Quantification of pharmaceutical peptides using selenium as an elemental detection label

Abstract

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.

Investigation of Elemental Mass Spectrometry in Pharmacology for Peptide Quantitation at Femtomolar Levels

“…That is why synthetic strategies based on the substitution of a proteinogenic sulfur-containing amino acid (cysteine or methionine) with its selenium analogue (selenocysteine or selenomethionine) are readily accessible. For instance, Gammelgaard and collaborators introduced a selenomethionine in place of methionine to follow by ICP-MS the cellular uptake of such modified peptide [ 9 ]. In another context, substitution of cysteine with selenocyteine in peptide is widely used, most notably by Alewood and co-workers [ 20 ] mainly to replace disulfide bridge by a diselenide bond engineered to better control the oxidative peptide cyclization and thereafter improve its proteolytic stability.…”

“…According to the calculations below, the LOD and LOQ were estimated from the mean values of 3 calibration curves to be 17 ng Se L -1 (22 femtomoles of injected Se) and 57 ng Se L -1 (72 femtomoles of injected Se), respectively: with a representing the slope and δ(b) the standard deviation estimated on 10 blanks. As a matter of fact, an LOD of 400 picomoles of injected Se was depicted for selenium-containing peptide quantitation using macrobore RP-LC-ICP-MS instrument equipped with a membrane desolvator system [ 9 ]. The described experimental set-up and methodology proved to be more sensitive with an LOD of about 20 femtomoles of injected Se.…”

“…It means that it can be considered as a relevant quantification probe as demonstrated by Gammelgaard et al . to estimate the cellular up-take of a cell-penetrating peptide [ 9 ]. Secondly, in contrast to metals which required chelates to be associated with bioactive compounds, this element can be covalently bound to the targeted biomarker (peptide/small molecule) according to conventional organic chemistry procedures.…”

Evaluation of a membrane desolvator for LC-ICP-MS analysis of selenium and platinum species for application to peptides and proteins

“…The column recovery for the selenopeptide was 101.5% AE 1.1% (n ¼ 3). The system was applied for cell uptake studies of a synthetic Se containing peptide 15 and the chromatogram shows examples of analysis of cell lysate containing the intact peptide and cell medium containing intact peptide as well as some degradation products eluting around 2 min. The content of the intact peptide in the lysate corresponded to 14 mg L À1 and the content of the cell medium corresponded to 406 mg L À1 , but it was not possible to quantify the degradation products due to lack of an authentic standard.…”

“…14 ), selenomethionine (SeMet) (Sigma-Aldrich, St. Louis, Missouri, USA), Se-methylselenocysteine (Se-MeSeCys) (Sigma-Aldrich), Se-methylselenogalactosamine (SeGalac) (synthesized at University of Copenhagen), Se-containing peptide (RQIKIWFQNRR(M Se )KWKK-NH 2 , PenM Se ) (prepared according to Møller et al15 ), sodium selenite pentahydrate (Merck, Whitehouse Station, New Jersey, USA) and sodium selenate (Fluka, Buchs, Switzerland) were prepared in Milli-Q water. Stock solutions of cisplatin (Sigma-Aldrich) and Human Serum Albumin (Sigma-Aldrich) for chromatography were prepared in 10 mM phosphate buffered saline, pH 7.4, (Sigma-Aldrich) added $150 mM NaCl (Merck).…”