Protocol for mass spectrometric profiling of lysine malonylation by lysine acetyltransferase in CRISPRi K562 cell lines

Lysine malonylation is a protein posttranslational modification. We present a protocol to generate stable gene-knockdown K562 cell lines through lentiviral infection of a CRISPR interference (CRISPRi) system followed by lysine malonylation measurement using mass spectrometry (MS). We detail guide RNA (gRNA) vector cloning, lentiviral infection, cell line purification, protein digestion, malonyl-lysine enrichment, desalting, and MS acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Zhang et al.1 and Bons et al.2 [Display omitted] •The CRISPRi system enables efficient and stable gene knockdown in K562 cells•CRISPRi K562 cell lines can be purified through FACS or antibiotic selection•Malonyl-lysine-specific antibody beads allow the enrichment of malonylated peptides•Malonylated peptides can be measured by data-independent acquisition mass spectrometry Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Lysine malonylation is a protein posttranslational modification. We present a protocol to generate stable gene-knockdown K562 cell lines through lentiviral infection of a CRISPR interference (CRISPRi) system followed by lysine malonylation measurement using mass spectrometry (MS). We detail guide RNA (gRNA) vector cloning, lentiviral infection, cell line purification, protein digestion, malonyl-lysine enrichment, desalting, and MS acquisition and analysis.