Tmt10plex

Reduction of disulfide
bridges in cysteine-containing proteins was performed with dithiothreitol
(56 °C, 30 min, 10 mM in 50 mM HEPES, pH 8.5). Reduced cysteines
were alkylated with 2-chloroacetamide (room temperature, in the dark,
30 min, 20 mM in 50 mM HEPES, pH 8.5). Samples were prepared using
the SP3 protocol,^{74 (link),75 (link)} and trypsin (sequencing grade,
Promega) was added in an enzyme to protein ratio of 1:50 for overnight
digestion at 37 °C.
Peptides were labeled with the TMT10plex^{76 (link)} isobaric label reagent (Thermo Fisher) according
to the manufacturer’s instructions. Samples were combined for
the TMT10plex, and for further sample cleanup, an OASIS HLB μElution
plate (Waters) was used. Offline high-pH reverse-phase fractionation
was carried out on an Agilent 1200 Infinity HPLC system, equipped
with a Gemini C18 column (3 μm, 110 Å, 100 × 1.0 mm,
Phenomenex).^{77 (link)}

All extracted samples were adjusted to 50 mM ammonium bicarbonate, heated for 10 min at 80 °C, followed by reduction with 5 mM dithiothreitol (60 °C, 30 min) and alkylation with 10 mM iodoacetamide (30 min, RT, in the dark). Subsequently, samples were incubated with trypsin, chymotrypsin or GluC (Roche; 37 °C, overnight, 1 µg enzyme per 100 µg protein). The following day, the enzyme reaction was quenched and Rapigest was precipitated by acidifying with trifluoroacetic acid (TFA). The solution was cleared by centrifugation (10,000 × g, 10 min) and peptides were purified on C18 Sep-Pak columns (Waters), and dried down using a SpeedVac vacuum concentrator (Thermo Fischer Scientific). If not already desialylated, the dried peptides were resuspended in 1 mL 50 mM sodium acetate (pH 5.5) containing 0.1 U/mL neuraminidase (Sigma, N3001) followed by incubation at 37 °C for 1 h, purified by C18 Sep-Pak columns and dried down.
In the case of rat tissues, 200 µg digest was labeled with TMTsixplex or TMT10plex (Thermo Fischer Scientific) according to manufacturer’s instructions (Supplementary Data 2 and 3: Rapigest).

LC-MS-grade water was purchased from VWR International (Radnor, PA, USA). Reagents for TMT10plex, the MicroBCA protein assay, and Pierce Concentrator 3KDa MWCO 0.5 mL were purchased from Thermo Fisher Scientific (Rockford, IL, USA). The protease inhibitors cocktail cOmplete^TM Mini EDTA-free EASYpack was acquired from Roche (Basel, Switzerland). Lysyl endopeptidase C (Lys-C), mass spectrometry grade, was bought from Wako (Neuss, Germany).
Furthermore, the 0.22 µm spin filters, AssayMap BRAVO 5 µL cartridges (C18 and RPS), and immunodepletion Multiple Affinity Removal Spin Cartridge MOUSE-3 were purchased from Agilent Technologies (Santa Clara, CA, USA).
Vivaspin 500 3 kDa MWCO filters were bought from Sartorius (Gottingen, Germany). Trypsin/Lys-C mix Mass Spec grade was purchased from Promega (Madison, WI, USA), and size Exclusion Chromatography columns (qEV-70 nm) were obtained from IZON (Christchurch, NZ, USA).
All other reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

The dried peptide samples were subsequently labeled with isobaric tag (TMT 10-plex, Thermo Fisher Scientific) according to the manufacturer’s instructions. To control for ratio distortion during quantification, a peptide calibration mixture consisting of six digested standard proteins mixed in different amounts were added to each sample before TMT labeling as recently described^{60 (link)}. After pooling the TMT labeled peptide samples, peptides were again desalted on C18 reversed-phase spin columns according to the manufacturer’s instructions (Macrospin, Harvard Apparatus) and dried under vacuum. TMT-labeled peptides were fractionated by high-pH reversed phase separation using a XBridge Peptide BEH C18 column (3,5 µm, 130 Å, 1 mm × 150 mm, Waters) on an Agilent 1260 Infinity HPLC system. Peptides were loaded on column in buffer A (ammonium formate (20 mM, pH 10) in water) and eluted using a two-step linear gradient starting from 2% to 10% in 5 min and then to 50% (v/v) buffer B (90% acetonitrile/10% ammonium formate (20 mM, pH 10) over 55 min at a flow rate of 42 µl/min. The elution of peptides was monitored with a UV detector (215 nm, 254 nm). A total of 36 fractions were collected, pooled into 12 fractions using a post-concatenation strategy as previously described^{61 (link)}, dried under vacuum and subjected to LC-MS/MS analysis.