Journal: Scientific Reports

Article Title: LPS-induced lipid alterations in microglia revealed by MALDI mass spectrometry-based cell fingerprinting in neuroinflammation studies

doi: 10.1038/s41598-022-06894-1

Figure Lengend Snippet: Workflow of MALDI MS microglia fingerprinting for investigation of lipopolysaccharide- (LPS-) driven changes in microglia lipids. Cultured cells are treated in 96-well format with LPS in combination with a compound or a solvent control. After removal of culture supernatants, 96-well plates are snap-frozen and stored at − 80 °C. Cells are resuspended in acetonitrile/water to 2000 cells/µl and 2 µl (corresponding to a total of 4,000 cells per spot) are spotted on a 384-well MALDI target plate in quadruplicate using a CyBio FeliX pipetting platform. DHB matrix is sprayed onto the target plate using an M3 sprayer. MS-spectra are acquired using a rapifleX MALDI-TOF mass spectrometer in automated fashion. Spectra are processed and computationally analyzed to reveal biomolecular patterns and putative markers. Ultra-high resolution mass spectrometry using a 7 T solarix XR MRMS, timsTOFfleX-MS and tandem MS are later used for identification of candidate marker lipids.

Article Snippet: MTPs were measured on a rapifleX MALDI-TOF MS (Bruker Daltonics, Bremen, Germany).

Techniques: Cell Culture, Solvent, Control, Mass Spectrometry, Marker

Journal: Scientific Reports

Article Title: LPS-induced lipid alterations in microglia revealed by MALDI mass spectrometry-based cell fingerprinting in neuroinflammation studies

doi: 10.1038/s41598-022-06894-1

Figure Lengend Snippet: Exemplary high resolution MS spectrum and molecular structural analysis of m/z 772.5253 [PC(16:0/16:0) + K] + . ( a ) RapifleX MALDI-TOF MS-spectrum of m/z 772.55. ( b ) Accurate mass determination of m/z 772.5253 by 7 T solarix XR FTICR MS. VEH-treated (red) and LPS-treated (2.5 ng/ml; green) microglial cells. ( c and d ) Extracted ion mobility with 1/K 0 = 1.407 ± 0.01 (CCS = 286.8 Å 2 ) depicts the presence of only one peak for m/z 772.525. ( e ) Tandem MS spectra and structural elucidation of m/z 772.5253 by FTICR-MS (top, black) and timsTOF fleX-MS (down, blue). Typical fragments considered for the phosphatidylcholine [M + K] + were the neutral loss of (CH 3 ) 3 N) [M + K-59] and [M + K − 183], the PC headgroup at m/z 184.0733 and cyclophosphane (124) + K + at m/z 162.9557. Additional fragment ions at m/z 313 and 551 correspond to the presence of fatty acid C16:0.

Article Snippet: MTPs were measured on a rapifleX MALDI-TOF MS (Bruker Daltonics, Bremen, Germany).

Techniques:

Journal: Scientific Reports

Article Title: LPS-induced lipid alterations in microglia revealed by MALDI mass spectrometry-based cell fingerprinting in neuroinflammation studies

doi: 10.1038/s41598-022-06894-1

Figure Lengend Snippet: SAHA treatment reduce IL-6 and TNF-α release and modulate inflammation-associated lipid markers in LPS-stimulated SIM-A9 microglial cells. SIM-A9 microglial cells were treated for 1 h with SAHA or vehicle control (VEH) prior to LPS-stimulation for 18 h (LPS 2.5 ng/ml). Culture supernatants were collected, IL-6 ( a ) and TNF-α ( b ) release was assessed by ELISA ( N = 2–4 per condition). Data represent the mean ± s.d. One-way ANOVA with Tukey’s post hoc test. **** P ≤ 0.0001 and ** P ≤ 0.01 to Vehicle; ### P ≤ 0.0001 to LPS-stimulated cells without HDAC inhibitor. ( c-h ) Effect of SAHA on LPS-induced lipid levels. MALDI-TOF mass spectra of cells were acquired using a rapifleX. One-way ANOVA with Tukey’s post hoc test. **** P ≤ 0.0001; *** P ≤ 0.001 and ** P ≤ 0.01 indicates a significant difference from VEH via Tukey’s post hoc test. # P ≤ 0.05; ## P ≤ 0.01; ### P ≤ 0.001 and #### P ≤ 0.0001 indicates a significant difference from LPS via Tukey’s post hoc test. Data are expressed as mean ± s.d. ( N = 6 biological replicates with 4 technical replicates each). DG (Diacylglycerol), TG (Triacylglycerol) and LysoPC (Lysophosphatidylcholine).

Article Snippet: MTPs were measured on a rapifleX MALDI-TOF MS (Bruker Daltonics, Bremen, Germany).

Techniques: Control, Enzyme-linked Immunosorbent Assay