Glycoprotein methods protocols - biotechnology 048-9-121-128.pdf

Glycoprotein methods protocols - biotechnology

Sequencing Glycopeptides 121

121

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

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

11

Identification of Glycosylation Sites in Mucin Peptides

by Edman Degradation

Natasha E Zachara and Andrew A Gooley

1 Introduction

Although it is possible to determine and characterize the total carbohydrate profile after release of the mucin oligosaccharides (usually by β-elimination), it is challeng-ing to assign the sites of glycosylation (macroheterogeneity) and the carbohydrate heterogeneity at a given glycosylation site (microheterogeneity) Typically, the char-acterization of macro- and microheterogeneity has been dependent on the isolation of small peptides with only one glycosylation site However, this is not possible with high molecular weight, heavily glycosylated domains such as those found in mucins The two methods for determining the sites of glycosylation in proteins are mass spectrometry and Edman sequencing Multiple sites of glycosylation cannot easily be detected using mass spectrometry; one strategy involves the β-elimination of the car-bohydrate, which results in the conversion of serine to dehydro-alanine and threonine

to α-amino butyric acid These amino acids have unique masses and can be used to

map glycosylation sites (1,2) However, the macro- and microheterogeneity of the

carbohydrates can not be determined unless the peptide has just one glycosylation site.

In 1950, Edman sequencing was introduced as a repetitive degradation of proteins

with phenylisothiocyanate (3) In the mid-1960s, the process was automated (4),

resulting in a machine where the N-terminal amino acid is derivatized, cleaved, and transferred to a separate reaction vessel, in which it undergoes a conversion to a

phenylthiohydantoin (PTH)-amino acid (Fig 1) It is the PTH-amino that is separated

by reversed-phase chromatography and detected.

Although the modern pulsed liquid sequenator is pmol sensitive, glycosylated

amino acids are only recovered in low yield Samples are sequenced (Fig 1) on

glass-fiber supports or membranes such as polyvinylidene difluoride (PVDF) Following cleavage, the released amino acid is transferred from the reaction cartridge to the con-version flask by nonpolar solvents such as ethyl acetate or chlorobutane The more

polar glycosylated amino acids are not soluble and remain in the support matrix (see

Fig 1, vessel 1) and a blank cycle is observed in the sequencer.

An alternative approach is to use solid-phase sequencing to extract glycosylated

amino acids (5) Peptides and proteins are covalently attached to solid supports (6,7),

allowing the delivery of polar reagents such as trifluoroacetic acid (TFA) and metha-nol, which facilitates the transfer of glycosylated amino acids More importantly, it has been shown that amino acids modified by different carbohydrates have different

retention times on high-performance liquid chromatography (HPLC) (8) Thus, for the

first time, both macro- and microheterogeneity can be determined for a glycopeptide

or glycoprotein in a single experiment (5) This technique has allowed mucin (and

mucin-like) glycopeptides and trifluoromethane sulfonic acid (TFMSA)-treated mu-cin tandem repeats to be sequenced through the heavily glycosylated regions and the

macroheterogeneity to be assigned and quantitated (9–11).

Simple modifications to most modern sequenators facilitate the identification of

glycosylated amino acids (9,11,12) Although it is possible to identify amino acids

substituted with a monosaccharide using standard programs, the glycosylated amino acids are not recovered quantitatively Modifications of the transfer solvents to more polar reagents, such as TFA or methanol, increase the recovery of glycosylated amino acids modified by monosaccharides and larger oligosaccharides (degree of polymer-ization [DP]=19) Elsewhere we have published alternative HPLC methods for the separation of PTH-glycosylated amino acids on a modified Beckman LF3600

sequenator (12) These methods were developed for two reasons: (1) to provide a

dis-tinct elution window for the PTH-glycosylated amino acids, and (2) to be of low enough salt concentration for the direct infusion of the PTH-glycosylated amino acid

Fig 1 Edman degradation Schematic version of Edman degradation in the modern sequenator (PITC, phenylisothiocyanate)

Sequencing Glycopeptides 123

into an electrospray ionisation mass spectrometer However, the chromatography

described in ref 12 is not commercially available and must be made in-house The

following examples from the Hewlett Packard G1000A and the Procise™ from PE

Biosystems show HPLC separations (Fig 2) of PTH-glycosylated amino acids

achieved with commercial kits.

2 Materials

2.1 Chemicals

1 Human Glycophorin A, blood type NN (Sigma, cat no G-9266)

2 Bovine κ-Casein glycopeptide (Sigma, cat no C-2728)

3 Sequencing grade TFA

4 HPLC grade solvents, Milli Q water

2.2 Reagents for Adsorption onto Hyperbond

1 Hyperbond membranes were from Beckman

2.3 Protein Coupling to Arylamine Membranes ( see Note 1)

1 Reagents are available in kit form from Beckman and PerSeptive Biosystems (MA; see

Note 2).

