Isotope-coded two-dimensional maps: tagging with deuterated acrylamide and 2-vinylpyridine.
Isotope-coded two-dimensional maps, with either D(0)/D(3)-acrylamide or D(0)/D(4) 2-vinyl pyridine, are described in detail. They have the advantage of running the two samples under investigation within a single slab gel, thus minimizing errors because of spot matching with software packages when samples are run in parallel maps. Labeling with deuterated acrylamide is very simple and inexpensive, because this chemical is commercially available. The experiment has to be carried out at alkaline pH values (pH 8.5-9.0) and with high molarities of alkylating agent (50-100 mM) to ensure good conversion efficiency. On the contrary, labeling with 2-vinyl pyridine (2-VP) can be performed in much lower alkylant molarities (20 mM) and at neutral pH values, thus ensuring essentially 100% conversion efficiency coupled with 100% specificity, because the reaction is sustained by the partial positive and negative charges on the 2-VP and -SH group, respectively. However, deuterated 2-VP is not commercially available and it has to be synthesized ad hoc.
Righetti
Pier Giorgio
PG
Polytechnic of Milano, Milan, Italy.
Sebastiano
Roberto
R
Citterio
Attilio
A
D020106:Acrylamide / Q000737:chemistry*; D000818:Animals; D020552:Comet Assay; D003903:Deuterium / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D011725:Pyridines / Q000737:chemistry*
D011506:Proteins; D011725:Pyridines; D020106:Acrylamide; D003903:Deuterium; C030953:2-vinylpyridine