Quantification of Casein in Baked Food Products by Selective Analysis of Phosphorylated Peptides Using Fluorous Derivatization with Liquid Chromatography-Tandem Mass Spectrometry Method.

Casein is one of the allergen proteins present in milk. Therefore, a quantification method for the selective analysis of casein using fluorous derivatization with LC-tandem mass spectrometry (LC-MS/MS) was developed. After two allergen proteins (αS1-casein and ß-casein) extracted from baked sugar cookies were tryptic digested, the obtained phosphorylated peptides were selectively derivatized by ß-elimination with Ba(NO3)2 under basic condition and Michael addition with perfluoroalkylthiol (1H,1H,2H,2H-perfluorooctanethiol, PFOT). In this study, YKVPQLEIVPN(pSer)AQQR (104-119 fragment from αS1-casein) and FQ(pSer)EEQQQTEDELQDK (33-48 fragment from ß-casein) obtained by tryptic digestion were selected as target peptides. The phosphorylated serine residue in each peptide was converted to a perfluoroalkyl group by derivatization. The obtained fluorous-derivatized peptides were analyzed by LC-MS/MS, to which a fluorous LC column was connected. Therefore, it was possible to analyze casein without being affected by the matrix components in the baked food sample. When the present method was applied to cookies with arbitrary amounts of αS1-casein and ß-casein, the obtained quantification values were in good agreement with the arbitrary amounts spiked. The quantification limits of αS1- and ß-casein in cookie analysis were 246 and 152 ng/g, respectively. Hence, this method can be used to analyze trace amounts of allergen proteins present in the baked food.

Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis.

Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested ß-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via ß-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to ß-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

A Pyridinium Derivatization Reagent for Highly Sensitive Detection of Poly(carboxylic acid)s Using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

Short-chain fatty acids are difficult to analyze with high sensitivity using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) owing to the high polarity of their carboxyl groups. Various derivatization methods have been developed; however, most are effective only for monocarboxylic acids and not for those having multiple carboxyl groups. Therefore, we successfully attempted to synthesize a derivatization reagent that could analyze both mono- and poly(carboxylic acid)s with high sensitivity. We optimized our derivatization reagent by modifying the structure of the reaction site, hydrophobicity of the derivatized compound, and linker structure connecting the reaction site to the permanently charged substructure. The reactivity toward carboxyl groups was improved by employing a piperidine moiety as the reaction site, and the ESI efficiency was improved by the highly hydrophobic and permanently charged triphenylpyridinium group. Furthermore, the incorporation of an alkyl linker enabled polylabeling. When the optimized reagent was applied to mono-, di-, tri-, and tetracarboxylic acids, the ESI efficiency increased with polylabeling; thus, our derivatization reagent outperforms existing derivatization methods and enables the analysis of poly(carboxylic acid)s with high sensitivity. Since this derivatization reagent can be applied to most carboxyl-containing compounds, it can be widely used for lipidomics, proteomics, and metabolomics.

Selective analysis of the okadaic acid group in shellfish samples using fluorous derivatization coupled with liquid chromatography-tandem mass spectrometry.

Okadaic acid (OA) group are diarrheal shellfish poison that accumulates in the midgut glands of shellfish. It is difficult to remove these poisons by normal cooking because they are thermally stable and hydrophobicity. Therefore, in order to prevent foodborne disease due to shellfish poison, analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) before shipment is necessary. Herein the selective analytical method for OA group in shellfish sample using fluorous derivatization coupled with LC-MS/MS was developed. OA group were derivatized with the fluorous alkylamine reagent by condensing agent, and the obtained derivatives were separated with fluorous LC column (Fluofix-II 120E, 250 × 2.0 mm i.d., 5 µm, Fujifilm Wako Pure Chemical). The derivatized OA group were selective retained by fluorous LC column and accurate analysis was enabled. The present method was applied to the analysis of OA and dinophysistoxin-1 (DTX-1) in scallop midgut gland which is the certified reference material provided by national metrology institute of Japan. As a result of analysis using the present method with DTX-2 as the internal standard, the quantitative value were in agreement with the certified value.

Assessment method for deamidation in proteins using carboxylic acid derivatization-liquid chromatography-tandem mass spectrometry.

An analytical method for the degree of protein deamidation has been developed by using carboxy group derivatization and liquid chromatography-tandem mass spectrometry (LCMS/MS). The fragment peptides (LGEYGFQNALIVR and YNGVFQECCQAEDK) obtained by digesting bovine serum albumin (BSA) with trypsin and their asparagine deamidated peptides (LGEYGFQDALIVR and YDGVFQECCQAEDK) were selected as model peptides, and their carboxy groups were derivatized with ethylamine. This derivatization enabled a clear distinction between natural peptides and deamidated peptides by mass, allowing for facile distinction by LCMS/MS before and after deamidation. Good linearity was confirmed for four peptides used in this study via isotope dilution mass spectrometry, showing that protein deamidation can be evaluated by the present method. To confirm the validity of this method for the evaluation of deamidation, natural peptides and deamidated peptides were mixed in arbitrary ratios, and degree of deamidation in these solution was analyzed. This confirmed that accurate evaluation was possible at deamidation degree values of ca. 10 %, 5 %, 2.5 %, and 1 %. Additionally, an accelerated storage test of BSA demonstrated that the deamidation of asparagine at position 404 of BSA progressed by 4 % in 9 weeks at 40 °C and pH 8 in the dark, and that the deamidation process can be traced over time.