290-297 LCE May05ready

2 There has been a wealth of recent interest in proteomics, which involves the global analysis of protein expression and function. Identification of complex protein mixtures is a difficult process that can first involve, for example, two-dimensional gel electrophoresis to resolve individual proteins. These separated proteins can then be hydrolysed with enzymes such as trypsin to their constituent peptides, which can be identified by high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS).1 Several factors must be taken into account to achieve high-resolution chromatographic separation and identification of peptides: • Low pH is preferred because the ionization of silanol groups on silica-based reversed-phase columns is suppressed, together with their detrimental interactions with the charged peptides. Also, sensitivity is enhanced in positive electrospray ionization MS (ESI–MS). • Volatile buffers–additives such as formic acid, trifluoroacetic acid or heptafluorobutyric acid are used because they do not lead to contamination of the source, which occurs with involatile inorganic buffers such as phosphate. • A low concentration of additives is preferred, which leads to less signal suppression in the mass spectrometer even when volatile substances are used. • Formic acid is generally considered to give less signal suppression in ESI–MS than trifluoroacetic acid. One of the reasons for this finding is the possibility of ion-pairing between charged peptides and trifluoroacetic acid, which contributes to this loss of sensitivity. Relatively little work has been published on the chromatographic effects of changing between the buffers conventionally used in HPLC–UV analysis (phosphate, for example) and these volatile buffers (particularly formic acid and trifluoroacetic acid) necessary for use with MS detection. For proteins, Huber and co-workers reported peak widths at half height 69–104% larger when using 0.5% formic acid compared with 0.1% trifluoroacetic acid. Peak shape was worse still when using 0.1% formic acid. However, formic acid was still recommended because of its reduced signal suppression effects.2 In a later paper,3 the same group recommended the use of trifluoroacetic acid because of the relatively poor resolution of peptides obtained with formic acid. They suggested that silanol activity might be the cause of poor peak shape and that these interactions were reduced when using trifluoroacetic acid. In this article, we describe a study of the origins of the differences in peak shape obtained when using different buffers–additives. We chose the Alberta test, a commercially available mixture of four basic peptides synthesized specifically for column evaluation4 and also a mixture of bradykinin and analogues. The Alberta mixture contains peptides with 1–4 basic lysine residues, whereas the bradykinins contained 1–3 basic arginine residues; the amino acid sequence of these peptides is shown in Table 1. These mixtures represent a rather severe test of silanol activity, because they contain solutes with multiple positive charges at low pH that could interact with dissociated negatively charged silanols on the column surface. We decided to compare the performance of a silica-based reversed-phase column with that of a totally polymeric column. The latter, being a polystyrene–divinylbenzene based material, has no silanol groups and allows the possible contribution of silanols to peak shape on the silica column to be deduced by comparison of results. However, the possibility of finding ionic groups on such polymeric surfaces cannot be entirely discounted and is considered later. Despite the frequent attribution of poor peak shapes of peptides and other protonated analytes to silanol activity, we have found some intriguing results in the study of peak shapes of basic drugs in reversed-phase chromatography. For example, Choice of Buffer for the Analysis of Basic Peptides in Reversed-Phase HPLC