Phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED) provides real-time phosphoproteomics data to aid in cancer drug selection

AbstractGlobal high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography –tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To addr ess these limitations, here we have optimized a new protocol,phospho-heavy-labeled-spiketide FAIMS Stepped-CVDDA (pHASED), that employs online phosphoproteome deconvolution using high-fieldasymmetric waveformionmobilityspectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows,...
Source: Clinical Proteomics - Category: Biochemistry Source Type: research