Microcapillary liquid chromatography/tandem mass spectrometry using alkaline pH mobile phases and positive ion...

Extending the dynamic range of microcapillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) peptide sequencing methods is essential for extracting new discoveries from proteomic studies. The complexity of global protein digests and in vivo processed peptide repertoires (as isolated from immunologically important HLA complexes) have led to the development of novel separation methods to increase the number of peptides identified by a single analysis. Separation of complex mixtures by multidimensional high-performance liquid chromatography (HPLC) decreases the number of isolated peptides contained in each fraction and increases the likelihood of detecting low abundant peptides in a background of dominant signals. In this study, we have evaluated the use of two dimensions of reversed-phase chromatography for resolving and sequencing naturally processed HLA-A2 presented peptide repertoires. The first dimension of separation was reversed-phase chromatography using the strong ion pairing reagent trifluoroacetic acid (TFA) to ensure the highest efficiency of peptide fractionation. The second dimension of reversed-phase chromatography was online with an electrospray ionization (ESI) ion trap mass spectrometer. Mobile phases used for the second dimension of chromatography were modified with volatile reagents including a contemporary acetate-modified acidic solvent, which was compared with mobile phases prepared with ammonium hydroxide at an alkaline pH. As expected, we demonstrate improved separation of the HLA-A2 presented fractions using the alkaline pH conditions. However, less obvious was the improved peptide signal-to-noise detected for peptide signals by positive ion ESI ion trap mass spectrometric detection, which was attributed to a reduced chemical background when using the alkaline pH mobile phases that allowed the ion trap to fill with the peptide ions until the automatic gain control detected a full trap. The term 'wrong-way-round ionization' has been used to describe intense [M+H](+) ions generated during ESI under strongly basic solutions. Ultimately, a larger number of the HLA-A2 peptide repertoire was sequenced by coupling TFA-modified reversed-phase fractionation with alkaline-modified microcapillary LC/MS/MS analysis of each fraction. In the present report, we compare the two second-dimension approaches and demonstrate the quality of data that was acquired using alkaline pH reversed-phase conditions.