iD-plus

iD-plus identifies proteins from reflectron-embedded MALDI-TOF mass spectrometry data by exploiting C-terminal peptide fragmentation patterns and metastable ions to determine peptide sequences and C-terminal residues.

Key Features:

• Metastable Ion Utilization: Capitalizes on prominent metastable ions b_{n-1} and [b_{n-1} + 18] that appear as slightly shifted, broad m/z peaks in MALDI-TOF spectra and can be distinguished from typical product ions.
• C-Terminus Fragmentation: Exploits the higher propensity of C-terminal peptide bonds to undergo cleavage, producing b_{n-1} ions that enable reliable identification of C-terminal amino acids.
• Enzyme Specificity (LysN): Uses LysN protease specificity (cleavage at the N-terminal side of lysine) to generate predictable peptide sets for analysis.
• Database and In Silico Analysis: Employs an in silico LysN-digested peptide database derived from SwissProt: 2017_03 containing 553,941 protein sequences and over 11 million possible peptide combinations for search and matching.
• Identification Without Probability-Based Scoring: Demonstrates that identification can be achieved using at least four peptides without relying on probability-based scoring methods.

Scientific Applications:

• Proteome-wide Protein Identification: Identification of proteins from MALDI-TOF spectra in proteomics studies using C-terminal fragmentation signatures.
• C-Terminal Residue Determination: Determination of C-terminal amino acids of peptides via detection of b_{n-1} and [b_{n-1} + 18] metastable ions.
• Analysis of Complex and Gel-Derived Samples: Identification of proteins in complex mixtures and gel-derived proteins where C-terminal-specific fragmentation aids assignment.
• Low-Scoring-Model Identification: Protein identification in workflows that avoid probability-based scoring by leveraging multiple peptide matches (≥4 peptides) for confident assignment.

Methodology:

iD-plus performs in silico searches of a LysN-digested peptide database (SwissProt: 2017_03; 553,941 sequences, >11 million peptides) and matches observed peptide masses and C-terminal residue information to spectra using enzyme specificity and C-terminal fragmentation signatures.