Dataset Information

Immune Isoform Atlas: Landscape of alternative splicing in human immune cells

ABSTRACT: This dataset contains the global and terminal proteome of THP-1 and LCL cell lines untreated or treated with phorbol 12-myristate 13-acetate (PMA) and interferon-α2 (IFN-α2), respectively. Extracted proteins were digested with trypsin or V8 protease and protein terminal peptides were isolated with strong cation exchange chromatography or metal oxide-based ligand-exchange chromatography. All peptides were labeled with TMT-tag, and a total of 16 channels were used with quadruplicates of untreated LCL, IFN-α2-treated LCL, untreated THP-1, and PMA-treated THP-1 peptides, respectively. The labeled peptides were fractionated into 24 vials by reversed-phase HPLC at high pH conditions.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD040962 | JPOST Repository | Mon Apr 08 00:00:00 BST 2024

REPOSITORIES: jPOST

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			Action	DRS
	220321HN_THP1NATMTproTrypticWholeFr01.raw	Raw
	220321HN_THP1NATMTproTrypticWholeFr02.raw	Raw
	220321HN_THP1NATMTproTrypticWholeFr03.raw	Raw
	220321HN_THP1NATMTproTrypticWholeFr04.raw	Raw
	220321HN_THP1NATMTproTrypticWholeFr05.raw	Raw

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Project description:Objective: ADP-ribosylation is a post-translational modification that plays an important role in cellular processes. Our previous work implemented multiple gas-phase separation strategies (e.g., FAIMS) and in-source CID on the quadrupole-Orbitrap (Exploris 480) to increase the yield and acceptor site confidence scores of HCD-dependent ADP-ribosyl (ADPr) peptide identifications. We evaluated whether FAIMS coupled on the quadruple-ion trap-Orbitrap (Fusion Lumos) also improves EThcD-dependent ADP-ribosyl peptide sequencing. Methods: ADPr peptides derived from the human macrophage-like cell line THP-1 (THP-1-Mφ) were analyzed on the Lumos fronted with a FAIMS Pro and EASY-Spray Source, coupled to an Easy-nLC1200 HPLC pump. Gas-phase segmentation (GPS) for the MS1 scan range, and single and multiple combined compensation voltages (CVs) for FAIMS were applied for HCD and EThcD properties. ADP-ribosyl peptide spectra were analyzed using Proteome Discoverer 2.5. The ptmRS function was used for calculating ADP-ribosyl site probabilities. Results: We evaluated the number of ADPr and non-ADPr PSMs using ADPr peptides pooled from PBS and IFN-γ treated THP-1-Mφ across a range of CVs (-40V to -85V) using FAIMS with HCD and EThcD. The peak CVs for ADPr and non-ADPr PSMs were shifted for HCD, while they represented similar distributions for EThcD. The net number of unique ADPr and non-ADPr peptides across the CVs increased by 3.2- and 3.8-fold more respectively for HCD, and 2.0- and 3.6-fold respectively for EThcD, compared to no FAIMS. We then tested 4 distinct MS methods: (method 1: m/z 400-900, method 2: m/z 400-655 and m/z 650-900, method 3: three CVs combination (-50V, -60V, -70V) with m/z 400-900, method 4: three different acquisitions using distinct CVs (-50V, -60V, -70V) with m/z 400-900) for identifying the maximum number of ADPr acceptor sites with high (>95%) confidence using EThcD. Method 4 was best with 562 ADPr PSMs, but only 54% were high confidence ADPr sites. Due to sample volume limitation, we used method 3 for analyzing ADPr peptides enriched from THP-1-Mφ treated with PBS or IFN-γ separately. We identified 324 and 196 unique ADPr peptides from PBS and IFN-γ, of which 257 and 139 unique acceptor sites were identified with high confidence. The most frequent ADPr acceptor site was lysine (>90%), followed by serine. Conclusion: Our data demonstrated that while FAIMS is valuable for EThcD-dependent sequencing of ADPr peptides, the gains are less for ADPr peptides or contaminant non-ADPr peptides when using HCD.

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Immune Isoform Atlas: Landscape of alternative splicing in human immune cells

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