Hydroxylamine

All CSF samples were balanced for diagnosis, race, age, and sex (in that order) across 16 batches using ARTS (automated randomization of multiple traits for study design) [37 (link)]. Using a 16-plex Tandem Mass Tag (TMTpro) kit (Thermo Fisher Scientific, A44520, Lot number: VH3111511), 13 channels of each batch were allocated to a CSF sample (127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N). The remaining 3 channels were occupied with a GIS pool (126), a standard biomarker negative pool (133C), and a standard biomarker positive pool sample (134N). Information regarding the origination of these pooled samples were reported previously [38 (link)]. Supplemental Table 2 provides the sample to batch arrangement. In preparation for labeling, each CSF peptide digest was resuspended in 75 μl of 100 mM triethylammonium bicarbonate (TEAB) buffer meanwhile 5 mg of TMT reagent was dissolved into 200 μl of ACN. Once, both were in suspension, 15 μl of TMT reagent solution was subsequently added to the resuspended CSF peptide digest. After 1 h, the reaction was quenched with 4 μl of 5% hydroxylamine (Thermo Fisher Scientific, 90,115) for 15 min. Then, the peptide solutions were combined according to the batch arrangement. Finally, each TMT batch was desalted with 60 mg HLB columns (Waters) and dried via speed vacuum (Labconco).

The SiMAG-Carboxyl (0.5 µm ϕ, Chemicell. Prod. No. 1402) particles were washed twice with 1 M MES buffer using a magnetic separator (Promega). After the second wash, the magnetic particles were resuspended in MES buffer containing 10 mg EDC (Thermo Fisher). Subsequently, only freshly prepared EDC was added to the particles, followed by mixing on a shaker for 10 min at RT. After this step, the EDC solution was removed and replaced by prepared NHS (Thermo Fisher), followed by fully mixing on a shaker and reaction at RT for 30 min. Then, the NHS was removed and an amine group containing ligand GABA (10 mg dissolved in double distilled water) was added to the activated particles and mixed on a shaker for 2-3 h at RT. Next, the particles were resuspended in blocking buffer (10 mm hydroxylamine [Thermo Fisher]), shaken, and reacted for 10 min at RT to terminate the reaction. Finally, the particles were resuspended in PBS for storage and later use.

To quantify the peptides, the peptide quantification kit (Thermo Scientific, Catalog number: 23275, Waltham, MA, USA) was used. Next, 50 µg of peptides were extracted from each sample and labeled using a TMT pro10-plex Label Reagent Set (Thermo Scientific, Catalog number: 90110, Waltham, MA, USA) following the manufacturer’s protocol. Three rats in the control group were labeled with TMT-126, while three rats in the model group were labeled with TMT-128N, and three rats in the amygdalin group were labeled with TMT-130C. The labeling reagent was dissolved in acetonitrile and incubated with peptides for 1 h. The incubation was terminated with 50% hydroxylamine (Thermo Scientific, Catalog number: 90115, Waltham, MA, USA). The labeled peptides were then pooled, desalted using peptide desalting spin columns (Thermo Scientific, Catalog number: 89852, Waltham, MA, USA), and freeze-dried under vacuum.

Dithiothreitol (DTT), iodoacetamide, ammonium bicarbonate (Ambic), ammonium hydroxide, sodium docecylsulphate (SDS), trifluoroacetic acid (TFA), sodium deoxycholate (SDC), tris(hydroxymethyl)aminomethane (Tris), formic‐acid, and urea were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Triethylamonium bicarbonate (TEAB) and hydroxylamine were from Thermo Fisher Scientific. Water and organic solvents were all LC‐MS grade and supplied by Merck (Darmstadt, Germany) or (Thermo Fisher Scientific). Endoproteinase Lys‐C was obtained from Wako (Osaka, Japan) and sequencing‐grade modified trypsin was purchased from Promega (Madison, WI, USA). Cell lines SK MEL2 (HTB‐68), SK MEL28 (HTB‐72), and RPMI‐7951 (HTB‐66) were obtained from the American Type Culture Collection (ATCC).

TMT labeling of digested Set 1 and Set 2 samples was performed as previously described (38 (link)). The discovery dataset (Set 1) was comprised of 36 individual samples and 3 GIS randomized based on age, sex and diagnosis into three batches and the replication dataset (Set 2) was comprised of 85 individual samples and 5 GIS randomized into five batches. Supplemental Tables 4 and 10 provides the sample-to-batch arrangement for each of these samples. These samples were then labeled using TMTpro kits (Thermo Fisher Scientific, A44520, Lot number: VH3111511 for Set 1; UK297033 for Set 2 with XB338618 for channels 134C and 135). First, each peptide digest was resuspended in 75 μl of 100 mM triethylammonium bicarbonate (TEAB) buffer, and 5 mg of TMT reagent was dissolved into 200 μl of anhydrous acetonitrile (ACN). After that, 15 μl of TMT reagent solution was subsequently added to the resuspended peptide digest and incubated for 1 hour at room temperature. Following that, the reaction was quenched with 4 μl of 5% hydroxylamine (Thermo Fisher Scientific, 90115) for 15 minutes. Then, the peptide solutions were combined according to the batch arrangement. Finally, each TMT batch was desalted with 60 mg HLB columns (Waters) and dried via speed vacuum (Labconco).