Details

Autor(en) / Beteiligte

• Jiang, Xuehui
• Shamshurin, Dmitry
• Spicer, Vic
• Krokhin, Oleg V.

Titel

The effect of various S-alkylating agents on the chromatographic behavior of cysteine-containing peptides in reversed-phase chromatography

Ist Teil von

• Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2013-02, Vol.915-916, p.57-63

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2013

Link zum Volltext

Quelle

ScienceDirect Journals (5 years ago - present)

Beschreibungen/Notizen

• ► Chromatographic behavior of cysteine-containing peptides with 6 different alkylation chemistries has been investigated under 3 different reversed-phase HPLC conditions ► Peptide's retention is strongly affected by the charge state of the side chain ► Peptide retention prediction models should be modified to accommodate changes in the alkylation status of Cys. We investigate the influence of various alkylation chemistries on the reversed phase (RP) HPLC behavior of Cys-containing peptides under the most popular RP-HPLC conditions used in proteomics: C18 phases with trifluoroacetic acid (TFA) or formic acid (FA) as the ion pairing modifiers, and separation at pH 10. Akylating agents studied are iodoacetamide (IAM), iodoacetic acid (IAA), 4-vinylpyridine (4-VP), acrylamide (AA) and methyl methanethiosulfonate (MMTS). These were compared against the retention of identical peptides without alkylation, i.e. free cysteines. The intrinsic hydrophobicity values of the Cys residue under formic acid conditions for these modifications were found to increase in the following order: 4-VP<IAM<AA<IAA<free Cys<MMTS. The retention contribution of the positively charged 4-VP derivative is affected by the nature of the ion-pairing modifier; this is the most hydrophilic residue for formic acid based eluents, and second most hydrophilic behind IAM-alkylated Cys using TFA eluent. Switching to a basic condition dramatically decreases the retention of free cysteine and IAA-alkylated analytes due to the ionization of side-chains. The opposite effect is observed for 4-VP, which become neutral at basic pHs. The careful measurement of the hydrophobic contributions for these residues is vital to the development of accurate peptide retention prediction models; the incorporation of these modifications into our Sequence Specific Retention Calculator model is presented.