Faims pro

LC‐MS/MS analysis was performed using a nanoflow EASY‐nLC 1200 system coupled to an Orbitrap Explroris 480 mass spectrometer with FAIMS Pro device (Thermo Fisher Scientific, USA). Samples were first loaded and analyzed on a homemade analytical column (75 µm i.d. × ≈20 cm; 1.9 µm, ReproSil‐Pur 120 C18‐AQ, Dr. Maisch GmbH, Germany) packed using the Flashpacking approach.^[42 (link)
^] The mobile phases consisted of solvent A (0.1% formic acid) and solvent B (0.1% formic acid in 80% ACN). The peptides were eluted using the following gradients: 5–44% B in 43 min, 44–60% B in 5 min, 60–100% B in 2 min, and 100% B for 10 min at a flow rate of 200 nL mi⁻¹n. Data acquisition mode was set to obtain one MS scan at a resolution of 120 000 (m/z 350–1600) and followed by MS/MS scans using HCD (cycle time of 3s; normalized collision energy (NCE) of 30; isolation width of 0.7 m/z; resolution of 7500; maximum injection time of 22 ms). Two FAIMS experiments of −45 CV and −65 CV were employed, and data were acquired using a top speed of 1 sec/CV for each FAIMS experiment. Dynamic exclusion of 30 s was applied with a precursor mass tolerance of 10 ppm.

Quantitative proteomics was conducted by Bio Miao Biological Technology Co, Ltd (Beijing, China). For DDA analysis, we performed on the pooled serum samples. The purified peptide mixtures were separated into 6 fractions using high-pH reversed-phase liquid chromatography and collected. The mass spectrometer used for analysis was the Orbitrap Exploris 480 in combination with the FAIMS PRO and Nanospray Flex Ion Source (Thermo Fisher Scientific, Bremen, Germany). Briefly, the peptide mixture was re-dissolved in buffer A and was loaded on a 25 cm column (150 um inner diameter, packed using ReproSil-Pur C18-AQ 1.9 um silica beads; Beijing Qinglian Biotech Co., Ltd, Beijing, China). Running buffer A was 0.1% FA in water, and running buffer B was 0.1% FA in 80% acetonitrile. Peptides were separated using a gradient starting from 8 to 12% buffer B in 5 min, followed by a gradient from 12 to 30% buffer B over 30 min, and stepped up to 40% buffer B in 9 min. A 16 min wash at 95% buffer B was performed afterward. The total duration of the run was 60 min. MS data were acquired using a DDA method in top speed mode, with the following parameters: full scan resolution 60,000 at m/z 200, mass range of full mass 350–1500; high-collision dissociation scans with resolution 15,000 at m/z 200.

Tryptic peptides were separated using a 60-min total data collection for peptide separation, with the Easy-nLC 1200 system connected online to an Orbitrap Eclipse mass spectrometer equipped with FAIMS Pro (Thermo). Scans were collected in data-dependent top-speed mode with dynamic exclusion at 90 s. Raw data were analyzed using MaxQuant version 1.6.17.0 search against the Human Fasta database, with label-free quantification and match-between-run functions enabled. The identification of proteins that were differentially expressed between patients with RA and HCs was conducted based on the empirical Bayes method using the limma R package. The p-values were obtained by using the t-test, and the p-values of comparisons were adjusted for multiple comparisons to preserve an error rate of 5%. To identify the significant pair(s), we used multiple comparisons. The Benjamini–Hochberg method was used for the multiple comparisons.

For each sample group, seven technical replicates of measurements were conducted to mimic the sample size of current label-free single-cell proteomics data. First, 120 pg of digests were injected and separated using a commercial chromatography column (Aurora, IonOpticks) by a nanoflow liquid chromatography (Ultimate 3000 RSLCnano, ThermoFisher) with a flow rate of 100 nl/min. Mobile phase A was 0.1% FA in 2% acetonitrile, while mobile phase B was 0.1% FA in 80%acetonitrile. The 70-min LC gradient was as follows: 5% to 6.2% B for 2 min, 6.2% to 31.2% B for 40 min, 31.2% to 42.5% B for 16 min, 42.5% to 99% B for 5 min, and then isocratic at 99% B for 10 min. Label-free quantification was performed using a tribrid mass spectrometer (Orbitrap Eclipse, ThermoFisher) with an ion mobility interface (FAIMS Pro, ThermoFisher). The FAMIS compensation voltages of −55 V and −70 V were used with a cycle time of 1 s. The MS spectra were collected by an Orbitrap analyzer and the MS2 spectra were collected by a linear ion trap analyzer with a max ion injection time of 200 ms.

100 μl of each lipid extract was dried in a 96 well plate (Eppendorf twin.tec PCR Plate 96, colourless) and then reconstituted in 50 μl isopropanol:methanol:chloroform 4:2:1 [v/v/v] containing 0.5 mm sodium acetate. The plate was sealed with Teflon Ultra-Thin sealing tape (Analytical Sales and Services, Pompton Plains, NJ). Samples were introduced to an Orbitrap Fusion Lumos ultra-high resolution/accurate mass spectrometer (Thermo Fisher Scientific) using an Advion Triversa Nanomate nano electrospray ionization (nESI) source (Advion). The nESI gas pressure was set to 0.3 psi and the spray voltage to 1.1 kV. The Orbitrap Fusion Lumos ion transfer capillary temperature was set to 150°C, the RF-value to 10%, the AGC target to 2 × 105 and mass resolving power at 500 000 (at 200 m/z). All spectra were recorded in negative ionization mode for a period of 3 min over a mass range of 350–1600 m/z. For cardiolipin (CL) analysis, a Field Asymmetric Ion Mobility Spectrometry (FAIMS) interface (FAIMS Pro, Thermo Fisher Scientific) was added to the Orbitrap Fusion Lumos and operated in standard resolution mode using a compensation voltage of 60 V (optimized for the transmission of the [M-2H]²- precursor ion charge states of cardiolipin species using authentic standards).

LC-MS analysis was performed on an Ultimate 3000 RSLC-nano system connected to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific, Bremen, Germany). Approximately 1 μg of each peptide sample was resuspended in 0.1% (v/v) formic acid and loaded onto a 25 cm fused silica column heated to 50°C. The internal diameter (75 μm) of the column was packed with 1.9 μm C18 particles. Peptides were separated over 70 min with a linear gradient of 3%–20% acetonitrile in 0.1% formic acid at a flow rate of 300 nL/min. Compensation voltages (−50 and −70 V) were applied from a FAIMS Pro interface (Thermo Scientific) to regulate the entry of ionized peptides into the mass spectrometer. MS scans (m/z 300–1500) were acquired at resolution 60,000 (m/z 200) in positive ion mode, with MS/MS scans of fragment ions measured at 15,000 resolution after applying 27.5% higher-energy collision dissociation. A dynamic exclusion period of 40 s was specified.