Timstof pro 2 mass spectrometer

Digested samples were resuspended in Buffer A (0.1% FA in water), and the peptide amount was determined by Pierce™ Quantitative Peptide Assays & Standards (Thermo Fisher Scientific) according to manufactures instructions. Samples were injected into a nanoElute UPLC autosampler (Bruker Daltonics) coupled to a timsTOF Pro2 mass-spectrometer (Bruker Daltonics). The peptides were loaded on a 25 cm Aurora ultimate CSI C18 column (IonOpticks) and chromatographic separation was achieved using a linear gradient starting with a flow rate of 250 nL/min from 2% Buffer B (0.1% FA in MeCN) and increasing to 13% in 42 min, followed by an increase to 23% B in 65 min, 30% B in 70 min, then the flow rate was increased to 300 nL/min and 80% B in 85 min, this was kept for 5 min. The mass-spectrometer operated in positive polarity for data collection using a data-dependent acquisition (ddaPASEF) mode. The cycle time was 1.17 s and consisted of one full scan followed by 10 PASEF/MSMS scans. Precursors with intensity of over 2500 (arbitrary units) were picked for fragmentation and precursors over the target value of 20,000 were dynamically excluded for 1 min. Precursors below 700 Da were isolated with a 2 Th window and ones above with 3 Th. All spectra were acquired within an m/z range of 100 to 1700 and fragmentation energy was set to 20 eV at 0.6 1/K0 and 59 eV at 1.60 1/K0.

Samples were acidified with
0.5% trifluoroacetic acid. The desalting and concentration step was
performed with ZipTip C18 (Millipore), the digested samples were speed-vacuum-dried,
and the total protein content was analyzed by a bicinchoninic acid
assay. LC-TIMS-MS/MS was carried out using a nanoElute nanoflow ultrahigh-pressure
LC system (Bruker Daltonics, Bremen, Germany) coupled to a timsTOF
Pro 2 mass spectrometer equipped with a CaptiveSpray nanoelectrospray
ion source (Bruker Daltonics). Details of the methodology can be found
in Montserrat-de la Paz et al.,^{16 (link)} although
in this case, the reference library is acquired from UniProt_proteome_Salvia-ssp_Feb22.

Digested peptides (0.1 μg/μL) were analyzed using an Easy-nLC 1000 system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a timsTOF Pro2 mass spectrometer (Bruker, Karlsruhe, Germany). The peptides were loaded onto a C18 column (75 μm × 25 cm × 1.6 μm, Thermo Fisher, Waltham, MA, USA) at 300 nL/min, and eluted with solvent A (0.1% formic acid and 2% acetonitrile) and solvent B (0.1% formic acid and 80% acetonitrile) over 60 min (0–45 min: 3% solvent B; 45–50 min: 28% solvent B; 50–55 min: 44% solvent B; 55–60 min: 90% solvent B). The separated samples were identified using a timsTOF Pro2 mass spectrometer in DDA mode with the MS scanning range set at 100–1700 m/z.

Samples were analyzed on a timsTOF Pro 2 mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to an Evosep one system (Evosep, Odense, Denmark) operating with the 30SPD method developed by the manufacturer. Briefly, the method is based on a 44-min gradient and a total cycle time of 48 min with a C18 analytical column (0.15 × 150 mm, 1.9-μm beads, ref. EV-1106) equilibrated at 40°C and operated at a flow rate of 500 nl/min. H₂O/0.1% FA was used as solvent A and ACN/ 0.1% FA as solvent B.
The timsTOF Pro 2 was operated in PASEF mode (86 (link)) over a 1.3-s cycle time. Mass spectra for MS and MS/MS scans were recorded between 100 and 1700 mass/charge ratio (m/z). Ion mobility was set to 0.75 to 1.25 V·s/cm² over a ramp time of 180 ms. The data-dependent acquisition was performed using six PASEF MS/MS scans per cycle with a near 100% duty cycle. Low m/z and singly charged ions were excluded from PASEF precursor selection by applying a filter in the m/z and ion mobility space. The dynamic exclusion was activated and set to 0.8 min, and a target value of 16,000 was specified with an intensity threshold of 1000. Collisional energy was ramped stepwise as a function of ion mobility.

Transmission electron microscopy (TEM) images were acquired on a JEOL JEM-2100F field emission electron microscope. Dynamic diameters and zeta potentials were assessed by a Malvern Zetasizer Nano ZS. UV-vis absorption spectra were detected by a Shimadzu 2600 UV-vis-NIR spectrophotometer. Fourier transform infrared (FTIR) spectra were determined by Bruker VERTEX 80V. Photoluminescence (PL) spectroscopy was obtained on a Shimadzu RF-6000 fluorescence spectrometer. Electron spin resonance (ESR) spectra were recorded on Brucker ELEXSYS spectrometer. OLYMPUS FV1000 was used to detect the confocal laser scanning microscopy (CLSM) images. Flow cytometry was performed by BD FACSCalibur Flow Cytometer. The fluorescence images of the mice were obtained on a two-dimensional InGaAs array (Princeton Instruments, NIRvana-640). UHPLC-MS/MS analyses were performed using a nanoElute UHPLC system (Bruker, Germany) coupled with a tims TOF pro2 mass spectrometer (Bruker, Germany) in Novogene Co., Ltd.