Strategies for Handling Polypeptides on a Microscale

Abstract

The challenge that comes with a sample for analysis is to prepare it with the minimum of contamination, modification, and loss. For the first two points it is necessary to work in clean conditions with the cleanest possible reagents. Contaminants may include amine-containing buffer components, such as glycine or Tris, so careful choice of buffers is advisable. Other reagents, too, may cause problems. For example, triton and other nonionic detergents may contain traces of reactive peroxide species, which may modify proteins (1). Such problems are minimized by the use of fresh, specially purified detergent stored under nitrogen (such as is available from commercial sources, such as Pierce, Rockford, IL). Polypeptide modification may also occur in conditions of low pH; for instance, N-terminal glutaminyl residues may cyclize to produce the blocked pyroglutamyl residue, glutamine and asparagine may become deamidated, or the polypeptide chain may be cleaved (as described in Chapter 6). Again, exposure of proteins to formic acid has been reported to result in formylation, detectable by mass spectrometry (2). Problems of this sort are reduced by minimizing exposure of the sample to acid and substitution of formic acid by, for example, acetic or trifluoroacetic acid for the purposes of treatment with cyanogen bromide (see Chapter 6).