Glycoprotein Methods and Protocols - P8

Amino Acid Analysis of Mucins 113

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From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A. Corfield © Humana Press Inc., Totowa, NJ

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Amino Acid Analysis of Mucins

Jun X. Yan and Nicolle H. Packer

1. Introduction

Amino acid analysis is a commonly used technique that provides quantitative esti￾mation of the amounts of proteins/amino acids present in a sample and/or qualitative

information on the amino acid composition of a protein. For protein analysis, the tech￾nique essentially involves acid hydrolysis of amino acid peptide bonds within the pro￾tein; chemical derivatization of hydrolysate (amino acids) of the protein; and high￾performance liquid chromatography (HPLC) separation, detection, and analysis of

those derivatized amino acids.

The commercially available amino acid analyzers (e.g., Waters Pico-Tag system

[Waters Corp., Milford, MA]; GBC AminoMate system [GBC Scientific, Dandenong,

Victoria, Australia]) have made amino acid analysis more practical and feasible in

routine protein analysis laboratories. The sensitivity of the analysis has been dramati￾cally increased to low picomole levels of proteins, including those low molecular

weight (10–20 kDa) ones (low amount of total amino acids analyzed) (1).

In this chapter, we describe a 9-fluorenylmethyl oxycarbonyl chloride (FMOC)-

based precolumn derivatization amino acid analysis that has been extensively vali￾dated (1,2). Although the detailed protocols on the use of the automated GBC

AminoMate (GBC Scientific) amino acid analyzer have been described elsewhere (3),

here, we emphasize the procedures that are used in a manual operation. Thus, this

technique can be easily adapted in any laboratory where an HPLC system with a fluo￾rescent detector and gradient controller is available.

Acid hydrolysis is the first and most important step to release the amino acids from

the proteins, and it must be carefully controlled in the analysis of mucins. The acid

hydrolysis described here recovers 16 amino acids (asparagine and glutamine are

deamidated to their corresponding acids, whereas tryptophan and cysteine are destroyed).

During the acid hydrolysis, the carbohydrate side chains on the mucins are degraded.

Because of the high carbohydrate content of mucins (up to 90% of the dry weight), the

sugars can be caramelized and further charcoaled, and the acid hydrolysis results in a

black residue. This residue appears to precipitate protein/amino acids, interferes with