Lift ms ms device

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)).

Statistically significant protein spots were collected manually, washed, and digested according to previously described methods [55 (link),56 (link)]. Trypsin was used to cleave protein peptides at 37 °C overnight. Next, 0.8 µL of tryptic peptides was loaded into a MALDI target plate (384 MTP Anchorchip, 800 µm Anchorchip, Bruker Daltonics, Bremen, Germany). MALDI-MS/MS spectra were measured on an UltraflexTerm TOF mass spectrometer connected to a LIFT-MS/MS device (Bruker Daltonics). MS data were translated using BioTools v3.2 (Bruker Daltonics). Each peptide sequence was aligned to the database to identify proteins by utilizing the Mascot engine (v2.0.04, updated on 9 May 2021; Matrix Science Ltd., London, UK). Only those proteins that showed a Mascot score greater than 56 with p ≤ 0.05 were considered for Ingenuity Pathway Analysis (IPA).

Coomassie-stained gel spots corresponding to the same spots that showed statistically significant differential abundance in the 2D-DIGE gels were excised manually. They were washed and digested according to previously described methods [47 (link),48 (link),49 (link)]. Finally, a 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) spectra were obtained using an Ultraflextreme time-of-flight (TOF) mass spectrometer equipped with a LIFT-MS/MS device (Bruker Daltonics) at reflector and detector voltages of 21 kV and 17 kV, respectively, as described previously [47 (link),48 (link)]. PMFs were calibrated against a standard (peptide calibration standard II, Bruker Daltonics, Bremen, Germany). The PMFs were assessed using Flex Analysis software (version 2.4, Bruker Daltonics, Bremen, Germany)). MS data were interpreted using BioTools v3.2 (Bruker Daltonics). The peptide masses were searched against the Mascot search algorithm (v2.0.04, updated on 9 May 2020; Matrix Science Ltd., London, UK). The identified proteins were screened for a Mascot score of higher than 56 and p < 0.05.