Super dhb

Lipid A was precipitated from GMMA as previously described [25 (link)] using mild acid hydrolysis with 1% acetic acid for 2 h at 100°C. Samples were centrifuged at 14,000 g for 15 min, the pellets resuspended in water, and washed twice with water. The pellets were dried overnight using a Speedvac, resuspended in chloroform-methanol 4:1, and mixed with an equal volume of Super DHB (Sigma-Aldrich) solution in water/acetonitrile 1:1 (vol/vol). Two μL of the mixture were loaded to the target plate (MTP 384 target plate ground steel BC, Bruker Daltonics) and analyzed by Ultraflex MALDI-TOF (Bruker Daltonics) in reflectron ion-negative mode. A Peptide Calibration Standard (Bruker Daltonics), mixed with the Super DHB solution, was included in each analysis. The m/z rations were determined by Flex Analysis software in comparison to the Peptide Standard.

All samples were mixed in a 1:1 ratio with sDHB (Super-DHB, Bruker) matrix solution (50 mg mL in 50% acetonitrile (ACN), 50% water, and 0.1% trifluoroacetic acid). Subsequently, 1-μL aliquots of the mixture were deposited on a BigAnchor MALDI target (Bruker) and allowed to dry and crystallize at ambient conditions.
MS spectra were acquired on a rapifleX MALDI-TOF/TOF (Bruker, Germany) in the mass range of 20,000–120,000 m/z in linear positive mode for intact protein measurements and in the mass range of 100–1,600 m/z in reflector positive mode for lipid measurements. The Compass 2.0 (Bruker) software suite was used for spectra acquisition and processing.

The rapifleX MALDI-TOF/TOF
instrument (Bruker) was used with a high mass acquisition method with
dedicated high mass calibrants. Calibrant proteins: insulin (no. I5500),
ubiquitin (no. U6253), and thioredoxin (no. T0910) were ordered from
Sigma-Aldrich and prepared as stock solutions at 50 pmol/μL
in TA30 (70% water, 30% acetonitrile (ACN), and supplemented with
0.1% trifluoroacetic acid (TFA)). Asialofetuin (no. A4781, Sigma)
was reduced (dithiothreitol), alkylated (iodoacetamide), and digested
using trypsin (Promega) according to standard vendor protocols. Each
protein was mixed with Super-DHB (sDHB, Bruker) matrix solution (50
mg/mL) and 1 μL directly spotted on a ground steel MALDI target
plate (Bruker).