Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation

Abstract

Protein prenylation is an important post-translational modification that regulates protein interactions, localizations, and signaling pathways in normal functioning of eukaryotic cells. It is also a critical step in the oncogenic developments of various cancers. Direct identification of native protein prenylation by mass spectrometry (MS) has been challenging due to high hydrophobicity and the lack of an efficient enrichment technique. Prior MS studies of prenylation revealed that prenyl peptides readily generate high-intensity fragments after neutral loss of the prenyl group (R group), and more recent investigation of oxidized prenyl peptides discovered more consistent neutral loss of the oxidized prenyl group (RSOH group). Here, a dual-stage neutral loss MS 3 (DS-NLMS3)-based strategy is therefore developed by combining both gas-phase cleavable properties of the prenyl thioether bond and mono-oxidized thioether to improve the large-scale identification of prenylation. Both neutral losses can individually and distinctively confirm the prenylation type in MS 2 and the sequence of the prenyl peptide upon targeted MS 3 fragmentation. This dual-faceted NLMS3 strategy significantly improves the confidence in the identification of protein prenylation from large-scale samples, which enables the unambiguous identification of prenylated sites of the spiked low-abundance farnesyl peptide and native prenyl proteins from mouse macrophage cells, even without prior enrichment during sample preparation. The ease of incorporating this strategy into the prenylation study workflow and minimum disruption to the biological lipidome are advantageous for unraveling unknown native protein prenylation and further developments in profiling and quantifying prenylome.

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene–Tetrazine Click Chemistry

“…The development of proteomic methods to comprehensively identify the prenylome has relied on the use of bioorthogonal probes for protein enrichment. While prenylomic analysis using substrates containing biorthogonal functionality has limitations, it remains the most widely used method in the field for characterizing the prenylome in a comprehensive manner. , Other methods focused on enrichment of prenylated proteins without metabolic labeling are being developed but have not been widely used to date. − Using alkyne-containing isoprenoid analogues followed by conjugation with biotin-azide allows proteins modified through metabolic labeling to be isolated via avidin enrichment. After confirming that C10NorOPP could be used to metabolically label protein targets of prenylation, chemical proteomics was used to identify which prenylated proteins were modified by C10NorOPP using the workflow shown in Figure A.…”