Towards mapping electrostatic interactions between Kdo₂-lipid A and cationic antimicrobial peptides via ultraviolet photodissociation mass spectrometry

Abstract

Cationic antimicrobial peptides (CAMPs) have been known to act as multi-modal weapons against Gram-negative bacteria. As a new approach to investigate the nature of the interactions between CAMPs and the surfaces of bacteria, native mass spectrometry and two MS/MS strategies (ultraviolet photodissociation (UVPD) and higher energy collisional activation (HCD)) are used to examine formation and disassembly of saccharolipid·peptide complexes. Kdo2-lipid A (KLA) is used as a model saccharolipid to evaluate complexation with a series of cationic peptides (melittin and three analogs). Collisional activation of the KLA·peptide complexes results in the disruption of electrostatic interactions, resulting in apo-sequence ions with shifts in the distribution of ions compared to the fragmentation patterns of the apo-peptides. UVPD of the KLA·peptide complexes results in both apo- and holo-sequence ions of the peptides, the latter in which the KLA remains bound to the truncated peptide fragment despite cleavage of a covalent bond of the peptide backbone. Mapping both the N- and C-terminal holo-product ions gives insight into the peptide motifs (specifically an electropositive KRKR segment and a proline residue) that are responsible for mediating the electrostatic interactions between the cationic peptides and saccharolipid.

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae

“…We used native mass spectrometry (32-36) coupled with UV photodissociation (UVPD) (37-42) to better understand how these peptides differ in their polysaccharide interactions. UVPD of noncovalent peptide•ligand complexes produces sequence ions that retain the ligand, termed holo ions, as well as ligand-free sequence ions termed apo ions (43). The resulting fragmentation patterns can be used to determine binding sites, essentially localizing residues or regions that interact with the ligand (37-43).…”

What Are We Missing by Not Measuring the Net Charge of Proteins?

“…The systematic absence of experimentally determined values of Z has likely impeded a rigorous understanding of most chemical processes in which proteins are involved including aggregation and self‐assembly, ligand binding, catalysis, electron transfer, protein crystallization, analytical separation, and protein engineering . It is tempting to assume that the formal net charge of a protein predicted from generalized residue p K a values ( Z seq ) is so similar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about a protein's net charge.…”

“…[1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering. [51][52][53][54][55][56] It is tempting to assume that the formal net chargeo faprotein predicted from generalized residue pK a values (Z seq )issosimilar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about ap rotein's net charge.…”

Polyproline peptide targets Klebsiella pneumoniae polysaccharides to collapse biofilms

“…UVPD of the peptide · polysaccharide complexes produces apo fragment ions (conventional sequence-type ions from the peptide) as well as holo fragment ions containing a portion of the peptide plus the bound polysaccharide. 50 Analyzing the holo-ions and ligand-free apo-ions allows identification of the key amino acids that may interact with polysaccharides and facilitates the assessment of the structural changes that accompany the formation or disruption of non-covalent interactions. 47 – 54 In essence, interaction of the polysaccharide with the peptide results in non-covalent interactions that may either stabilize regions of the peptide and suppress release of fragment ions upon UVPD or enhance the production of other fragment ions not favored for the apo peptide.…”

“…High-resolution measurements for the peptide-oligosaccharide complexes were generated using a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer coupled to a Coherent 193 nm excimer laser for UVPD in the high-pressure linear ion trap. 50 , 80 A Au/Pd-coated pulled static emitter was loaded with each stachyose peptide solution and individually infused into the mass spectrometer. The peptide · stachyose complexes (bac7 (1–35) in 5+ charge state and bac7 (10–35) in the 4+ charge state) were isolated and subjected to UVPD (1 laser pulse at 3 mJ).…”