2 Milli Q water and analytical grade methanol are required for wash steps

2.4 Edman Degradation

1 Reagents are obtained in the form of quality-assured sequencing kits, from the manufac-turers of the sequenators

2.5 Apparatus

1 Heating blocks at 80°C and 50°C

2 Speed-Vac™ vacuum centrifuge (Savant Instruments, NY)

3 Beckman LF3600 protein sequenator, or Hewlett Packard G1000A protein sequencer, or

PE Biosystem’s Procise™

3 Methods

3.1 Sample Preparation

Edman sequencing is a molar-dependent chemistry, and it is necessary to determine

how many moles of glycopeptide are to be sequenced (see Note 3) Samples for Edman degradation (see Note 4) must be salt free and not contain sialic acid (see Note 5) Samples can be electroblotted (see Note 6) or adsorbed onto Hyperbond or PVDF (see

Note 7), or covalently coupled to an arylamine-derivatized solid support (see Note 8).

For qualitative analysis of glycosylation sites, as little as 10 pmol of material is required, whereas for quantification of the macro- and microheterogeneity, more material is required, depending on the length of the peptide and the variation in the

structures present (see Note 9).

3.1.1 Desialylation of Glycopeptides

1 Place glycopeptide (5–50 pmol/µL) in salt-free buffer (up to 20% organic modifier [v/v])

in a screw-capped polypropylene Eppendorf tube

Fig 2 Glycosylated PTH-amino acid profiles (A) The glycosylated amino acid window with the

elution profiles of PTH-threonine-GalNAc-Gal (black arrowheads) and PTH-threonine-GalNAc (gray arrowheads) from Hewlett Packard G1000A protein sequencer Standard amino acids are shown for comparison (threonine, open arrowhead) These amino acids were separated using the manufacturer’s

suggested solvent system and program according to the Program 3.1 chemistry (B) The glycosylated

amino acid window with the elution profiles of serine-GalNAc-Gal (black arrowheads) and PTH-serine-GalNAc (gray arrowheads) from Hewlett Packard G1000A protein sequencer Standard amino acids are shown for comparison (serine, open arrowhead) These amino acids were separated using the

manufacturer’s suggested solvent system and program according to the 3.1 chemistry (C) The

glycosylated amino acid window with elution profiles of PTH-threonine-GalNAc-Gal (black arrow-heads) and PTH-threonine-GalNAc (gray arrowarrow-heads) from the Applied Biosystems Procise Stan-dard amino acids are shown for comparison (threonine, open arrowhead) These amino acids were separated using the manufacturer’s suggested solvent system

Sequencing Glycopeptides 125

2 Add an equal volume of 0.2 M TFA.

3 Incubate samples at 80°C for 30 min

4 Remove TFA by lyophilization in a vacuum centrifuge (see Note 10).

5 Resuspend desialylated glycopeptide in 10–20% (v/v) acetonitrile

3.1.2 Electroblotting

Glycopeptides can be desialylated before or after electrophoresis

Electro-blotted glycopeptides are desialylated as in Subheading 3.1.1 with the

follow-ing precautions:

1 Prior to addition of TFA, place membranes in the reaction vessel and wet with 10 µL of methanol

2 Add enough 0.1 M (not 0.2 M as in Subheading 3.1.1.) TFA to cover the membrane.

3 Following incubation at 80°C, remove the membrane from the TFA, wash in Milli Q water (three times) and dry

3.1.3 Adsorption of Glycopeptides onto Hyperbond or PVDF

1 Place a piece of Hyperbond or PVDF (a round membrane is preferable, diameter ~0.5 cm;

see Note 11) in a lid of an Eppendorf tube and place on a hot plate at 50°C

2 Wet with 10 µL of methanol

3 When the excess methanol has evaporated, but before the membrane has dried, apply the sample in 10-µL aliquots (see Note 12).

4 When all of the sample is applied dry the membrane

3.1.4 Covalent Attachment of Glycopeptides

Glycopeptides are covalently attached to arylamine-derivatized membranes via the

activation of peptide carboxyl groups using water-soluble EDC (see Note 13)

Cova-lent coupling of the peptide to arylamine-derivatized membrane is as described in the kit user’s guide.

1 Place an arylamine-derivatized membrane in an Eppendorf tube lid at 50°C

2 Apply samples in 10-µL aliquots, allowing the membrane to come to near dryness between

each aliquot (see Note 14).

