Tmt10plex kit

Eyes from five animals were used to study proteome changes following DEX implant intervention in CRVO, comparing CRVO + DEX (n = 5) vs. CRVO + sham (n = 5) with proteomic analysis by tandem mass tag (TMT) based mass spectrometry. Sample preparation was performed with the suspension-trapping method [26 (link)] using S-Trap mini columns from Protifi (Farmingdale, NY, USA), as described in detail in a previous study [6 (link)]. TMT-based mass spectrometry was performed essentially as described in a recent paper [27 (link)]. Eleven micrograms from each sample were used for TMT labeling with TMT10-plex kit (Lot number: RG234624A, Thermo Scientific, Waltham, MA, USA). High-pH reversedphase peptide fractionation into 8 fractions was performed as described in a previous study [28 (link)].

After trypsin digestion was completed, the desalting of peptides was conducted with the Strata-X C18 SPE column (Phenomenex, Los Angeles, CA, United States), followed by vacuum drying. The peptides were reconstituted in TEAB (0.5 M) and processed on the basis of the protocol of the manufacturer of the TMT-10plex kit (Thermo Fisher Scientific, Waltham, MA, United States). Generally, the thawing and reconstituting of one unit of TMT reagent were conducted in acetonitrile (ACN). The incubation of peptides was performed at ambient temperature for 2 h, followed by pooling, desalting, and drying during vacuum centrifuging.

The IP elutes were verified by Western blot (5% of total material) and the remaining material was precipitated using methanol and chloroform. The pellet was washed with methanol to remove excess detergent. Protein was then dissolved in 5μL fresh 1% RapiGest SF Surfactant (Waters Cat. #186001861) and diluted with 10μL 0.5M HEPES pH 8.0 and 32.5μL H2O. 0.5μL of a 0.5M TCEP was added to reduce disulfide bonds. 1μL of a 0.5M iodoacetamide was added to acetylate free sulfhydryl groups for 30min at room temperature in the dark. Trypsin digestion was performed overnight at 37℃ with rigorous shaking. Trypsinized samples were labeled with TMT using a TMT10plex kit (Thermo Scientific catalog #90110). Excess label was neutralized with ammonium bicarbonate at a final concentration of 0.4% for 1h. Samples were mixed and acidified to pH 2 with formic acid. Sample volume was reduced to 1/6th of the original volume using a SpeedVac and restored to the original volume using Buffer A (5% acetonitrile, 0.1% formic acid). Rapigest was cleaved by incubating at 42°C for 1h. Samples were spun at 14,000 rpm for 30 min and supernatant was transferred to a new tube and stored at −80°C.

The total protein was extracted from 170 mg of tissue collected from six-day-old seedlings grown on a Murashige and Skoog (MS) medium without sugar (three samples each from the WT, z1, and z2), in accordance with the method described by Roitinger et al. [17 (link)]. The proteins were precipitated using the TCA/acetone method, collected via centrifugation, and processed for trypsin digestion using filter-aided sample preparation. Digestion was carried out overnight at 37 °C using 0.22 µg/µL MS-grade trypsin (Promega, PRV5111, Madison, WI, USA), and the samples were purified on peptide desalting spin columns (ThermoFisher 89851). The phosphopeptides were labeled with nine isobaric tandem mass tags using the TMT10plex kit (ThermoFisher, 90110) and enriched via sequential enrichment of metal oxide affinity chromatography (High-Select SMOAC; ThermoFisher, A32993).

LWAC isolation of Tn and T‐O‐glycopeptides was performed as described previously 27, 30. Briefly, proteins were reduced (5 mM DTT 45 min 60°C) and alkylated (10 mM IAA 30 min RT), digested with trypsin (Roche), and neuraminidase‐treated to remove sialic acid residues. For experiments in COSMC KO (SC), these steps were followed by labeling with light or medium isotopomeric dimethyl labels 42. The labeled digests were mixed in a 1:1 ratio in the following sets: HaCaT^SC (light) and HaCaT^SCΔT1 (medium), HaCaT^SC (light) and HaCaT^SCΔT2 (medium), and HaCaT^SC (light) and HaCaT^SCΔT3 (medium). The mixed pairs of digests were enriched using agarose‐bound VVA LWAC and eluted with GalNAc. For experiments in WT, digests of three clones of each ΔT1, ΔT2, and ΔT3, and one WT were labeled using the TMT‐10plex Kit (Thermo Scientific) according to the manufacturer's instructions. Labeled digests were mixed in an equimolar ratio and enriched using Jacalin LWAC, followed by elution with D‐galactose. Isoelectric focusing was performed on VVA‐enriched glycopeptides by a 3100 OFFGEL fractionator (Agilent) using pH 3–10 strips (GE Healthcare) 79. High pH fractionation was performed on TMT‐labeled Jacalin‐enriched glycopeptides, as well as peptides before enrichment. All glycopeptide samples were desalted by self‐made Stage Tips (C18 sorbent from Empore 3 M) and submitted to LC‐MS and HCD/ETD‐MS/MS.