Glycoprotein methods protocols - biotechnology 048-9-111-119.pdf

Glycoprotein methods protocols - biotechnology

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

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

10

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 propro-tein; 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

chromatography, and leads to a significantly lower recovery of all the amino acids, especially serine and threonine Strong acid, high temperature, and short time acid

hydrolysis (12 N HCl at 155 °C for 1 h) is generally used in our laboratory for less

glycosylated proteins (1) We have found that weaker acid, lower temperature, and

longer time (ensuring the completion of the hydrolysis) acid hydrolysis (6 N HCl at

105 °C for 24 h) is a must to obtain reproducible quantitative analysis of mucin

gly-coproteins The hydrolysate of mucin is then derivatized with Fmoc on the α-amino groups under alkaline conditions The derivatized amino acids are separated by a C18 reversed-phase column and detected by fluorescence Our routine analyses have shown that the method described here is reliable and useful for both quantitative and qualitative mucin glycoprotein amino acid analysis.

2 Materials

2.1 Apparatus

2.1.1 Hydrolysis Equipment

1 Hydrolysis vessel: the design can be viewed on the World Wide Web at http://www.bio

mq.edu.au/APAF (see Note 1).

2 Vacuum pump: Savant Speedvac (Savant Industries, Farmingdale, NY) (or equivalent) with a vacuum gauge

3 Two-way line connection to an argon line and a vacuum pump

4 Autosampler glass vials made from inert Chromacol Gold™ grade glass (Chromacol, cat

no 02-MTVWG, Herts, UK)

2.1.2 HPLC System ( see Note 2)

1 LC-pump with ternary gradient controller

2 Fluorescence detector: excitation, λ = 270 nm; detection, λ = 316 nm

3 Sample injector

4 Degasser: alternatively, buffers can be degassed by continuous flow of helium

5 Hypersil C18 reversed-phase column: 150× 4.6 mm inner diameter 5 µm (Keystone, Bellefonte, PA)

6 In-line filter (2 µm) (Upchurch, cat no 100-10)

2.2 Chemicals

2.2.1 Hydrolysis and Derivatization Reagents

1 Hydrochloric acid: constant boiling temperature 6 N (Pierce, cat no 24309).

2 Ultrapure phenol (ICN, cat no 800672) kept at 4°C

3 Borate buffer: 250 mM boric acid (analytical reagent grade [AR] grade) in water, adjusted

to pH 8.5 with NaOH Buffer may be kept up to 1 mo at 4°C

4 Fmoc reagent: 4 mg/mL fluorenylmethyl chloroformate (Sigma, cat no F0378) in aceto-nitrile (HPLC grade) Reagent may be stored up to 1 wk at 4°C

5 Cleavage reagent: 680 µL of 0.85 M NaOH (AR grade), 150 µL of 0.5 M hydroxylamine

hydrochloride (Sigma, cat no H2391), and 20 µL of 2-methylthio-ethanol (Sigma, cat

no M9268) Stock solution can be kept for 1 mo at 4°C Working cleavage reagent must

be made freshly prior to each use

6 Quenching reagent: 2 mL of acetic acid (AR grade) and 8 mL of acetonitrile

2.2.2 Chromatography Reagents

1 Amino acid standards H: protein hydrolysate standard in 0.1 N HCl, containing a solution

of 17 amino acids (Sigma, cat no A9781)

2 L-hydroxyproline used as internal standard (Sigma, cat no H1637)

3 Phosphate buffer (2 M): 2 M anhydrous ammonium monohydrogen phosphate (AR grade) solution, adjusted to pH 6.5 with 2 M anhydrous dihydrogen phosphate (AR grade)

solu-tion Buffer may be kept up to 6 mo at 4°C

4 Mobile phase A (30 mM ammonium phosphate [pH 6.5]): Into a 1000 mL volumetric flask, add 15 mL of 2 M phosphate buffer and dilute to volume with mobile phase B.

