TY - JOUR
T1 - Quantification of pharmaceutical peptides using selenium as an elemental detection label
AU - Møller, Laura Hyrup
AU - Gabel-Jensen, Charlotte
AU - Franzyk, Henrik
AU - Bahnsen, Jesper Søborg
AU - Stürup, Stefan
AU - Gammelgaard, Bente
PY - 2014/9
Y1 - 2014/9
N2 - The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating peptide, penetratin, were synthesized, one with selenomethionine (SeMet) added at the N-terminal of the peptide (N-PenM(Se)) and the other with the internal methionine (Met) replaced with SeMet (i-PenM(Se)). The purity of the synthesized peptides was 92% for N-PenM(Se) and 89% for i-PenM(Se) as determined by liquid chromatography (LC)-ICP-MS. The selenium-labelled peptides were investigated by cell uptake studies in HeLa WT cells. The stability of the peptides was monitored in water, cell medium and during cell uptake studies. Total uptake of selenium was quantified by flow injection (FI)-ICP-MS. Speciation analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation was observed during cell uptake studies. The major degradation products were determined by LC-electrospray ionization mass spectrometry (ESI-MS). The labelling method in combination with FI-ICP-MS, LC-ICP-MS and LC-ESI-MS techniques provided detailed information on the fate of penetratin in cellular uptake studies. Most pharmaceutical peptides, including penetratin, are synthetic analogues of endogenous peptides, and incorporation of selenium may improve the critical assessment of the native drug or drug delivery candidate early in the drug development process.
AB - The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating peptide, penetratin, were synthesized, one with selenomethionine (SeMet) added at the N-terminal of the peptide (N-PenM(Se)) and the other with the internal methionine (Met) replaced with SeMet (i-PenM(Se)). The purity of the synthesized peptides was 92% for N-PenM(Se) and 89% for i-PenM(Se) as determined by liquid chromatography (LC)-ICP-MS. The selenium-labelled peptides were investigated by cell uptake studies in HeLa WT cells. The stability of the peptides was monitored in water, cell medium and during cell uptake studies. Total uptake of selenium was quantified by flow injection (FI)-ICP-MS. Speciation analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation was observed during cell uptake studies. The major degradation products were determined by LC-electrospray ionization mass spectrometry (ESI-MS). The labelling method in combination with FI-ICP-MS, LC-ICP-MS and LC-ESI-MS techniques provided detailed information on the fate of penetratin in cellular uptake studies. Most pharmaceutical peptides, including penetratin, are synthetic analogues of endogenous peptides, and incorporation of selenium may improve the critical assessment of the native drug or drug delivery candidate early in the drug development process.
U2 - 10.1039/c4mt00085d
DO - 10.1039/c4mt00085d
M3 - Journal article
C2 - 25027387
SN - 1756-5901
VL - 6
SP - 1639
EP - 1647
JO - Metallomics : integrated biometal science
JF - Metallomics : integrated biometal science
ER -