JCGGDB TOP GlycoScience Protocol Online Database

Enrichment of sulfated glycopeptides by sulfate emerging method
Text Search

Enrichment of sulfated glycopeptides by sulfate emerging method

Authors:
Introduction Protocol References Credit lines
Category
Large scale preparation of glycans, glycoproteins & glycolipids
Protocol Name

Enrichment of sulfated glycopeptides by sulfate emerging method

Authors
Toyoda, Masaaki
Research Center for Medical Glycoscience, AIST

Kameyama, Akihiko *
Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST)
*To whom correspondence should be addressed.
KeyWords
Reagents

Carboxypeptidase B, (Sigma-Aldrich, St. Louis, MO)

Sep-Pak C18 cartridge, (Waters Corp., Milford, MA)

Acetohydrazide, (Sigma-Aldrich)

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), (Wako Pure Chemical Industries, Ltd., Osaka, Japan)

Instruments

TSKgel QAE-2SW column (Tosoh Corp., Tokyo, Japan)

Methods
1.

A mixture of tryptic peptides was used as a starting material. Reduction and alkylation of proteins by a conventional protocol is recommended prior to trypsinization.

1) 

 Tryptic peptides (1 mg) were suspended in 50 μL of 20 mM Tris-HCl (pH 7.8) containing 10 mM CaCl2.

Comment 0
2) 

 Carboxypeptidase B (1 μg) was added to the suspension.

Comment 0
3) 

 The mixture was incubated at 37°C for 60 min.

Comment 0
4) 

 The protease was then inactivated by boiling the mixture for 5 min.

Comment 0
5) 

 The mixture was loaded onto a Sep-Pak C18 cartridge* (50 mg, 1 mL) that was pre-equilibrated with 0.1% trifluoroacetic acid (TFA). After washing the column with 0.1% TFA the bound material was eluted with 0.1% TFA in 80% acetonitrile (ACN). Eluates were then dried in vacuo.

Comment 1
6) 

 The obtained mixture was dissolved in 50 μL of 2.5 M acetohydrazide.

Comment 0
7) 

 The solution was adjusted to pH 2.5* with 1N HCl.

Comment 1
8) 

 One microliter of 4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added to the solution.*

Comment 1
9) 

 The mixture was incubated at room temperature for 1 h.

Comment 0
10) 

 These addition and incubation procedures (i.e., steps 8 and 9) were then repeated four times.

Comment 0
11) 

 After the last incubation, the mixture was loaded onto a Sep-Pak C18 cartridge, and the modified peptides were obtained as described in step 5.

Comment 0
12) 

 The obtained peptides were dissolved in 20 mM Tris-HCl containing 30% acetonitrile, pH 8.6 (solvent A), and injected onto the column (TSKgel QAE-2SW column) pre-equilibrated with a solvent A.

Comment 0
13) 

 The chromatography was run at a flow rate of 0.7 mL/min at 32°C.

Comment 0
14) 

 After running with solvent A for 10 min, the mobile phase was switched stepwise to 0.75 M NaCl in solvent A (solvent B).

Comment 0
15) 

 The solvent B flow was collected for 30 min (i.e., fraction containing sulfated glycopeptides).

Comment 0
16) 

 The collected fraction was adjusted to pH 2.5 with 1 N HCl,* dried in vacuo, and desalted by a Sep-Pak C18 cartridge as described in the step 5.

Comment 1
17) 

 The enriched sulfated glycopeptides were detected from the obtained mixture by MALDI-TOF MS.*, **

Comment 1
18) 

 If required, the sulfated glycopeptides can be further purified by reversed-phase HPLC.*

Comment 1
Notes

Peptides containing a phosphate group are also enriched by this procedure. Phosphate groups should be removed using IMAC resins or a phosphatase prior to starting the SE procedure. This is particularly important when the sample includes relatively large amounts of glycopeptides containing phosphate groups (e.g., samples prepared from the fractions including lysosomes).

Figure & Legends

Figure & Legends

Fig. 1. Outline of SE procedure

Red color indicates residue having a negative charge. In the SE procedure, basic amino acid residues (Arg and Lys) are removed by carboxypeptidase B, and carboxyl groups are modified with acetohydrazide. Eventually, only sulfated glycopeptides have a negative charge in the SE processed mixture.

 

Fig. 2. Enrichment of sulfated glycopeptides using ion exchange chromatography

 

Fig. 3. Reaction scheme for the modification of the carboxyl group

Copyrights Creative Commons License   Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
Date of registration:2014-06-19 13:44:16
©2010 - 2023 Ritsumeikan University, AIST & JCGGDB. All Rights Reserved