Qe hf mass spectrometer

Tryptic peptides were resuspended in 0.1% formic acid (Sigma) at a concentration of 0.5mg/mL for instrumental analysis. Samples were analyzed using a Dionex UltiMate 3000 UPLC coupled to a Thermo Scientific QE-HF mass spectrometer. Solvents consisted of 0.1% formic acid in water as buffer A and 0.1% formic acid in 80% acetonitrile as buffer B. LC gradients consisted of a trapping phase from 0 to 18 minutes at 4% B moving to 40% at 120 minutes, 75% B from minutes 120.5 to 130, 97% B from minutes 130.5 to 140, and 4% B from minutes 140.5 to 155. Survey scans of peptide precursors from 300 to 1500 m/z were performed using a resolving power of 60,000 with an AGC target of 1 x 10⁶ and maximum injection time of 150 ms. The top 20 precursors were then selected for higher energy collisional dissociation fragmentation with a normalized collision energy of 30, an isolation width of 2.0 Da, resolving power of 15,000, an AGC target of 5 x 10⁴, a maximum injection time of 150 ms and a lower mass limit of 120.0 m/z. Precursors were subject to dynamic exclusion for 15 seconds with a 10-ppm mass tolerance. Each sample was acquired in technical triplicate.

The detailed proteomics methodology is provided as supplementary information. Serum and synovial fluid samples were prepared for liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis by solubilization, reduction, and alkylation followed by digestion with trypsin. The desalted samples were randomized and injected onto a Thermo QEHF mass spectrometer and collected using data independent acquisition. Spectronaut (version 15) [27 (link)] was employed for protein identification and quantification, using the reference Canus Lupus Familiaris proteome from Uniprot. Perseus (1.6.14.0) [28 (link)] was employed for data processing and statistical analysis using the MS2 intensity values generated by Spectronaut. The data were log2 transformed and normalized by subtracting the median for each sample. Statistical analysis of acquired data was performed as described below. The proteins that were exclusively detected in one experimental group were also reported for further bioinformatics analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for both synovial fluid and serum results.

The SubAP-MALDI source was coupled with a QE-HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) for all data acquisition. A software tool (Target-ng, MassTech, Columbia, MD) was used to control the XY stage motion in the ion source, while iFunnel software (MassTech, Columbia, MD) was utilized to control the ion funnel operation. In the dried droplet sample preparation method, peptide standards were premixed with matrix solutions and then applied onto an ABI Opti-TOF 192 target plate (Applied Biosystems, Foster City, CA) and analyzed using a spiral motion mode for the laser spot travel across the sample surface. For MSI, constant speed raster motion was used. The mass spectrometer was operated in the MS mode with positive polarity. A mass range of m/z 400–4,000 was used, and the resolution was set at 120,000 unless otherwise specified. Automatic gain control (AGC) target was set to 3×10⁶ with 300 ms maximum injection time. The AGC value was high enough to ensure that in every scan the ion accumulation took place exactly during 300 ms injection time which, with the constant scan speed of the MALDI target plate, ensured each pixel was the same size and prevented the Orbirap cell from being overloaded.