Dataset Information

TMT-based proteomic analysis of Atg5-/- mouse brain

ABSTRACT: The brains of neonatal mice (Atg5+/+ in triplicate, Atg5-/- in quadruplicate, and Atg5-/-;NSE-Atg5 in triplicate) were lysed in 500 µL of 6 M guanidine-HCl containing 100 mM Tris-HCl, pH8.0, and 2 mM DTT. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 g for 15 min at 4 ºC. Proteins (100 µg each) were reduced in 5 mM DTT at room temperature for 30 min, alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark, and subjected to methanol/chloroform precipitation. After solubilization with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate, the proteins were digested with 1 µg of trypsin/Lys-C mix (Promega) for 16 h at 37 ºC. The peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay. Approximately 25 µg of peptides for each sample was labeled with 0.2 mg of TMT-10plex reagents (Thermo Fisher Scientific) for 1 h at 25 ºC. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using the Pierce high pH reversed-phase peptide fractionation kit. Eight fractions were collected using 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile (ACN). Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis of the resultant peptides (1 µg each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a 75 µm inner diameter X 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear gradient of 4–20% ACN for 0–180 min and 20–32% ACN for 180–220 min, followed by an increase to 80% ACN for 220–230 min. The mass spectrometer was operated in a data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375–1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.4 m/z, a maximum injection time of 100 ms, and a normalized collision energy of 32. Dynamic exclusion was set to 30 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.2 (Thermo Fisher Scientific) with Mascot search engine version 2.5 (Matrix Science) for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as a variable modification. Peptides were filtered at a false-discovery rate of 1% using the percolator node.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD049220 | JPOST Repository | Sat Jun 01 00:00:00 BST 2024

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	171004_kuma_atg5_tmt10_2_9.pdResult	Other
	171004_kuma_atg5_tmt10_2_9.xlsx	Xlsx
	171_kuma_atg5_tmt10_2.raw	Raw
	173_kuma_atg5_tmt10_3.raw	Raw
	175_kuma_atg5_tmt10_4.raw	Raw

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Publications

YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

Kitta Shinri S Kaminishi Tatsuya T Higashi Momoko M Shima Takayuki T Nishino Kohei K Nakamura Nobuhiro N Kosako Hidetaka H Yoshimori Tamotsu T Kuma Akiko A

The EMBO journal 20240531 14

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and G ...[more]

PMID: 38822137

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