MALDI-TOF analysis

Samples were re-suspended in TFA (30% Acetonitrile and 0.1% Trifluoroacetic Acid) and diluted to ≤ 1 ug/ul. The MALDI matrix 2,5-Dihydroxybenzoic Acid (DHB) was re-suspended in TA30 with a working concentration of 40ug/ul and combined with the sample in a 1:1 ratio that was spotted on a Bruker MTP 384 ground steel target plate and allowed to dry. All spectra were recorded using a Bruker Autoflex Max MALDI-TOF mass spectrometer equipped with a smartbeam-II solid-state laser operating in positive reflectron mode and controlled with Bruker’s flexControl (V3.4) instrument software. For each sample, spectra were acquired within a mass range of 500−3,500 Da at an acquisition rate of 2,000 Hz. The remaining instrument parameters were configured as follows: ion source 1 = 19.00 kV, ion source 2 = 16.75 kV, lens = 8.50 kV, reflector = 21.00 kV, reflector 2 = 9.60 kV, pulsed ion extraction time = 130 ns, and matrix suppression ≤ 450 Da. All raw spectra were processed with Bruker’s flexAnalysis (V3.4) to include automated baseline subtraction and smoothing.