Mass Spectrometry Studies of Anti-Biofilm Peptides
Poster Number
22A
Faculty Mentor Name
Jianhua Ren
Research or Creativity Area
Other
Abstract
KR-12-a5, an engineered peptide derived from the human cathelicidin LL-37, exhibits potent antimicrobial and anti-biofilm activity against oral pathogens linked to endodontic infections. Notably, incorporating D-amino acids into KR-12-a5 has been demonstrated to significantly enhance cell selectivity while preserving its potent anti-inflammatory activity. So, to optimize the properties of KR-12-a5, we synthesized a set of derivatives by incorporating a D-amino acid at the 5/6 positions or attaching an N-terminal 5(6)-FAM label through linkers of varying lengths. This approach enabled us to identify the optimal spacer that best preserved the native peptide structure while minimizing functional deviations from the untagged form. These optimized peptides will be used to assess their antimicrobial potency against biofilms, and we will investigate whether the D-amino acid substitution and the 5(6)-FAM/linker architecture influence peptide fragmentation patterns in mass spectrometry. The doubly and triply protonated peptides underwent extensive and consistent fragmentation. Peptides with four charges fragmented at specific positions, predominantly producing low- to medium-intensity y-ions. Strong fragmentation was observed on the N-terminal side of Trp (W), generating corresponding y-ions. The presence of a single D-amino acid had minimal impact on fragmentation. Fluorescent tags with varying linker lengths exhibited similar effects on fragmentation; however, they promoted the formation of Tag-b1 ions. Lowly charged peptides fragmented more extensively, producing a wide range of b- and y-ions. Overall, the single D-amino acid residue exerted minimal influence on the fragmentation pattern, and the FAM tag and linker had comparable yet negligible effects on peptide fragmentation.
Purpose
D-amino acid substitution and N-terminal 5(6)-FAM labeling with optimized linkers can improve KR-12-a5 properties without significantly altering its MS/MS fragmentation behavior.
KR-12-a5 derivatives were synthesized with D-amino acids (positions 5/6) or FAM/linkers and analyzed by MS/MS to evaluate fragmentation patterns and structural integrity.
Both modifications had minimal impact on fragmentation, supporting their use for optimizing peptide function while preserving analytical and structural consistency.
Results
KR-12-a5 derivatives containing D-amino acid substitutions (positions 5/6) and N-terminal 5(6)-FAM labels with varying linker lengths were successfully synthesized and analyzed by MS/MS. Doubly and triply protonated peptides showed extensive and consistent fragmentation, while quadruply charged species fragmented at more specific sites, predominantly yielding low- to medium-intensity y-ions. Strong fragmentation was consistently observed N-terminal to Trp (W), producing corresponding y-ions. The incorporation of a single D-amino acid produced no significant change in fragmentation patterns. Similarly, FAM labeling with different linker lengths showed comparable fragmentation behavior, although Tag-b1 ions were observed. Lower charge states resulted in broader fragmentation with diverse b- and y-ion series. Overall, both D-amino acid substitution and FAM/linker modifications had minimal impact on peptide fragmentation.
Significance
This work is important because it explores how chemical modifications can optimize KR-12-a5 for therapeutic use without compromising its structural and analytical properties. It contributes to peptide drug design, improves understanding of structure–function relationships, and may support the development of more effective antimicrobial agents against biofilm-related infections.
Location
University of the Pacific, DeRosa University Center
Start Date
24-4-2026 11:00 AM
End Date
24-4-2026 2:00 PM
Mass Spectrometry Studies of Anti-Biofilm Peptides
University of the Pacific, DeRosa University Center
KR-12-a5, an engineered peptide derived from the human cathelicidin LL-37, exhibits potent antimicrobial and anti-biofilm activity against oral pathogens linked to endodontic infections. Notably, incorporating D-amino acids into KR-12-a5 has been demonstrated to significantly enhance cell selectivity while preserving its potent anti-inflammatory activity. So, to optimize the properties of KR-12-a5, we synthesized a set of derivatives by incorporating a D-amino acid at the 5/6 positions or attaching an N-terminal 5(6)-FAM label through linkers of varying lengths. This approach enabled us to identify the optimal spacer that best preserved the native peptide structure while minimizing functional deviations from the untagged form. These optimized peptides will be used to assess their antimicrobial potency against biofilms, and we will investigate whether the D-amino acid substitution and the 5(6)-FAM/linker architecture influence peptide fragmentation patterns in mass spectrometry. The doubly and triply protonated peptides underwent extensive and consistent fragmentation. Peptides with four charges fragmented at specific positions, predominantly producing low- to medium-intensity y-ions. Strong fragmentation was observed on the N-terminal side of Trp (W), generating corresponding y-ions. The presence of a single D-amino acid had minimal impact on fragmentation. Fluorescent tags with varying linker lengths exhibited similar effects on fragmentation; however, they promoted the formation of Tag-b1 ions. Lowly charged peptides fragmented more extensively, producing a wide range of b- and y-ions. Overall, the single D-amino acid residue exerted minimal influence on the fragmentation pattern, and the FAM tag and linker had comparable yet negligible effects on peptide fragmentation.
