Dataset for "Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells"

Data collection method:

Samples were generated as described in "Development of methodology to investigate the surface SMALPome of mammalian cells." In summary, 3T3-L1 mouse fibroblasts were surface biotinylated prior to extraction with either styrene maleic acid (SMA) or detergent buffer control (SDS/Sodium deoxycholate/NP-40). Purification of biotinylated surface membrane proteins using NeutrAvidin beads was conducted, prior to three different wash protocols. Samples underwent nano-LC MS/MS with further proteomic analysis to determine differences between proteins extracted using SMA and detergent control. The raw data files were processed and quantified using Proteome Discoverer software v2.1 (Thermo Scientific) and searched against the UniProt Mouse database (downloaded February 2020; 83561 sequences) using the SEQUEST HT algorithm. The outputs from the Proteome Discoverer were filtered to identify transmembrane proteins and proteins that have a signal sequence. Firstly, any non-mouse contaminants were removed (i.e. contaminants = TRUE) from the data sets. Next, proteins with ≤ 1 unique peptide were removed from the data sets. The filtered data sets were compared with mouse proteins (86521 proteins) in the Uniprot database.

Data processing and preparation activities:

Uniprot lists for mouse protein searches; To identify integral membrane proteins: Organism [OS], Mus musculus (mouse) AND Keyword [KW] Transmembrane helix (18359 proteins, downloaded December 2020). To identify proteins with a signal sequence but not a transmembrane domain: Organism [OS] mus musculus (mouse) AND PTM Processing>molecule processing.signal peptide, NOT Keyword [KW] transmembrane helix (6683 proteins, downloaded April 2021). To identify GPI anchor proteins associated with lipid rafts: keyword:"GPI-anchor [KW-0336]" AND organism:"Mus musculus (Mouse) [10090]" (226 proteins, downloaded July 2021). To identify mitochondrial transmembrane proteins: keyword:"Transmembrane [KW-0812]" keyword:"Mitochondrion [KW-0496]" AND organism:"Mus musculus (Mouse) [10090]" (810 proteins, downloaded June 2021).

Technical details and requirements:

Surface biotinylation following the protocol from the Pierce™ Cell Surface Protein Isolation Kit (#89881). Analysis of peptides by nano-LC MSMS using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) controlled by Xcalibur 3.0 software (Thermo Scientific) and operated in data-dependent acquisition mode.

Additional information:

Supplementary table 1: Contains the raw datasets and subsequent filters, legends are supplied for each tab to describe the dataset. Supplementary table 2: Contains datasets referring to mitochondrial membrane protein comparisons. Legends are supplied to further describe the dataset. Supplementary table 3: Contains datasets referring to Glycosylphosphatidylinositol (GPI) anchour protein comparisons. Legends are supplied to further describe the dataset.