LL-37: A Cationic Helix and What It Does to the Chromatography
LL-37 is a 37-residue cationic peptide, and almost every difficulty in handling and analysing it follows from two facts: it carries a large net positive charge, and it folds into a helix whose degree of folding depends on what it is dissolved in.
Sequence and origin
LL-37 is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, the C-terminal 37 residues of the human cathelicidin precursor protein hCAP18. The name comes from the two leucines it begins with and its length. It is C205H340N60O53, average mass approximately 4493.3 Da, CAS 154947-66-7.
It is a defined fragment of a human protein rather than a designed sequence, which is why the residue numbering in the literature sometimes refers to positions within hCAP18 instead of within the fragment. A paper describing residue 33 may mean the thirty-third residue of LL-37 or a position in the precursor.
A strongly cationic peptide
The sequence contains five arginines and six lysines against two aspartates and three glutamates. Net charge at neutral pH is around plus six.
On a silica-based reversed-phase column this produces severe peak tailing unless an ion-pairing agent is present, because the protonated guanidinium and ammonium groups bind residual silanols. Trifluoroacetic acid suppresses that interaction and is standard for this peptide. Switching to formic acid, as mass-spectrometry-friendly methods often do, usually degrades the peak shape noticeably.
The same charge makes the peptide adsorb to glass and to some plastics from dilute solution. Low concentrations lose material to container walls, which shows up as poor recovery rather than as a visible problem.
No tryptophan, no tyrosine
LL-37 contains four phenylalanines and no tryptophan or tyrosine. Phenylalanine absorbs weakly near 257 nm and contributes almost nothing at 280 nm, so as with other aromatic-poor sequences, detection is at 214 nm.
The practical consequence is the same one that applies to any 214 nm method: the purity figure counts every peptide-bond-containing species in the sample, including truncated fragments that would be invisible at 280 nm. That makes a 214 nm area percentage more conservative and more informative than a 280 nm figure on the same material.
Conformation depends on the solvent
In plain water at low ionic strength, LL-37 is largely unstructured. In the presence of salts, in buffers at physiological ionic strength, or in the presence of anionic surfaces, it adopts an amphipathic alpha-helix in which the hydrophobic and charged residues segregate onto opposite faces.
This is not a curiosity. Helical content affects aggregation behaviour, adsorption and retention, which means the same lot can behave differently in two solvents. Circular dichroism is the measurement that reports helicity, and it is occasionally included on characterisation documents for this peptide where it would be pointless for a small linear one.
Aggregation and solubility
The amphipathic arrangement that produces the helix also drives self-association at higher concentrations. Concentrated stock solutions can become turbid or form oligomers that pass a visual check but change the chromatogram, usually by broadening the main peak or producing an early-eluting shoulder.
Dissolving in slightly acidic water before dilution into a buffer generally gives better results than dissolving directly into buffer, because the peptide is more soluble and less helical at low pH.
Degradation routes
There is no cysteine and no methionine, so the usual oxidation targets are absent. The vulnerabilities are elsewhere: asparagine at position 30 can deamidate, the two aspartates can cyclise to a succinimide and isomerise to isoaspartate, and the many arginines make the peptide a ready substrate for trypsin-like contamination if the sample is not clean. Each of these produces a small mass shift, one dalton or less for deamidation and zero for isomerisation, which means chromatography rather than mass is what detects them.
Mass spectrometry at 4493 daltons
A peptide this basic ionises readily and produces a broad charge envelope, commonly from four to eight protons. The five-plus ion appears near m/z 899.7 and the six-plus near m/z 749.9. Deconvolution to a neutral mass is standard, and the reported figure should be compared against the calculated average mass rather than the monoisotopic one, because at this size the monoisotopic peak is a minor component of the isotope cluster.
What confirms a lot
A deconvoluted average mass near 4493.3 Da, a 214 nm chromatogram run with trifluoroacetic acid as the ion-pairing additive, and an acknowledgement of which solvent the sample was prepared in. Purity measured on an aggregated sample is not purity; it is a measurement of the aggregate.
Related reading
The behaviours described here follow from charge and from conformation rather than from anything specific to this sequence. The general cases are set out in isoelectric point and solubility and in peptide aggregation and solution behaviour.
