Direct determination of naturally occurring biologically active compound–serum albumin conjugate by matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

Opium alkaloids were conjugated with bovine serum albumin (BSA) to give individual antigen conjugates which were analyzed using BSA as an internal standard by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It became clear that 9 molecules of thebaine were conjugated in a thebaine–BSA conjugate. Codeine and morphine contents in individual conjugates were determined to be 12 and 6 molecules, respectively. Using a relative molecular mass of BSA, 11.5 molecules of forskolin conjugated with BSA. Tetrahydrocannabinolic acid (THCA)–carrier protein conjugates usingβ-alanine or withoutβ-alanine as a linker were analyzed. From these results it became evident that the hapten molecules of THCA–β-alanine–BSA, THCA–BSA, THCA–β-alanine–human serum albumin (HSA) and THCA–HSA were 17.2, 12.7, 27.8 and 54.4, respectively. Crocin–HSA conjugates were synthesized by two methods using succinate and NaIO4cleavage of sugar moiety. A broad molecular peak of crocin–HSA conjugate appeared at aroundm/z98,342 resulting in 30 molecules of crocin-hemisuccinate conjugated with HSA. The crocin–HSA synthesized via NaIO4oxidation showed a sharp molecular peak around 69,504 compared to that of hemisuccinate preparation. The number of crocin combined with HSA was, however, smaller 3.1 molecules.Analysis of monoclonal antibody by MALDI mass spectrometry determined the exact molecular weight and their purities.

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites

“…In addition to the method to prepare hapten-carrier protein conjugates, the number of hapten molecules bound to carrier proteins affects the specificity of antibodies. The hapten numbers are typically evaluated via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) using sinapinic acid as the matrix [ 41 ]. The relationship between the number of hapten molecules and antibody specificity has been investigated using a mercaptopropionic acid derivative of atrazine: high antibody titers with moderate antibody specificity are induced from 15–30 hapten molecules per carrier protein, while a lower number of hapten molecules exhibits a slower immune response with higher specificity [ 42 ].…”

A Quantitative ELISA Using Monoclonal Antibody to Survey Paeoniilorin and Albiflorin in Crude Drugs and Traditional Chinese Herbal Medicines

“…Determination of Hapten Number in PF-Carrier Protein Conjugates by Matrix-Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) Mass Spectrometry As an effective method routinely employed in our laboratory, 22) a small amount of (1-10 pmol) hapten-conjugate was mixed with a 1000-fold molar excess aqueous solution of saturated sinapinic acid containing about 30% acetonitrile in 0.15% trifluoroacetic acid. The mixture (0.5-1.0 ml) was deposited on the gold-coated sample plate and air-dried.…”

Precision Localization of Lipid‐Based Nanoparticles by Dual‐Fluorescent Labeling for Accurate and High‐Resolution Imaging in Living Cells

“…The mass spectral analysis of the singly charged species [M + H] + , doubly charged species [M + 2 H] 2+ , and triply charged species [M + 3 H] 3+ of pure BSA was present at 66.6, 33.3, and 22.1 kDa, respectively (Figure 1d), which is consistent with earlier observations. [ 26 ] Intriguingly, at this detection level, BSA fragment ions were absent in elution volumes 3–5 mL (fractions 6–10, 0.5 mL per fraction) and began to appear in volumes 5.5–10 mL (Figure 1d, S3a, Supporting Information), with volume of 7 to 8 mL exhibiting the highest abundance. Although this method cannot be used to determine quantitatively the amount of BSA involved in label dissociation, the mass distribution of BSA in elution volumes did match with the second peak of the SEC elution profile of FLPs (Figure S3b, Supporting Information).…”