Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.
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 solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA). The present matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis demonstrates that thiourea is an effective scavenger 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 the cyclic compound thiazolinidone monoimine. This reaction strongly competes 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, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.
Galvani
M
M
GlaxoWellcome SpA, Medicines Research Center, Verona, Italy.
Rovatti
L
L
Hamdan
M
M
Herbert
B
B
Righetti
P G
PG
D000478:Alkylation; D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007460:Iodoacetamide; D007700:Kinetics; D007768:Lactalbumin / Q000032:analysis / Q000737:chemistry; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013890:Thiourea; D014508:Urea
D007782:Lactoglobulins; D020543:Proteome; D014508:Urea; D007768:Lactalbumin; D013890:Thiourea; D007460:Iodoacetamide