Dataset Information

LFQ of neo-amino terminal peptides using Tryp-N

ABSTRACT: To determine candidate OMA1 substrates during mitochondrial stress, OMA1 KO MEFs or its rescued cells expressing OMA1-Myc were treated with or without 40 uM FCCP for 2 h, then washed once with 1× PBS (pH 7.4), and cell pellets were collected. The pellets were then lysed in a guanidine buffer [6 M guanidine-HCl, 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP (Sigma Aldrich), and 40 mM 2-chloroacetamide (CAA, Sigma Aldrich)]. After heating and sonication, 30 ug of proteins was purified by methanol-chloroform precipitation and resuspended in 20 uL of PTS buffer [100 mM Tris-HCl (pH 8.0), 12 mM sodium deoxycholate, and 12 mM sodium lauroylsarcosinate]. The protein solution was diluted 10-fold with 10 mM CaCl2 and digested with 600 ng of Tryp-N (LysargiNase, Merck Millipore) at 37° C for overnight. After acidification with 0.5% TFA (final conc.), an equal volume of ethyl acetate was added to each sample, followed by centrifugation at 15,700g for 2 min to separate the ethyl acetate layer. The aqueous layer was collected and desalted using GL-Tip SDB, and the elutes were evaporated and dissolved in 50 uL of 2.5% formic acid and 30% acetonitrile. Enrichment of protein N-terminal peptides was performed using GL-Tip SCX (GL Sciences Inc.) based on the previous report (Chang et al, 2021). Flow-through fractions were evaporated and dissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. Peptides were separated on a 75 µm inner diameter × 150 mm C18 reverse phase column (Nikkyo Technos, Tokyo, Japan) with a linear gradient of 4%-32% acetonitrile for 0-100 min followed by an increase to 80% acetonitrile for 100-110 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were analyzed directly against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer version 2.4 for identification and label-free precursor ion quantification. Search parameters were as follows: (a) Tryp-N as a semi-specific enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) cysteine carbamidomethylation as a fixed modification; and (e) protein N-terminal acetylation and methionine oxidation as variable modifications.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Takumi Koshiba

PROVIDER: PXD063017 | JPOST Repository | Sat Jan 31 00:00:00 GMT 2026

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	173_Koshiba_Nterm_1_KO_FusionIT.raw	Raw
	175_Koshiba_Nterm_2_KO_FCCP_FusionIT.raw	Raw
	177_Koshiba_Nterm_3_WT_FusionIT.raw	Raw
	179_Koshiba_Nterm_4_WT_FCCP_FusionIT.raw	Raw
	210206_Koshiba_Nterm_mOMA1_FCCP_LFQ_FusionIT.xlsx	Xlsx

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Publications

Stress-induced OMA1-mediated cleavage of AIFM1 suppresses cell growth by controlling mitochondrial OXPHOS activity.

Nishigori Mitsuhiro M Hirata Serina S Kosako Hidetaka H Ichinohe Takeshi T Nolte Hendrik H Riemer Jan J Langer Thomas T Koshiba Takumi T

The EMBO journal 20260324 11

Mitochondrial proteases regulate dynamic properties of organelle morphology and ensure functional plasticity at the cellular level. The metalloprotease OMA1 mediates constitutive and stress-inducible processing of its mitochondrial substrates, although only a few of its direct functional targets have been characterized. Using in vitro and in vivo multiproteomic and biochemical approaches, we here demonstrate that the membrane-anchored intermembrane space (IMS) protein AIFM1 serves as a mitochond ...[more]

PMID: 41876740

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