Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

<div><p>Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca²⁺-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only <i>in vitro</i>, i.e., for serum or plasma stability assays, but of lower importance <i>in vivo</i>.</p></div

Degradation of oncocin derivatives.

<p>Onc18, Onc72, and Onc112 were analyzed after one hour incubation in blood (dark red), direct serum (red), activated serum (orange), commercial serum (yellow), heparin plasma (dark blue), and EDTA plasma (blue). Peptides were separated by RP-HPLC in the presence of 0.1% trifluoroacetic acid and detected by absorbance at 214 nm. Peptide amounts were calculated relative to the quantities determined at time point zero.</p

Degradation of elongated Onc112 derivatives.

<p>AAYR-Onc112 (left) and LVPR-Onc112 (right) were analyzed after one hour incubation in blood (dark red), direct serum (red), activated serum (orange), commercial serum (yellow), heparin plasma (dark blue), and EDTA plasma (blue). Peptides were separated by RP-HPLC in the presence of 0.1% formic acid and detected by absorbance at 214 nm. Peptide amounts were calculated relative to the quantities determined at time point zero. Onc112 (white) was released from both constructs, whereas metabolites YR-Onc112 (squared) and R-Onc112 (striped) were detected only for AAYR-Onc112.</p

Stability of eight peptides in fresh blood, serum, and plasma.

<p>Stability of eight peptides in fresh blood, serum, and plasma.</p

Fibrinopeptide A and its degradation products.

<p>Peak areas obtained from extracted ion chromatograms of triply protonated murine fibrinopeptide A (DTEDKGEFLSEGGGVR, purple; <i>m/z</i> 565.9) and its triply protonated degradation products TEDKGEFLSEGGGVR (green; <i>m/z</i> 527.6) and EDKGEFLSEGGGVR (white; <i>m/z</i> 493.9). Six to nine samples were analyzed for each matrix (blood, B; direct serum, DS; activated serum, AS) and time point.</p

Degradation of apidaecin derivatives.

<p>Peptides in blood (dark red), direct serum (red), activated serum (orange), commercial serum (yellow), heparin plasma (dark blue), and EDTA plasma (blue) were analyzed after 10 min (Api88) and one hour (Api134 and Api137) incubation. Separation was performed by RP-HPLC in the presence of 0.1% trifluoroacetic acid and detected by absorbance at 214 nm. Peptide amounts were calculated relative to the quantities determined at time point zero.</p

Sequences of all degradation products identified in blood, serum, and plasma samples.

<p>Sequences of all degradation products identified in blood, serum, and plasma samples.</p

Simultaneous Detection of Low and High Molecular Weight Carbonylated Compounds Derived from Lipid Peroxidation by Electrospray Ionization-Tandem Mass Spectrometry

Reactive oxygen species (ROS) and other oxidative agents such as free radicals can oxidize polyunsaturated fatty acids (PUFA) as well as PUFA in lipids. The oxidation products can undergo consecutive reactions including oxidative cleavages to yield a chemically diverse group of products, such as lipid peroxidation products (LPP). Among them are aldehydes and ketones (“reactive carbonyls”) that are strong electrophiles and thus can readily react with nucleophilic side chains of proteins, which can alter the protein structure, function, cellular distribution, and antigenicity. Here, we report a novel technique to specifically derivatize both low molecular and high molecular weight carbonylated LPP with 7-(diethylamino)­coumarin-3-carbohydrazide (CHH) and analyze all compounds by electrospray ionization-mass spectrometry (ESI-MS) in positive ion mode. CHH-derivatized compounds were identified by specific neutral losses or fragment ions. The fragment ion spectra displayed additional signals that allowed unambiguous identification of the lipid, fatty acids, cleavage sites, and oxidative modifications. Oxidation of docosahexaenoic (DHA, 22:6), arachidonic (AA, 20:4), linoleic (LA, 18:2), and oleic acids (OA, 18:1) yielded 69 aliphatic carbonyls, whose structures were all deduced from the tandem mass spectra. When four phosphatidylcholine (PC) vesicles containing the aforementioned unsaturated fatty acids were oxidized, we were able to deduce the structures of 122 carbonylated compounds from the tandem mass spectra of a single shotgun analysis acquired within 15 min. The high sensitivity (LOD ∼ 1 nmol/L for 4-hydroxy-2-nonenal, HNE) and a linear range of more than 3 orders of magnitude (10 nmol/L to 10 μmol/L for HNE) will allow further studies on complex biological samples including plasma

Sequences and monoisotopic masses of all studied peptides.

<p>Sequences and monoisotopic masses of all studied peptides.</p

Carbonylated Plasma Proteins As Potential Biomarkers of Obesity Induced Type 2 Diabetes Mellitus

Protein carbonylation is a common nonenzymatic oxidative post-translational modification, which is often considered as biomarker of oxidative stress. Recent evidence links protein carbonylation also to obesity and type 2 diabetes mellitus (T2DM), though the protein targets of carbonylation in human plasma have not been identified. In this study, we profiled carbonylated proteins in plasma samples obtained from lean individuals and obese patients with or without T2DM. The plasma samples were digested with trypsin, carbonyl groups were derivatized with O-(biotinylcarbazoylmethyl)­hydroxylamine, enriched by avidin affinity chromatography, and analyzed by RPC-MS/MS. Signals of potentially modified peptides were targeted in a second LC-MS/MS analysis to retrieve the peptide sequence and the modified residues. A total of 158 unique carbonylated proteins were identified, of which 52 were detected in plasma samples of all three groups. Interestingly, 36 carbonylated proteins were detected only in obese patients with T2DM, whereas 18 were detected in both nondiabetic groups. The carbonylated proteins originated mostly from liver, plasma, platelet, and endothelium. Functionally, they were mainly involved in cell adhesion, signaling, angiogenesis, and cytoskeletal remodeling. Among the identified carbonylated proteins were several candidates, such as VEGFR-2, MMP-1, argin, MKK4, and compliment C5, already connected before to diabetes, obesity and metabolic diseases