Identifying probe-modified peptides in chemical proteomics experiments is challenging due to the production of only one modified peptide per probe reaction at a specific site on a protein and the possibility of inconclusive proteomic identification. To address these issues, we propose a principle of Single-Sequence Identification (SSI) and a method for identifying probe-modified peptides in chemical proteomics experiments. The new approach creates multiple versions of the modified peptide in the proteomic identification space to increase identification probability and confidence. The SSI method, tagging-triplication, is implemented using a one-pot triplex tagging approach, attaching three homologous biotinyl azides with different PEG linker lengths to clickable proteins. The identification of modified peptides carrying two forms of tags was assigned as confident, with tag diagnostic ions and elution time dependent on the linker length further enhancing confidence. Variability in CuAAC-tagged proteins was also identified, and a split-and-pool strategy was recommended to minimize the variability. The tagging-triplication method can be easily integrated into existing chemical proteomics workflows to identify peptides modified by alkynyl covalent probes confidently. Our findings demonstrate the potential of the SSI principle to significantly enhance modified peptide identification for proteomes with post-translational and chemical modifications. Data are available through ProteomeXchange: identifier PXD037770.
Supporting Information Cover
Text S1. Offset search parameters for identifying MeLacA-modified peptides with the desthiobiotin-PEG3-azide tag.
Text S2. Open search parameters for identifying MeLacA-modified peptides with biotin-PEGx-azide tags.
Text S3. Offset and labile search parameters for identifying MeLacA-modified peptides with biotin-PEGx-azide tags.
Table S1 List of MeLacA-desthiobiotin-modified peptides.
Table S2. List of probe-modified peptides identified using triple tagging (PEGx sequence)
Table S3. Comparison of probe-modified peptides identified with two or three PEGx tags using Offset search with and without labile mode
Table S4. Comparison of FDR filtering at the PSM level and both PSM and peptide levels
Table S5. List of probe-modified peptides identified for each click reaction replicate of MixClick and Analysis-and-Group workflows.