Agilent Identification User Manual

Identification of Pyridylethylated Cysteine Residues in Peptide Maps Using Diode-array Detection

Cysteine and cystine residues are important amino acid residues because they are involved in the correct formation of the tertiary and quaternary structure of peptides and proteins. The full biological acitivity relies on correctly synthesized intra- and intermolecular disulfide bridges between two particular cysteine residues. As a consequence of disulfide bridge formation and hence a globular structure many proteins are hardly succeptible to proteolytic attack. Therefore, the protein chemical characterization of these proteins is often not possible without prior modification of the cysteine/cystine residues.

Column

0.3 ˘ 250 mm Vydac C-18

Mobile phase

A = water, 0.05 % TFA B = acetonitrile, 0.045 % TFA

Flow rate

4 µl/min after split from 100 µl/min

Gradient

at 0 min 2 % B at 140 min 45 % B

UV detector

diode-array detector 206 nm, 254 nm, 280 nm spectra 200–320 nm 500 nl micro flow cell

Temperature

ambient

Conditions

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Figure 1 Tryptic digest of a bacterial neurotoxin protein after reduction and alklation with vinylpyridine

Biopharmaceutical

[mAU]

8 4 0

VP-Cys +Trp

240 280

[nm]

[mAU]

4 2 0

240 280

VP-Cys

[nm]

[mAU]

40 20

240

280

Absorbance [mAU]

120

80 40

0 20 40 60 80 100 120 140

Time [min]

Tyr

[nm]

Equipment

Agilent 1100 Series

• Binary pump

• Vacuum degasser

• Standard autosampler

• Diode-array detector micro flow cell 10-mm path length, 500 nl

• Agilent ChemStation + 3D software LC Packings flow splitter, IC-100-VAR precolumn microflow processor*

HPLC Performance

Typically, reduction with a variety of reagents, such as mercaptoethanol or dithiodithreithol is carried out followed by alkylation using iodoacetic acid, iodoacetamide or vinylpyridine. Afterwards peptides are separated by HPLC and identified by Edman sequencing or MS-MS sequencing.

Prior to sequence analysis peptide maps are routinely separated by microbore or capillary HPLC. Identification of modified cysteine residues is best performed using vinylpyridine as alkylation reagent, since it adds additional spectral characteristics to this residue. As shown in figure 1, using diode-array detection vinylpyridinilated cysteine residues can be unambiguously identified in peptides separated by reversed-phase capillary HPLC by their characteristic absorption maximum at 254 nm They can thus be easily distinguished from tyrosine and tryptophan residues which have an absorption maximum around 280 nm, even if they are present in the same peptide. With this approach collected peptide fractions can be easily analyzed further by protein sequencing or alternatively online by MS-MS sequence analysis.

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LC Packings 80 Carolina Street, San Francisco, CA 94103, USA