Iodoacetamide

The peptides from preterm and term hUC-MSC CM were reduced with 10 mM DL-dithiothreitol (DTT; Promega, WI, USA) for 1 h at 56 °C and alkylated with 55 mM iodoacetamide (Promega) for 1 h in the dark at RT. Thereafter, precooled acetone was added, and the peptides were precipitated over 3 h at − 20 °C. After centrifuging for 20 min at 20,000×g and 4 °C, the precipitate was dissolved in 300 μl of the following buffer: 50% triethylamine borane (Sigma) and 0.1% SDS (Sigma). Next, the peptide solution was desalted using a Strata-X C18 column (Phenomenex, Torrance, CA, USA) and dried and labeled with TMT reagent (TMT 6-plex Label Reagent; Thermo Fisher Scientific) for 1 h [36 (link)]. Next, the preterm and term samples were mixed at a 1:1 ratio on the basis of the total peptide amount. Analysis of labeled peptides was performed on a Q Exactive Orbitrap LC-MS/MS system (Thermo Fisher Scientific). Qualitative and relative quantitative analyses of the detected peptides were performed using the SWISSPROT_human database and Mascot software (version 2.3.01). Peptides with absolute fold change ≥ 1.5 and P value < 0.05 were considered differentially expressed.

The excised FFPE brain regions were deparaffinized with n-heptane followed by ethanol washes and lysed in 100 mM triethyl ammonium bicarbonate (TEAB) and 1% w/w sodium deoxycholate (SDC) buffer, boiled at 95 °C for 60 min antigen retrieval. Samples were sonicated with a probe sonicator for 10 pulses to destroy DNA/RNA.
The excised fresh frozen brain regions were lysed in 100 mM TAB and 1% w/w SDC buffer, boiled at 95 °C for 5 min and sonicated with a probe sonicator for 10 pulses. For proteome analysis 20 µg for phosphopeptide enrichment (FF only) 200 µg of protein of each sample was taken and disulfide bonds reduced in the presence of 10 mM dithiotreitol (DTT) at 60 °C for 30 min. Cysteine residues were alkylated in presence of 20 mM iodoacetamide at 37 °C in the dark for 30 min and tryptic digestion (sequencing grade, Promega) was performed at a 100:1 protein to enzyme ration at 37 °C over night. Digestion was stopped and SDC precipitated by the addition of 1% v/v formic acid (FA). Samples were centrifuged at 16.000 g for 5 min and the supernatant was transferred into a new tube. Samples were dried in a vacuum centrifuge.

SCAPs and BMSCs were seeded on 100 mm plates at 20,000 cells/cm². When they reached 90% confluence, the medium was changed to growth medium or mineral-inducing medium. Six days later, the cells were washed five times with phosphate-buffered saline and cultured in serum-free medium for 24 h. The conditioned media were collected after centrifugation at 1,000 rpm for 10 min to remove the cellular debris and passed through a 0.22 μm filter. The samples were concentrated, air-dried, re-dissolved in triethylammonium bicarbonate (Promega, United States), and reduced with dithiothreitol at 55°C for 1 h. Next, iodoacetamide (Promega) was added to the samples, which were maintained for 1 h at room temperature in the dark. The protein concentration was determined using bicinchoninic acid (Thermo Fisher Scientific, United States) assay.