Orbitrap eclipse tribrid

Samples were reconstituted in 0.1% formic acid and then injected into a nano-LC system (EASY-nLC™ 1200, Thermo Fisher, San Jose) using trap-elute mode. Solvent A was 0.1% formic acid in water, while solvent B was 0.1% formic acid in 80% acetonitrile. After loading into the trap column (Thermo Scientific Acclaim PepMap 100 C18, 75 μm-i.d., 2 cm-long, 3 μm, 100 Å), all of the peptides were further separated by a home-packed 75 µm-i.d., 25 cm-long C18 (1.9 µm, Dr. Maisch GmbH, Ammerbuch, Germany) column at flow rate of 300 nL/min with different gradient settings based on sample types. An Orbitrap Eclipse Tribrid mass spectrometer supplied with a FAIMS Pro Interface (Thermo Fisher, San Jose) was employed for MS analysis. Spray voltage was set to 2.0 kV and ion transfer tube temperature at 320 °C. Combination of different FAIMS CVs (compensation voltage) were set to run data-dependent acquisition (DDA) mode of the most intense precursors for 1 s cycle to build a big cycle of 2 s or 3 s. The detailed parameters were shown in the supplementary Data 4. In general, the proteome samples were separated with longer gradient, and fragment ions were acquired using ion trap for deeper profiling.

LC–MS/MS analysis was carried out on an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific, San Jose, CA) connected online to a Dionex RSLC3000 liquid chromatography system (Thermo Scientific, San Jose, CA). Peptides were suspended in solvent A (0.1% formic acid) and loaded on a trap column (PepMap C₁₈ 2 cm × 100 µm, 100 Å) followed by high resolution separation column (EasySpray 50 cm × 75 µm, C₁₈ 1.9 µm, 100 Å, Thermo Scientific, San Jose, CA). The mass spectrometer was operated in data dependent mode with a cycle time of 2 s. Survey MS scan was acquired in Orbitrap mass analyzer with 120 K resolution, 4×e5 AGC target and 50 ms injection time. Monoisotopic precursor ions with charge state 2–4 were subjected to MS/MS with top scan priority followed by precursor with charge 1. Precursor ions (z = 2–4) were fragmented with 28% HCD normalized collision energy and acquired in orbitrap mass analyzer with 15 K resolution. Precursor ions (z = 1) with a mass range of 700–1400 m/z were fragment with 32% HCD NCE and analyzed in orbitrap analyzer. Dynamic exclusion was enabled with 30 s exclusion duration. Additional filters included monoisotopic precursor selection and intensity threshold of 2.5 × 10⁴.

The eluted peptides were
ionized using a Thermo Scientific Nanospray Flex ion source and injected
into a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer
operated in positive mode equipped with a FAIMSpro interface. Spectra
were acquired using data-dependent acquisition mode where a 120 k
resolution full MS1 scan was followed by sequential MS2 scans at a
resolution of 15,000 to utilize the remainder of the 3 s cycle time.
MS/MS spectra were collected using a 1.5 m/z window for precursor ion quadrupole isolation and normalized
HCD collision energy of 30% with a dynamic exclusion of 10 s and monoisotopic
peak determination set to peptide. The “auto” maximum
injection time was selected to allow the orbitrap to calculate the
maximum injection time available to maximize sensitivity while maintaining
the maximum scan rate. FAIMS separations were performed using FAIMS
mode on a standard resolution set to static gas mode with a nitrogen
carrier gas flow of 0 L/min and inner and outer electrode temperatures
of 100 °C with an asymmetric dispersion voltage (DV) of −5000
V. To selectively filter ions that enter the mass spectrometer, individual
compensation voltages (CVs) between the range of −25 and −70
V were applied to sequential survey scans and MS/MS cycles.

Recombinant A2ML1 was digested with trypsin alone or both trypsin and chymotrypsin in solution. Digested samples were purified using homemade C18 stage tips before analysis by LC-MS/MS. Online RP-HPLC fractionation using an EASY-nLC 1200 (Thermo Fisher Scientific) was performed with a 5–45% gradient of acetonitrile (ACN) over 30 min. Mass spectrometry was performed on an Orbitrap Eclipse Tribrid (Thermo Fisher Scientific) running a glycan-triggered MS2(HCD)_MS2(EThcd) data-dependent acquisition method with a resolution of 120,000 on the MS1 level and 60,000 on both MS2 levels. HCD fragmentation used stepped collison energies (27%, 30%, 32%) whereas EThcD used automatically adjusted ETD fragmentation augmented with 10% HCD. Data were searched with the Byonic search engine (Version 3.7.13. Protein Metrics) with a standard human glycosylation database and a PEP 2D score threshold/cut-off of 0.05.