BEH SEC200 4.6 × 30 mm, Waters) and a denaturing isocratic mobile phase (30% acetonitrile, 0.1% formic acid, 0.02% TFA) controlled by a Vanquish Horizon UHPLC (Thermo Scientific) with the eluent coupled directly to an Orbitrap Eclipse MS system (Thermo Scientific), using charge reduction of the eluted intact protein ions to improve spectral fidelity as previously reported (66 (link)). Raw LC-MS data were deconvolved and annotated using BioPharma Finder software (version 4.1, Thermo Scientific). Deconvolved masses were annotated using a maximum mass tolerance of 100 ppm relative to the theoretical mass of each identified species.

Glycan and glycopeptide compositional analysis was performed from m/z features extracted from LC–MS data using in-house written SysBioWare software (72 (link)). For m/z feature recognition from full MS scans, Minora Feature Detector Node of the Proteome discoverer 2.2 (Thermo Fisher Scientific) was used. The list of precursor ions (m/z, charge, peak area) was imported as ASCII data into SysBioWare, and compositional assignment within 3 ppm mass tolerance was performed. The main building blocks used for the compositional analysis were theoretical monoisotopic mass increment of NeuAc, Hex, HexNAc, dHex, and the theoretical monoisotopic mass increment of the most prominent peptide corresponding to each potential glycosites. To generate the potential glycopeptide list, all the glycoforms with an abundance higher than 5% of the most abundant glycoform were used for glycan feature analysis. Raw spectra for intact mass analysis were deconvoluted to zero charge by BioPharma Finder Software (Thermo Fisher Scientific) using default settings. Glycoproteoforms were annotated by in-house written SysBioWare software (72 (link)) using average masses of hexose, N-acetylhexosamine, and the known backbone mass of mucin1 TR reporter increment.

Kits were stored between 2 and 8 °C with automated temperature monitoring over a 12-month period. At selected time points (every 2 months), kits (in triplicate) were reconstituted in 5 mL ultra-pure water (VWR International, Fontenay-sous-Bois, France, Hypersolv Chromanorm for HPLC 83645.320) and subjected to UV-HPLC analysis (Phenomenex, Le Pecq, France, Luna C18; 250 mm × 4.6 mm × 5 µm; 0–1 min 95/5/1–7 min 5/95/7–8 min 5/95/8–9 min 95/5/9–10 min 95/5; 2.5 mL/min; H₂O 0.1% TFA/Acetonitrile; 10 min) and liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis (in singulet) (Lumos^®, ThermoFischer Scientific, Waltham, MA, USA, injection volume 10 µL, pre-column 300 µm internal diameter × 5 mm C18 PepMapTM; column 75 µm internal diameter x 50 cm nanoViper C₁₈, 2 µm, 100 Å—Acclaim^® PepMap RSLC; solvent A 95/05/0.1 H₂O/ACN/HCOOH; solvent B 20/80/0.1 H₂O/ACN/HCOOH; gradient 4–40% B in 50 min). Raw data were analyzed with BiopharmaFinder software (ThermoFischer Scientific, Waltham, MA, USA) allowing deconvolution of MS spectra during LC analysis. Fragments <1% were not considered. The sum of all significant fragments was set at 100%

Glycopeptide compositional analysis was performed from m/z features extracted from LC–MS data using in-house written SysBioWare software^{96 (link)}. For m/z feature recognition from full MS scans Minora Feature Detector Node of the Proteome discoverer 2.2 (ThermoFisher Scientific) was used. The list of precursor ions (m/z, charge, peak area) was imported as ASCII data into SysBioWare and compositional assignment within 3 ppm mass tolerance was performed. The main building blocks used for the compositional analysis were: NeuAc, Hex, HexNAc, dHex, and the theoretical mass increment of the most prominent peptide corresponding to each potential glycosites. Upon generation of the potential glycopeptide list, each glycosite was rank for the top 10 most abundant candidates and each candidate structure was confirmed by doing targeted MS/MS analysis followed by manual interpretation of the corresponding MS/MS spectrum. For intact mass analysis raw spectra were deconvoluted to zero-charge by BioPharma Finder Software (ThermoFisher Scientific, San Jose) using default settings. Glycoproteoforms were annotated by in-house written SysBioWare software^{96 (link)} using average masses of Hexose, N-acetylhexosamine, and the known backbone mass of mucin TR reporter sequence.