Protein calibration standard

The band corresponding to the enzyme identified as ACPase II was excised from polyacrylamide gels and digested with trypsin Gold-Mass V582A (Promega), according to Schevchenko et al [39 (link)]. The resulting peptides of each spot were submitted to mass spectrometric analyses using an UltraFlexIII MALDI-TOF/TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight), controlled with Flex Control 3.0 software (Bruker Daltonik). The sample was mixed with α-cyano-4hydroxycinnamic acid matrix solution (3:1, v/v) directly applied onto an MTP AnchorChip 400/384 target plate (Bruker Daltonik) and dried at room temperature. Peptides presenting mono isotopic masses were obtained in reflector mode with external calibration using the Protein Calibration Standard (Bruker Daltonik). Peptide MS/MS spectra were obtained by means of LIFT fragmentation. The software Flex Analysis 3.0 (Bruker Daltonik) and PepSeq (Waters) were used for mass spectrometric data analysis. Peptide primary structures were inferred by means of manual interpretation of fragmentation. The obtained sequences were then searched against the NCBI_nr protein database (www.ncbi.nlm.nih.gov) using the algorithm Blastp. The identified protein sequences were analyzed using the Peptide Mass tool from the ProtParam Server (www.expasy.org) in order to predict theoretical molecular weight [40 ].

MALDI-TOF/TOF studies were performed on a Bruker Autoflex III MALDI-TOF/TOF spectrometer (Bruker, Billerica, MA, USA) equipped with a 200-MHz smart-beam pulsed N2 laser (λ 337 nm). Aliquots of 1 μL of mixtures containing 10 μM or 5 μM protein (Hfra, Yfh1 and/or α-syn), 70 μM AA and 2.5 μM Cu²⁺ or Fe³⁺ prepared in buffer B1, were taken after 0 and 150 min of incubation (25 °C) and supplemented by trifluoroacetic acid (TFA) (0.2% v/v). Samples were then combined with 1 μL of matrix solution (10 μg of sinapinic acid in a solution water:acetonitrile (70:30) containing 0.1% TFA), and a 0.5 μL aliquot of this mixture was spotted onto a steel target plate (MTP 384), air-dried and subjected to mass determination. The IS1 and IS2 voltages were 20 kV and 18.5 kV respectively, and the lens voltage was 7.5 kV. Measurements were performed using a positive reflector mode with matrix suppression below 400 Da. The spectra were calibrated externally using a protein calibration standard (3600–17,000 Da) from Bruker. The experiments were performed in duplicate.

To measure the molecular mass of the selected fraction, a matrix-assisted ionization time-of-flight (MALDI-ToF) mass spectrometer (AutoFlex III) Smartbeam (Bruker Daltonics, Bremen, Germany) controlled by Flex Control 3.0 software was used (Bruker Daltonics, Bremen, Germany). A 0.37 µM sample was solubilized in Ultrapure water, mixed (1:1 v:v) in a saturated solution of sinapinic acid, as matrix, and applied to the target plate (Bruker Daltonics, Bremen, Germany) to dry at room temperature. The compound had its molecular mass obtained in the positive linear mode after external calibration, with Protein Calibration Standard (Bruker Daltonics, Bremen, Germany). The MALDI-ToF spectra were processed with Flex Analysis 3.0 software (Bruker Daltonics, Bremen, Germany).