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Data-Independent Acquisition Enhancement of a Competitive Activity-Based Protein Profiling Platform for Kinase Inhibitor Screening

ABSTRACT: Kinase inhibitors represent a vital class of therapeutic agents widely used in cancer research, immunology, and other disease areas. Mass spectrometry (MS) employing specially designed small molecule kinase-binding probes has become an essential strategy for identifying novel kinase drug targets. While traditional MS approaches often rely on targeted proteomics (e.g., multiple reaction monitoring, MRM) or data-dependent acquisition (DDA), data-independent acquisition (DIA) offers broader and more reproducible quantification, especially for low-abundance peptides. In this study, we systematically developed an activity-based protein profiling (ABPP) platform leveraging DIA, through integrated in-house informatics tools for data filtering and motif analysis, to provide an effective kinase profiling workflow. Compared to DDA, the DIA approach yielded more than a 100% increase in identified biotinylated peptides and over 40% improvement in kinase peptide coverage, while reducing the analysis time by half (90 min vs. 180 min per sample). Additionally, there was a modest improvement to the coefficient of variation (CV) in kinase peptide quantification (11.41% to 10.70%; mean CV). Shorter liquid chromatography (LC) gradient times (60, 45, and 30 min) were evaluated as a means for increasing sample analysis throughput. Notably, no significant loss in kinase peptide coverage was observed due to shorter gradients, highlighting the capability of DIA to significantly enhance the efficiency and scalability of kinase profiling workflows.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dingyin Tao

LAB HEAD: Dingyin Tao

PROVIDER: PXD072729 | Pride | 2026-02-23

REPOSITORIES: Pride

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Project description:Porphyra/Pyropia seaweeds are promising sources for functional foods development, offering a rich macro- and micronutrient profiles. In New Zealand (NZ), endemic Porphyra/Pyropia species (karengo), exhibit considerable variability driven by geography, seasonality, and climate, which may influence their nutritional quality. Despite their use as traditional foods, the NZ Porphyra/Pyropia remain underutilized commercially, in part due to the lack of biomolecular characterisation, particularly their bioactive protein components, hindering evidence-based species selection for seaweed farming commercialisation and functional food development. This study presents the first proteomic characterization of three NZ Porphyra/Pyropia species: Pyropia virididentata, Pyropia cinnamomea, and Porphyra GRB complex. Mass spectrometry-based proteomics analysis identified differences in the phycobiliprotein composition among the species, with the Porphyra GRB complex containing higher levels of phycocyanin. Using the protein sequence information, in silico gastrointestinal digestion analysis predicted that phycobiliproteins from NZ Porphyra/Pyropia seaweeds can potentially release bioactive peptides capable of inhibiting angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) activities. Sequence-based allergenicity prediction indicated possible cross-reactivity between NZ Porphyra/Pyropia β-phycoerythrin and β-phycocyanin against the β-phycocyanin allergen from spirulina, which is associated with a low incidence of allergy. Proximate analysis revealed that NZ Porphyra/Pyropia seaweeds have high protein (26–30.2 %) and carbohydrate (48.3–50.9 %) contents, and low fat and free sugar levels. Amino acid profiling further showed that NZ Porphyra/Pyropia seaweeds are relatively rich in sulphur-containing amino acids and umami-associated amino acids. Overall, these findings highlight the potential of NZ Porphyra/Pyropia seaweeds as a novel plant-based protein source for functional food applications.

			Action	DRS
	ADPProbeDDA.xlsx	Xlsx
	ADP_20uM_Cpd411_01_3uL.raw	Raw
	ADP_20uM_Cpd411_02_3uL.raw	Raw
	ADP_20uM_DMSO_01_3uL.raw	Raw
	ADP_20uM_DMSO_02_3uL.raw	Raw

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Data-Independent Acquisition Enhancement of a Competitive Activity-Based Protein Profiling Platform for Kinase Inhibitor Screening

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