Implementation of an enriched membrane protein carrier channel for enhanced detection of membrane proteins in mass spectrometry-based thermal stability assays

Abstract

In this work, we developed a membrane-enriched stable isotope isobaric-labeled carrier channel (meSIILCC) for mass spectrometry-based thermal stability assay (MS-TSA). A proof-of-concept study demonstrated that the meSIILCC method could modestly improve membrane protein (MP) detection in MS-TSA experiments. An enhancement of 10% in identifications of membrane-proteins was observed in the meSIILCC group. Hydrophobicity analysis of the identified and quantified peptides using the grand average of hydropathy index confirmed the meSIILCC approach enriched for peptides of higher hydrophobicity characteristic of membrane-associated proteins.To further improve meSIILCC, four membrane-protein enrichment approaches were compared. Using the selected and optimized workflow that utilized isobaric labeling-mass spectrometry, 8,662 protein groups were quantitatively characterized and then annotated based on their subcellular localization. The corresponding reporter ion intensities were used to construct a heatmap, which revealed an increased representation of proteins corresponding to the “plasma membrane” gene ontology term.In a separate DMSO-only MS-TSA experiment, the optimally performing meSIILCC was added at 10-fold the protein content of the lowest heated aliquot from the MS-TSA, and isotope interference was found to be the highest in the 134N channel, while to a much lesser degree in other channels that were left empty.To further assess the performance of meSIILCC in the DMSO-only MS-TSA experiment, an over-representation analysis was performed, which demonstrated that proteins exclusive to the meSIILCC group had more than a five-fold increase in gene ontology cellular component terms related to the “membrane” term.We found 496 proteins from the DMSO-only MS-TSA experiment, which were identified across all replicates and shared between the meSIILCC and control that were annotated with “plasma membrane.” A close to 28% increase in the set corresponding to unique peptides was realized, using the meSIILCC approach, with a median value of 6.3 peptides per protein, compared to 4.7 in the control.