Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

Wen Chu Yang; Hamid Mirzaei; Xiuping Liu; Fred E. Regnier

doi:10.1021/ac0600510

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

Wen Chu Yang, Hamid Mirzaei, Xiuping Liu, Fred E. Regnier

Research output: Contribution to journal › Article › peer-review

119 Scopus citations

Abstract

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (C _n-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the C_n-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of C_n-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C₃-NA and C₄-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C₄-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C ₄-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the C_n-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the C_n-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.

Original language	English (US)
Pages (from-to)	4702-4708
Number of pages	7
Journal	Analytical Chemistry
Volume	78
Issue number	13
DOIs	https://doi.org/10.1021/ac0600510
State	Published - Jul 1 2006

ASJC Scopus subject areas

Analytical Chemistry

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10.1021/ac0600510

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@article{47d7418eff824f829f323c52d5e51a44,

title = "Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry",

abstract = "A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (C n-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C 4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.",

author = "Yang, {Wen Chu} and Hamid Mirzaei and Xiuping Liu and Regnier, {Fred E.}",

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T1 - Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

AU - Yang, Wen Chu

AU - Mirzaei, Hamid

AU - Liu, Xiuping

AU - Regnier, Fred E.

PY - 2006/7/1

Y1 - 2006/7/1

N2 - A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (C n-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C 4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.

AB - A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (C n-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C 4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.

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DO - 10.1021/ac0600510

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JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 13

ER -

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

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