3 Dry the membrane after all of the sample has been applied

4 Mix approx 1 mg of EDC in 50 µL of reagent attachment buffer (see Note 15) and

care-fully pipet 10–50 µL onto the arylamine-derivatized membrane

5 Incubate the sample at 4°C for 30 min (see Note 16).

6 Wash the membrane alternately in 1 mL of methanol and 1 mL of Milli Q water three times and dry

3.2 Edman Sequencing

3.2.1 Glycopeptides Electroblotted or Adsorbed onto Hyperbond or PVDF

Glycopeptides adsorbed onto PVDF or Hyperbond can be sequenced with conven-tional Edman degradation (Program 3.1 PVDF on the Hewlett Packard G1000A or the PVDF routine for the blot cartridge on the Procise, or a modified program 40 recom-mended by Beckman for PVDF on the Beckman LF series) While amino acids modi-fied by monosaccharides will be identimodi-fied, their recovery (as well as that of the larger oligosaccharides) is not quantitative.

3.2.2 Glycopeptides Covalently Attached to Membranes

Covalently attached glycopeptides can be sequenced using a modified Hewlett Packard G1000A routine 3.1 PVDF that includes methanol rather than ethyl acetate

for the extraction of the cleaved amino acid from the cartridge (Fig 1, vessel 1; see

Note 17) to the converter (Fig 1, vessel 2; see Note 18).

4 Notes

1 The arylamine-coated membranes are stable at –20°C; the

N-ethyl-N'-dimethylamino-propylcarbodiimide (EDC reagent is stable at 4°C in a dry environment, and the buffer is stable at 4°C

2 Arylamine-derivatized membranes, Sequelon AA™, were originally produced by Milligen Biosearch, a division of Millipore Although Millipore no longer manufactures these membranes, both PerSeptive Biosystems and Beckman supply this product

3 Quantitation of glycopeptides is quite difficult, and we recommend that 10% of the sample

be first sequenced using the standard program prior to glycosylation site mapping

4 Mucins must be digested extensively by proteases and the glycopeptide purified,

desialylated (see Note 5), and desalted before sequencing For heavily glycosylated

pep-tides substituted with large oligosaccharides it may be necessary to treat with TFMSA

(13) or glycosidases before sequencing.

5 There are two principle reasons for desialylating glycopeptides:

a If the peptide is to be covalently bound to an arylamine-derivatized support, the sialic acid must be removed to prevent the formation of an amide bond between the car-boxyl group of the sialic acid and the amine of the support Although the alternative

of using diisothiocyanate-derivatized membranes is possible, we have found them of little practical use, since the peptides must contain lysine, preferably at the C-termi-nus, and this residue is uncommon in mucins

b For glycopeptides adsorbed onto Hyperbond or PVDF supports, we have observed poor recovery of sialylated glycosylated amino acids They elute as a series of multiple peaks and, in many cases, coelute with standard amino acids (unpublished results)

6 Electroblotting is not a usual method for the preparation of mucin glycopeptides since they are large in apparent molecular weight and are rarely separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) However, if glycopeptides can

be separated by SDS-PAGE, the glycopeptides can be electroblotted onto hydrophobic membranes such as Hyperbond or PVDF and stained with amido black

7 Hyperbond performs better in this application than PVDF

8 Covalent coupling gives a quantitative recovery of amino acids modified by more than one carbohydrate residue Other methods only identify an amino acid modified by one carbohydrate residue

9 Because mucin glycopeptides are usually of very high molecular weight, an initial yield

of 10 pmol in the sequenator represents approx 100 µg of a 200-kDa glycopeptide

Quan-titating the carbohydrate component (see Notes 3 and 8) and the length of sequence

obtained is dependent on the number and distribution of proline residues Proline cleaves

with reduced efficiency in Edman sequencing (Fig 1, vessel 1), and a significant lag is

introduced when several prolines occur in a cluster and more than one signal is detected

in each cycle

10 Freeze-drying mucins is not recommended because the solid is often poorly soluble The sample should not be taken to complete dryness

Sequencing Glycopeptides 127

11 Round pieces of PVDF or Hyperbond have better surface tension and allow larger aliquots

to be loaded Alternatively, a strip of PVDF or Hyperbond can be used, approx 2 × 10 mm Care must be taken when loading to maintain surface tension on the strip

12 Membranes should not be allowed to dry between application of sample aliquots

13 Although small peptides couple well, the high molecular weight of mucin glycopeptides reduces the efficiency of coupling to ~5%

14 Amine buffers such as Tris interfere with coupling to arylamine and should be avoided

15 The EDC reagent should be mixed with the reaction buffer just prior to coupling

16 Incubation at 4°C increases the initial yield (7).

17 To incorporate a methanol extraction into the routine, methanol is placed in an unused bottle position, and solvent delivery from this bottle is substituted for ethyl acetate during the extraction procedure

18 Poor yields of aspartic and glutamic acid will be observed with arylamine-coupled peptides

or proteins because of the coupling of the χ- and δ-carboxyl groups to the membrane

Acknowledgments

NEZ was supported by an Australian Postgraduate Award AAG acknowledges the support of the National Health and Medical Research Council The authors would like

to acknowledge the support and useful comments of Dr Nicolle Packer and Prof Keith Williams (Macquarie University).

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