5 Mobile phase B: 15% (v/v) methanol in water

6 Mobile phase C: 90% (v/v) acetonitrile in water

3 Methods

3.1 Sample Preparation and Hydrolysis

1 Add an aliquot of a solution of a sample of mucin into an autosampler glass vial and dry

under vacuum (see Note 3).

2 Place the vials, 400 µL of HCl (6 N), and a crystal of phenol into the bottom of the

hydrolysis vessel

3 Assemble the vessel tightly Connect the vessel to a two-way argon and vacuum line

4 Evacuate the vessel to 3 torr, and flush with argon Repeat this step twice, and seal the

vessel after the third evacuation step (see Note 4).

5 Place the vessel in a 105°C oven for 24 h

6 Remove the vessel from the oven and open the vacuum tap immediately within a fume

hood (see Note 4).

7 Place the vials into a vacuum centrifuge for 10 min to evaporate excess HCl

3.2 Derivatization ( see Note 5)

1 Dissolve the mucin hydrolysate in 10 µL of 250 mM borate buffer, pH 8.5.

2 Add 10 µL of Fmoc reagent, mix, and then wait 1 min

3 Add 10 µL of cleavage reagent, mix, and then wait 4 min

4 Add 10 µL of quenching reagent mix

3.3 Chromatography ( see Note 6)

1 Set up the fluorescent detector with excitation λ = 270 nm and detection λ = 316 nm

2 The mobile phase is a ternary solution system using the gradient shown in Table 1.

3 The stationary phase is a 5 µm Hypersil C18 column that is temperature controlled at

38°C Flow rate is 1.0 mL/min Allow the column to equilibrate with two gradient runs before the sample injection

4 Inject an aliquot (≥5 µL) of each derivatized sample (see Note 6) Run time is 35 min.

Allow at least 2 min for pump and column equilibration with the initial composition of the mobile phase

5 Figures 1 and 2 show typical chromatograms of the separation of amino acid standards

and hydrolyzed bovine conjunctival mucin (10–20 µg)

6 After the run, the peaks can be integrated and the picomoles of each mucin amino acid calculated by comparison with the peak areas given by the amino acid standard Since the total number of amino acids (or the molecular mass) in a mucin glycoprotein is unknown (or difficult to determine), the actual number of picomoles of mucin protein cannot be determined by this analysis In practice, the amount of mucin can usually be referred to as

Table 1

Gradient for the Baseline Separation

of 16 Fmoc–Derivatized Amino Acids Within 35 min

Fig 1 Typical chromatogram of separation of 16 standard Fmoc-amino acids RFU = rela-tive fluorescence units; Amino acid code: D = aspartic acid, E = glutamic acid, HP = hydroxy proline, S = serine, H = histidine, G = glycine, T = threonine, A = alanine, P = proline,

Y = tyrosine, R = arginine, V = valine, M = methionine, I = isoleucine, L = leucine, F = phenylalanine, K = lysine

the amount or ratio of certain amino acids, so a relative comparison in quantitative analy-sis, e.g., carbohydrate analyanaly-sis, can be done The amino acid composition of the mucin

(%pmol) = pmol of mucin amino acid/total pmol of total amino acids x 100% (see Table 2 for an example) (see Note 7).

4 Notes

1 The hydrolysis vessel can be made by any glassblower It has to withstand high tempera-ture in the presence of strong acid vapor, and hold positive and negative gas pressure during each hydrolysis run

2 The detection of amino acids can be achieved by any HPLC system However, if Fmoc is used as the derivatization reagent, a fluorescence detector is essential The HPLC must consist of a ternary gradient controller in order to run the gradient program necessary for the baseline separation of 16 amino acids We have investigated different types of C18 reversed-phase column and have found that the Hypersil C18 column manufactured by Keystone provides the best separation and resolution We use the GBC automated AminoMate HPLC system controlled by GBC WinChrom Windows software that

pro-vides an automatic integration of the chromatogram and assignment of peaks (3).

