384 mtp anchorchip

Purified protein complexes were precipitated by the addition of 20% trichloroacetic acid and resolved with 50 mm ammonium bicarbonate solution. To mask the thiol group, samples were treated with 10 mm DTT at 60 °C for 30 min and then reacted with 10 mm iodoacetamide. Protein digestion was performed by the addition of 1 U·mL⁻¹ Trypsin Gold (Promega, Madison, WI, USA) overnight. Digested peptides were prepared by a treatment with de‐salting by Zip‐Tip (Merck Millipore, Burlington, MA, USA). Peptides were then separated by silica‐based reverse‐phase chromatography (gradient with acetonitrile: 0–60% in 0.1 m trifluoroacetic acid) and spotted on MTP AnchorChip 384 (Bruker Daltonics, Billerica, MA, USA) with mixing with α‐cyano‐4‐hydroxy cinnamic acid. Spotted peptides were analyzed by Autoflex speed (Bruker Daltonics), an analyzer for MALDI‐TOF/TOF with the protocol, Protein Scape (Bruker Daltonics), and proteins were identified by Mascot server (Matrix Science, Tokyo, Japan).

For the MALDI-MS experiments 1,5-diamminonapthalene (DAN) was used as matrix. DAN solution (7 g/l) was prepared in acetonitrile. The LPS samples have been mixed with acetonitrile, 5 mg/ml. 1 μL of analyte and 1 μl of matrix were mixed, 1 μl of the analyte-matrix solution was spotted directly on the target plate (MTP Anchor Chip 384, Bruker, Bremen, Germany) and analyzed by MALDI-TOF-MS.

Manually excised Coomassie stained gel spots were washed and digested according previously reported studies (20 (link), 21 (link)). The mixture of tryptic peptides (0.8 μL) derived from each protein was spotted onto a MALDI target (384 MTP Anchorchip; 800 μm Anchorchip; Bruker Daltonics, Bremen, Germany). MALDI-MS(/MS). As previously mentioned, spectra were obtained using an UltraflexTerm time-of-flight (TOF) mass spectrometer equipped with a LIFT-MS/MS device (Bruker Daltonics) at 21 kV reflector and 17 kV detector voltages, respectively (20 (link), 21 (link)). PMFs were calibrated against a standard (peptide calibration standard II, Bruker Daltonics). Flex Analysis software was used to assess the PMFs (Bruker Daltonics v.2.4). Interpretation of MS data was done using BioTools v3.2 (Bruker Daltonics). The peptide masses were searched against the Mascot search algorithm (v2.0.04, updated on 09/05/2021; Matrix Science Ltd., UK). Identified proteins were accepted as correct if they the Mascot score > 56. Because some proteins were in low abundance and did not give sufficiently powerful mass fingerprints, not all spots of interest could be recognized; some spots were mixtures of multiple proteins (30 (link), 31 (link)).