Orbitrap fusion ms

All of the TMT batches were analyzed on the same Orbitrap Fusion MS instrument (Thermo Fisher Scientific). Between each individual TMT experiment, one blank was run, followed by analysis of a 15 peptide Retention Time Calibration (RTC) standard, to evaluate retention time drift. This was followed by analysis of an MCF10a total cell digest standard to evaluate peptide and protein identifications. The last step consisted of analysis of two blanks, one with an oscillating gradient and one with the gradient matching the samples to be run.

Digested peptides were put in auto sampler vials and loaded into an Easy-nLC 1200 (Thermo Fisher Scientific) coupled to an Orbitrap Fusion MS (Thermo Fisher Scientific). Chromatographic separation was carried out in commercially packed C18 columns (Acclaim PepMap 2 mm, 100 Å, 75 mm, 15 cm; Thermo Fisher Scientific). Peptides were loaded in buffer A (5% of acetonitrile and 0.1% of formic acid) and eluted with a 1 h linear gradient from 5 to 30% of buffer B (80% of acetonitrile and 0.1% of formic acid). Three biological replicates were sequentially injected with two 15-min wash runs and a 1 h blank run alternated between distinct ‘treatments’. Mass spectra were acquired using a Top20 data-dependent method with an automatic switch between full MS and MS/MS (MS2) scans. The Orbitrap analyzer parameters for the full MS scan were resolution of 120,000 mass range of 350 to 1800 m/z, and AGC target of 4e5 ions, whereas those for MS2 spectra acquisition were resolution of 30,000, AGC target of 5e4 ions, an isolation window of 2 m/z and dynamic exclusion of 30 s. Column chromatographic performance was routinely monitored with intermittent injections of 50 fmol of a commercially available Bovine serum albumin (BSA) peptide mix (Waters Inc.), as well as evaluating double-wash runs for carryover peptides.

Global proteomics analysis involved Tandem Mass Tag (TMT) isobaric labeling of peptides for the identification and relative quantitation of proteins by multiplexing the 50 discovery cohort participants (Fig. 1) using 11-plex TMT reagents (randomized into 5 sets). The samples were analyzed using a Thermo Orbitrap Fusion MS. A list of biomarker targets was compiled using statistical significance between DO and HV, DO and DF, DO and NDO, NDO and HV (Fig. 2b).

Proteins were dissolved in 25 ammonium acetate at a concentration of 10 uM, the drugs dissolved DMSO were diluted by 25 ammonium acetate to 100 uM. Then proteins were incubated with an equal volume of the drugs.
The above-mixed solutions were then injected into Orbitrap Fusion MS (Thermo Scientific) through direct injection. The MS was operated in intact protein mode. Data were analyzed with BioPharma Finder (Thermo Scientific) software (Marcoux et al., 2015 (link)).

Online 2D-nano LC/MS/MS was used to perform MudPIT mass spec analysis of S. feltiae ESPs. The mass spec apparatus consisted of a 2D nanoAcquity UPLC (Waters, Milford, MA) configured with an Orbitrap Fusion MS (Thermo Scientific, San Jose, CA). LC solutions/fractionation and MS parameters were as previously described [5 (link)]. The raw mass spec data was processed/analyzed with the Proteome Discoverer 2.2 software (Thermo Scientific, San Jose, CA) with the Sequest HT search engine running against the S. feltiae protein profile, steinernema_feltiae.PRJNA204661.WBPS11.protein.fa (Parasite.Wormbase.org). Duplicate genes were removed and only genes with FDR <5% were considered for further analysis. The raw mass spec data have been uploaded to the ProteomeXchange repository and can be accessed with the following links.
0 hr: ftp://massive.ucsd.edu/MSV0000829936 hr: ftp://massive.ucsd.edu/MSV000082997