Proteomics analysis of Control and Palbociclib-treated HeLa cells

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the proteomes of Control and Palbociclib-treated HeLa cells. For protein identification and quantification, mass spectrometry data from fractionated samples (data-dependent acquisition, DDA; 6 fractions) and single-shot samples (data-independent acquisition, DIA) were utilized to construct a DDA library and a direct-DIA library, respectively. These libraries were computationally merged into a hybrid spectral library using Spectronaut software (Biognosys). Subsequent functional annotation was performed through Gene Ontology (GO) and InterPro (IPR) analyses using the InterProScan-5 tool against the non-redundant protein database. Additionally, protein family and pathway analyses were carried out using the Clusters of Orthologous Groups (COG) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.