Maldi mtp 384 target plate

Standards of citric acid (Sigma-Aldrich, St. Louis, MO, USA), glyoxalic acid (Sigma-Aldrich, St. Louis, MO, USA), hydroxyacetone (Sigma-Aldric, St. Louis, MO, USA), and pyruvic acid (Sigma-Aldrich, St. Louis, MO, USA) were prepared by dissolving each of them individually in Milli-Q water to a final concentration of 0.01 mg/mL. For each EDC/4-APEBA reaction, 10 µL of each standard was diluted in 400 µL of 6 mg/mL EDC and 400 µL of 2 mg/mL 4-APEBA. For each 4-APEBA reaction without EDC, each standard was diluted in 400 µL of Milli-Q water and 400 µL of 2 mg/mL 4-APEBA. Reactions were quenched after 2 h, and 1 µL of each reaction was spotted onto a MALDI MTP 384 target plate (Bruker Daltonics, Billerica, MA, USA) and mixed with 1 µL of DHB matrix (40 mg/mL in 70% MeOH).

Thawed saliva and gingival fluid samples were vortexed. Subsequently, 1 µL of sample was spotted onto a MALDI MTP 384 target plate of polished steel (Bruker Daltonics GmbH, Germany). The air-dried sample was covered with 1 µL of matrix solution: α-cyano-4-hydroxycinnamic acid (HCCA) (Sigma Aldrich, St Louis, MO, USA) diluted in acetonitrile/water, 1:1, v/v with 2.5% TFA (Sigma-Aldrich, St. Louis, MO, USA).

Lipids were extracted from frozen frontal lobe specimens (50 ± 5 mg) by the Folch method [33 (link)] after steel bead-based homogenization in sterile deionized water using a TissueLyser (Qiagen, Valencia, CA). After evaporating the organic phase to dryness in a SpeedVac vacuum centrifuge (Thermo Fisher Scientific, Waltham, MA), the pellets were dissolved in 100 μL HPLC grade methanol and stored at −80°C.
Saturated α-Cyano-4-hydroxycinnamic acid (HCCA, Sigma Aldrich, St. Louis, MO) prepared in TA50 (Acetonitrile/0.1% trifluoroacetic acid (TFA) in water, 50:50 v/v) was used as matrix and mixed 1:1 (v/v) with lipid extract. 1 μl aliquots were spotted into a 384-well ground steel MALDI Target Plate (MTP 384) (Bruker Daltonics, Bremen, Germany) and air-dried. The samples were analyzed in the negative ion mode of an Ultraflextreme MALDI-time-of-flight (TOF)/TOF (Bruker Daltonics, Bremen, Germany). Spectra data with the mass range set to 60–3500 Da were collected with rasterizing across each well and acquiring 150000 shots/well. Flex Analysis and tandem Mass Spectrometry (MS/MS) were performed on parent ions and lipid ion identifications were made based on assignments in the LIPID MAPS database (http://www.lipidmaps.org/tools/index.html). Statistical analysis was performed using ClinProTools, Version 3.0.