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pmic7791-sup-0001-figureS1.pdf269K

Fig. 1. Protein profiling of mouse whole brain enriched membrane fractions. Mouse whole brains were homogenized and enriched g membrane fractions were prepared using differential ultracentrifugation. Membrane proteins were extracted using homemade extraction buffer [1.5 M 6-aminocaproic acid, 300 mM Bis-Tris, pH 7.0, protease inhibitor cocktail, 1% n-dodecyl β-D-maltoside (DDM)] or commercial membrane protein extraction kits. Extracted proteins were subjected to BN-PAGE or SDS-PAGE followed by visualization using Colloidal Coomassie Blue staining. The indicated gel areas (Fig. 2) were excised, and peptides were prepared by reduction/alkylation and chymotrypsin/trypsin digestion. Extracted peptides were analyzed via nano-LC-ESI-MS/MS using Orbitrap Velos Pro. LC-MS/MS data were analyzed with Mascot 2.4 against the UniProtKB database. Identified proteins were divided into three groups, Anchored/Integral; Integral; None (neither anchored nor integral). The number of proteins identified from each enzyme digestion and overlapped protein number are shown in Venn diagram.

Fig. 2. Efficiency of TMD containing protein identification between BN- and SDS-PAGE within same extraction methods. Mouse whole brains were homogenized and enriched membrane fractions were prepared using differential ultracentrifugation. Membrane proteins were extracted using homemade extraction buffer [1.5 M 6-aminocaproic acid, 300 mM Bis-Tris, pH 7.0, protease inhibitor cocktail, 1% n-dodecyl β-D-maltoside (DDM)] or commercial membrane protein extraction kits. Extracted proteins were subjected to BN-PAGE or SDS-PAGE followed by visualization using Colloidal Coomassie Blue staining. The indicated gel areas (Fig. 2) were excised, and peptides d b d ti / lk were prepared by reduction/alkylation and chymoptrypsin/trypsin digestion. Extracted peptides were analyzed via nano-LC-ESI-MS/MS using Orbitrap Velos Pro. LC-MS/MS data were analyzed with Mascot 2.4 against the UniProtKB database. The numbers of identified proteins extracted using 6-ACA or commercial kits were shown in Venn diagram to compare the efficiency of separation methods.

Fig 3. Comparison of identified protein profiles between two protein separation methods. Mouse whole brains were homogenized and enriched membrane fractions were prepared using differential ultracentrifugation. Membrane proteins were extracted using homemade extraction buffer [1.5 M 6-aminocaproic acid, 300 mM Bis-Tris, pH 7.0, protease inhibitor cocktail, 1% n-dodecyl β-D-maltoside (DDM)] or commercial membrane protein extraction kits. Extracted proteins were subjected to BN-PAGE or SDS-PAGE followed by visualization using Colloidal Coomassie Blue staining. The indicated gel areas (Fig. 2) were excised, and peptides were prepared by reduction/alkylation and multi-enzyme digestion. Extracted peptides were analyzed via nano-LCESI-MS/MS using Orbitrap Velos Pro. LC-MS/MS data were analyzed with Mascot 2.4 against the UniProtKB database. Identified proteins from the same protein separation methods (BN-PAGE or SDS-PAGE) were combined and categorized into three groups Anchored/Integral; Integral; None (neither anchored nor integral).

Fig. 4. Comparison of identified protein profiles between two different protein extraction methods. Mouse whole brains were homogenized and enriched membrane fractions were prepared using differential ultracentrifugation. Membrane proteins were extracted using homemade extraction buffer [1.5 M 6-aminocaproic acid, 300 mM Bis-Tris, pH 7.0, protease inhibitor cocktail, 1% n-dodecyl β-D-maltoside (DDM)] or commercial membrane protein extraction kits. Extracted proteins were subjected to BN-PAGE or SDS-PAGE followed by visualization using Colloidal Coomassie Blue staining. The indicated gel areas (Fig. 2) were excised, and peptides d b d ti / lk were prepared by reduction/alkylation and chymoptrypsin/trypsin digestion. Extracted peptides were analyzed via nano-LC-ESI-MS/MS using Orbitrap Velos Pro. LC-MS/MS data were analyzed with Mascot 2.4 against the UniProtKB database. Identified proteins from the same protein extraction methods (6-ACA extraction or commercial kits) were combined and categorized into three groups Anchored/Integral; Integral; None (neither anchored nor integral).

Fig. 5. Distribution of number of transmembrane domains (TMDs) from identified proteins. Membrane proteins were prepared from mouse whole brain enriched membrane fractions using homemade extraction buffer or commercial protein extraction kits. Extracted membrane proteins were subsequently subjected to BN-PAGE or SDS-PAGE followed by LC-MS/MS analysis using Orbitrap Velos Pro. The number of transmembrane domains of identified proteins were analyzed using UniProtKB database.

Fig. 6. Comparison of distribution of number of transmembrane domains (TMDs) from identified proteins between 6-ACA and commercial kit extraction method. Membrane proteins were prepared from mouse whole brain enriched membrane fractions using homemade extraction buffer or commercial protein extraction kits. Extracted membrane proteins were subsequently subjected to BN-PAGE or SDS-PAGE followed by LC-MS/MS analysis using Orbitrap Velos Pro. The number of transmembrane domains of identified proteins were analyzed using UniProtKB database.

Fig. 7. Comparison of distribution of number of transmembrane domains (TMDs) from identified proteins between BN-PAGE and SDS-PAGE. Membrane proteins were prepared from mouse whole brain enriched membrane fractions using homemade extraction buffer or commercial protein extraction kits. Extracted membrane proteins were subsequently subjected to BN-PAGE or SDS-PAGE followed by LC-MS/MS analysis using Orbitrap Velos Pro. The number of transmembrane domains of identified proteins were analyzed using UniProtKB database.

pmic7791-sup-0002-tableS1.pdf2573KTable 1. The list of the identified proteins and peptides from in-gel chymotrypsin digestion
pmic7791-sup-0003-tableS2.pdf6096KTable 2. The list of the identified proteins and peptides from in-gel trypsin digestion

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