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

Kawasue, Shimba; Sakaguchi, Yohei; Koga, Ritsuko; Hayama, Tadashi; Yoshida, Hideyuki; Nohta, Hitoshi

doi:10.1016/j.ab.2021.114247

Cited by 6 publications

(4 citation statements)

References 37 publications

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“…Indeed, concerning all articles devoted to the development of analytical methods to characterize phosphopeptides and phosphosites, CASK is never used as a phosphoprotein model. Until recently, a new chemical dephosphorylation/Michael addition-based method leading to the conversion, from multiple phosphorylated peptides of multiple phosphate groups into perfluoroalkyl groups, has been reported for the identification and the quantification of mono-and multi-phosphorylated peptides by a fluorous HPLC column coupled to mass spectrometry [32]. Although the non-coelution of peptides and fluorous-derivatized peptides is clearly evidenced, unfortunately this article does not discuss the monoisotopic profile of fluorous-derivatized peptides and does not report the MS/MS spectra of fluorous-derivatized peptides.…”

Section: Cpps and Phosphositesmentioning

confidence: 99%

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

Deracinois

Matéos

Romelard

et al. 2021

Foods

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The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

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Section: Cpps and Phosphositesmentioning

confidence: 99%

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

Deracinois

Matéos

Romelard

et al. 2021

Foods

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“…On the contrary, a novel derivatization method has been developed using the specific fluorous affinity − between perfluoroalkyl-containing compounds to eliminate matrix-induced effects in LC-MS/MS analysis. − Because the fluorous derivatives of the target analytes obtained with perfluoroalkylated reagents are selectively retained and separated using a perfluoroalkyl-modified stationary phase LC column, they can be clearly distinguished from endogenous components that cause matrix-induced effects. Furthermore, we previously developed a selective extraction method for phosphate-containing compounds using a simple liquid–liquid extraction system using a fluorous solvent, such as perfluoroalkanes. − Combining this method with metal chelate affinity techniques enables the selective extraction of nonfluorous phosphate compounds. − Briefly, the immobilized metal ion with perfluoroalkyliminodiacetic acid as the perfluoroalkylated chelating reagent was coordinated to negatively charged phosphate groups and the resulting fluorous chelate complex was then extracted into a fluorous solvent from a nonfluorous solution.…”

Section: Introductionmentioning

confidence: 99%

Accurate LC-MS/MS Analysis of Diacylglycerols in Human Plasma with Eliminating Matrix Effect by Phospholipids Using Fluorous Biphasic Extraction

Nishijo,

Hayama,

Tomita

et al. 2023

Anal. Chem.

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We developed an accurate method for determining diacylglycerols (DAGs) in human plasma using a fluorous biphasic liquid–liquid extraction method, followed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The lipid mixture in the plasma was first extracted with chloroform by using the Bligh–Dyer method. The resulting solution was subjected to fluorous biphasic liquid–liquid extraction to remove phospholipids, which are known to cause matrix effects during the LC-MS/MS analysis. In this method, phospholipids in a lipid mixture solution (nonfluorous solvent) were selectively extracted to tetradecafluorohexane (fluorous solvent) via the specificity of fluorous affinity by forming a complex with a perfluoropolyethercarboxylic acid-lanthanum(III) salt. The remaining DAGs in the nonfluorous solvent could be directly injected into the LC system through the positive electrospray ionization-MS/MS mode. The removal rate of the phospholipids through the fluorous biphasic extraction was more than 99.9%; thus, the matrix-effect-eliminating analysis of DAGs in human plasma with LC-MS/MS was enabled. Furthermore, the applicability of this method and the possibility of using DAGs as biomarkers were evaluated by applying this method to human plasma samples obtained from major depressive disorder as a related disease.

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“…20) In recent years, fluorous derivatization LC-MS methods almost unaffected by matrix components, such as biological samples and food samples, have been reported. [21][22][23][24][25][26] The derivatization method utilizes the specific affinity of perfluoroalkyl groups. Therefore, selective analysis with a reduced background is possible by derivatizing the analytes with fluorous reagents and separating them on a fluorous LC column.…”

Section: Introductionmentioning

confidence: 99%

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

Kawasue

Sakaguchi

Koga

et al. 2022

CHEMICAL & PHARMACEUTICAL BULLETIN

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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 (α S1 -casein and β-casein) extracted from baked sugar cookies were tryptic digested, the obtained phosphorylated peptides were selectively derivatized by β-elimination with Ba(NO 3 ) 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 α S1 -casein) and FQ(pSer)EEQQQTEDELQDK (33-48 fragment from β-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 α S1 -casein and β-casein, the obtained quantification values were in good agreement with the arbitrary amounts spiked. The quantification limits of α S1 -and β-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.

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

Cited by 6 publications

References 37 publications

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

Accurate LC-MS/MS Analysis of Diacylglycerols in Human Plasma with Eliminating Matrix Effect by Phospholipids Using Fluorous Biphasic Extraction

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

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