Nanoviper c18

Each obtained fraction was injected for nano Liquid chromatography-mass spectrometry analysis. The peptide mixture in buffer A (0.1% formic acid) was loaded onto a reverse phase trap column (Acclaim PepMap100, 100 μm×2 cm, nanoViper C18, Thermo Scientific) connected to the C18-reversed phase analytical column (Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin, Thermo Scientific). Then, the peptide was separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl min⁻¹ controlled by IntelliFlow technology. The linear gradient was: 0–35% buffer B for 50 min, 35–100% buffer B for 5 min and hold in 100% buffer B for 5 min.
LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific). MS data were obtained from the survey scan (300–1800 m/z) for higher-energy-collisional dissociation fragmentation. Automatic gain control target was set to 3e6 and maximum inject time to 10 ms. Dynamic exclusion duration was 40 s. Survey scans were acquired at a resolution of 70 000 at 200 m/z and resolution for higher-energy-collisional dissociation spectra was set to 17 500 at 200 m/z, and isolation width was 2 m/z. Normalized collision energy was 30 eV and the underfill ratio was defined as 0.1%.

Digested HDL proteins (50 ng) were loaded onto a trap column (nanoViper C18, 3 μm, 75 μm × 2 cm, Thermo Scientific) and eluted onto a C18 column (nanoViper, 2 μm, 75 μm × 15 cm, Thermo Scientific). Peptides were analyzed using an Easy-nLC 1200 UHPLC system (Thermo Scientific) coupled to an Orbitrap Fusion Lumos (Thermo Scientific) equipped with a nanospray FlexNG ion source (Thermo Scientific) in a 44 min gradient and normalized collision energy of 30 for HCD fragmentation. For untargeted analysis (DDA), peptides were analyzed using MS1 resolution of 120,000 (at m/z 200) with AGC target set to 4 × 10⁵, m/z range of 350–1550, and maximum injection time of 50 ms. MS2 resolution was set at 30,000 (at m/z 200) with AGC target of 5 × 10⁴ and maximum injection time of 54 ms. For targeted analysis (DIA), peptides were quantified using Orbitrap resolution of 30,000 (at m/z 200) with AGC target of 5 × 10⁵, precursor m/z range of 400–900, scan range of product ions between m/z 100 and 1000, maximum injection time of 54 ms, and isolation windows of 25 m/z with 0.5 m/z margins.

The HPLC liquid phase system Easy nLC system was used to separate samples. The injection volume is 2 μg peptide. The chromatographic column was balanced with a 95% solution of buffer A (aqueous 0.1% formic acid), with the sample first passed through the trap column (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18), and then separated by the analytical column (Thermo Scientific EASY column, 10 cm, ID 75 μm, 3 μm, C18-A2) with a flow rate of 300 nL/min. The separated samples were analyzed by a Q Exactive mass spectrometer. The detection mode was set to positive ion, with AGC (automatic gain control). Target and maximum IT were 1e6 and 50 ms, respectively. Other settings were used as previously described [47 (link)]. The scanning range of the precursor ions was 300 - 1800 m/z; the resolution of MS1 was 70,000 at 200 m/z. The mass charge ratio of peptides and polypeptide fragments was collected according to the following methods: 20 fragment maps (MS2 scan, HCD) were acquired after each full scan. MS2 have a resolution of 17,500 at m/z 200. The software MaxQuant (version 1.5.3.17) was used for database searches, while the LFQ (label free quantitation) algorithm was used for quantitative analysis [48 ]. The P17036_NCBI_ Nostoc_flagelliforme_18909_ 20,171 228 database was used in this study.

The peptide mixture was loaded onto the Thermo EASY-nLC System equipped with Acclaim PepMap100 trap column (100 μm × 2 cm, nanoViper C18, Thermo Fisher Scientific, Shanghai, China). Peptides were separated on analytical EASY column (75 μm × 10 cm, 3 μm, Thermo Scientific) over 60 min at a flow rate of 300 nL/min consisting of buffer A (0.1% formic acid) and buffer B (84% v/v acetonitrile and 0.1% v/v formic acid). The liquid-phase linear-gradient program was as follows: 0–35% buffer B for 50 min, 35–100% buffer B for 5 min, and hold in 100% buffer B for 5 min.
MS data were acquired using Q Exactive MS (Thermo Scientific) in the positive ion mode over 300–1800 m/z at a resolution of 70,000 at m/z 200. The automatic gain control (AGC) target was set to 3 × 10⁶, and the maximum inject time to 10 ms. Precursor ions for higher-energy collisional dissociation (HCD) fragmentation were dynamically selected according to a data-dependent top 10 method. Values for MS/MS analysis were set as follows: resolution for HCD spectra was 17,500 at m/z 200, isolation width was 2 m/z, normalized collision energy was 30 eV, dynamic exclusion duration was 40 s, and underfill ratio was defined as 0.1%.

The enriched malonylated peptides were separated by an Easy nLC system (Thermo Fisher Scientific, Waltham, MA, USA). In brief, peptides were dissolved in 0.1% formic acid, and then loaded onto sample column (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) with an automatic sampler. The samples were separated by an analytical column (Thermo Scientific EASY column, 10 cm, 75 μm ID, 3 μm, C18-A2) with a flow rate of 300 nL min⁻¹. The peptide separation gradient parameters were set according to previously described methods [14 (link)]. The separated peptides were then analyzed by a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For MS scans, the scanning range of the precursor ion was 300–1800 m/z. The resolution of the MS1 spectrum was 70,000 at 200 m/z, the target of AGC (automatic gain control) was 1 × 10⁶, the Maximum IT was 50 ms, and the dynamic exclusion was 60.0 s. The mass charge ratios of peptides and polypeptide fragments were determined as described by Li [42 (link)].