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A High‐Throughput Absolute‐Level Quantification of Protein‐Bound Amino Acids in Seeds

A High‐Throughput Absolute‐Level Quantification of Protein‐Bound Amino Acids in Seeds In this unit, we describe a high‐throughput absolute quantification protocol for 16 protein‐bound amino acids (PBAAs) that combines a microscale protein hydrolysis step and an absolute quantification step using multiple reaction monitoring—based liquid chromatography–tandem mass spectrometry detection. The approach facilitates analysis of a few hundred samples per week by using a 96‐well‐plate extraction setup and avoiding use of additives. Importantly, the method uses only ∼3 mg of tissue per sample and includes 12 heavy‐amino‐acid internal standards to enable quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues. © 2018 by John Wiley & Sons, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Protocols in Plant Biology Wiley

A High‐Throughput Absolute‐Level Quantification of Protein‐Bound Amino Acids in Seeds

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References (37)

Publisher
Wiley
Copyright
© 2018 John Wiley & Sons, Inc.
ISSN
2379-8068
eISSN
2379-8068
DOI
10.1002/cppb.20084
Publisher site
See Article on Publisher Site

Abstract

In this unit, we describe a high‐throughput absolute quantification protocol for 16 protein‐bound amino acids (PBAAs) that combines a microscale protein hydrolysis step and an absolute quantification step using multiple reaction monitoring—based liquid chromatography–tandem mass spectrometry detection. The approach facilitates analysis of a few hundred samples per week by using a 96‐well‐plate extraction setup and avoiding use of additives. Importantly, the method uses only ∼3 mg of tissue per sample and includes 12 heavy‐amino‐acid internal standards to enable quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues. © 2018 by John Wiley & Sons, Inc.

Journal

Current Protocols in Plant BiologyWiley

Published: Dec 1, 2018

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