3 Sample preparation is an important step in amino acid analysis Ideally, samples should

be dried and free of salts, amines, and detergents The sample of mucin must be desalted (e.g., by using a size-exclusion desalting column), or the mucin can be dot-spotted onto polyvinylidene difluoride (PVDF) membrane, or gel separated by electrophoresis and electroblotted onto PVDF Note that mucins are often difficult to bind to PVDF Wilkins

et al (3) have described a detailed protocol for PVDF-bound protein samples.

4 During the evacuation and argon flush of the hydrolysis vessel, the bottom of the vessel will become cold and the acid will boil under vacuum These are the signs that the vessel

is sealed tightly Following hydrolysis, the vessel should be opened as soon as it is removed from the oven Acid condensation inside the vessel is an indication of a com-plete hydrolysis Handling the vessel requires heat-resistant gloves, and safety goggles must be worn at all times

Fig 2 Typical chromatogram of separation of amino acids from bovine conjunctival mucin

hydrolysate RFU = relative fluorescence units Amino acid code as defined in Fig 1.

5 The mucin hydrolysate can be analyzed for its amino acid composition by any other chemical derivatization procedure and HPLC separation, e.g., phenylisothiocyanate derivatization with ultraviolet detection If an autosampler is available, the derivatization procedure can be automated Nevertheless, manual derivatization is adequate when thor-ough mixing after the addition of each reagent is practiced

6 The HPLC separation is highly reproducible, requiring no modification of the gradient or conditions from day to day, and only minor maintenance throughout the life of the column,

which is usually capable of 800 injections Wilkins et al (3) describe detailed

trouble-shooting of the chromatography

7 Note that this amino acid analysis does not take into account the amount of cysteine and tryptophan We are currently developing a method for quantitating cysteine by amino acid analysis

Table 2

Example of Quantitative Calculation of Mucin Amino Acid Composition

Bovine conjunctival

aThe on-column amount of each standard amino acid is generally 125 pmol For example, for a

10µL injection of 50 µM stock solution, on-column pmol of standard amino acid = 50 µM × 10 µL/4,

where 4 is the dilution factor in derivatization

bThe pmol of mucin amino acid = peak area of mucin amino acid/peak area of standard amino acid× 25 pmol × 2, where 2 is the dilution factor from the half of the sample injected

c%pmol = pmol mucin amino acid/total mucin amino acids× 100%, where total mucin amino ac-ids is the sum of mucin amino acac-ids (except cysteine and tryptophan; asparagine and glutamine are recovered as their acid form: aspartic acid and glutamic acid, respectively)

The authors acknowledge support for their research on amino acid analysis through grants from the Australia Research Council, Macquarie University Research Grants, National Health and Medical Research Council, and GBC Scientific Equipment (Dandenong, Victoria, Australia) Jun Yan acknowledges financial support through an MUCAB scholarship The authors also thank Malcom Ball for donating mucin samples and Dr Andrew Gooley and Prof Keith Williams for their support.

References

1 Yan, J X., Wilkins, M R., Ou, K., Gooley, A A., Williams, K L., Sanchez, J.-C., Golaz, O., Pasquali, C., and Hochstrasser, D.F (1996) Large-scale amino-acid analysis

for proteome studies J Chromatogr 736, 291–302.

2 Ou, K., Wilkins, M.R., Yan, J.X., Gooley, A.A., Fung, Y., Sheumack, D., and Wil-liams, K L (1996) Improved high-performance liquid chromatography of amino acids

derivatized with 9-fluorenylmethyl chloroformate J Chromatogr 723, 219–225.

3 Wilkins, M R., Yan, J X., and Gooley, A A (1999) 2-DE spots amino acid analysis with

9-fluorenylmethyl chloroformate, 2-D Protein Gel Electrophoresis Protocols (Link, A J.,

ed.), Humana, Totowa, NJ, 445–460