Quantitation of a 36-amino-acid peptide inhibitor of HIV-1 membrane fusion in animal and human plasma using high-performance liquid chromatography and fluorescence detection

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

“…Conventional peptide quantification is performed by, e.g., liquid chromatography combined with fluorescence or ultraviolet (UV) detection [31,32,33], capillary electrophoresis combined with UV detection [34,35,36], immunostaining when monitoring peptide distribution in tissues or on cell surfaces [37,38], or, most often, by immunoassays [7,39,40,41,42,43,44,45]. More recently developed methods such as quantitative matrix-assisted laser-desorption/ ionization-mass spectrometric (MALDI-MS) approaches [46,47,48,49,50] or surface-enhanced laser desorption/ionization (SELDI) technologies [16,51,52] have been discussed in the literature.…”

“…Adsorption by pipette tips, reaction vials, test and storage tubes, charging devices used in instrumental analysis, tubing and flexible tube connections, extrusion and injection syringes or by chromatographic columns reduces the analyte concentration. It has occasionally been shown to be advantageous to add proteins [10,68,69,70,71], detergents [32,67], or structurally related analogs [72] to the sample solutions to avoid or minimize loss of analyte. In our laboratory we designed a simple quantitative experiment consisting of seven consecutive steps for transfer of a peptide solution from one polypropylene reaction vial (0.5 mL) to another followed by LC-MS quantification.…”

“…These solvents can be cooled to less than 4°C to avoid thermal stress of the analyte [31,44,75,76,77,78]. The time required for precipitation varies from less than 1 min [32,48,67,77] to 1 h [31,44,75,76]. After precipitation, the samples are usually centrifuged between 2000 and 15,000 g and the supernatant is further processed by evaporation and reconstitution before LC-MS analysis.…”

“…Recovery therefore ranges from, for example, 50% for the 36-amino acid peptide T-20, an HIV fusion inhibitor [32], to 95% for the tripeptide glutathione (GSH), a physiological antioxidant [31]. In our laboratory, for instance, we determined the recovery of our model peptide MP1 (MW 2304 Da) after precipitation of spiked plasma samples with increasing volumes of ACN (Fig.…”

Sample preparation and fractionation for proteome analysis and cancer biomarker discovery by mass spectrometry

“…Precipitation in acetone or combination of TCA/acetone also removed lipids efficiently, although lipids' elimination was more effective when they were associated with proteins in membranes [13]. Precipitation employing ACN supplemented with 1% trifluoroacetic acid (TFA) and 1% n-nonyl-b-D-glucopyranoside was helpful in dissolving membrane proteins and lipids [45].…”