Protein alkylation in the presence/absence of thiourea in proteome analysis: A matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation

Citation
M. Galvani et al., Protein alkylation in the presence/absence of thiourea in proteome analysis: A matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation, ELECTROPHOR, 22(10), 2001, pp. 2066-2074
Citations number
24
Categorie Soggetti
Chemistry & Analysis
Journal title
ELECTROPHORESIS
ISSN journal
01730835 → ACNP
Volume
22
Issue
10
Year of publication
2001
Pages
2066 - 2074
Database
ISI
SICI code
0173-0835(200106)22:10<2066:PAITPO>2.0.ZU;2-#
Abstract
Although it is highly recommended that reduction and alkylation of free -SH groups in proteins should be performed prior to any electrophoretic step ( including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubiliza tion cocktail adopted recently (namely thiourea) strongly quenches such alk ylation process las typically carried out with iodoacetamide, IAA). The pre sent matrix assisted laser desorption/ionization-time of flight-mass spectr ometry (MALDI-TOF-MS) analysis demonstrates that thiourea is an effective s cavenger of IAA, since its sulfur atom reacts as efficiently as the ionized , free SH group of Cys in proteins at alkaline pH values (pH 8.5-9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form t he cyclic compound thiazolinidone monoimine. This reaction strongly compete s with the direct addition reaction of IAA onto the -SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, af ter proper sample reduction and alkylation, thiourea can be incorporated in to the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their p/ values and scavenging the excess of free IAA .