Rationale: Following digestion of proteins with trypsin, digests are typically subjected to further 'clean-up' prior to liquid chromatography/mass spectometry (LC/MS) analysis, in order to reduce the complexity of the digested matrix, as well as helping to remove residual denaturants and reduction/alkylation reagents prior to injection onto the analytical HPLC column. Often, this is carried out using off-line techniques, and is not ideally suited to high-throughput workloads, for example in clinical laboratories.
Methods: Bovine serum albumin (BSA) was used as a model protein. Following denaturation with urea, reduction/alkylation, and digestion with trypsin, the analytical recovery of a selection of proteotypic BSA peptides was assessed using a two-dimensional, turbulent flow chromatography method. Peptides were identified using a Q Exactive™ mass spectometer operating in full-scan mode.
Results: Total analysis time (including the on-line sample clean-up) was 15 min per injection. Aside from the most hydrophilic peptide selected, ATEEQLK, recovery using the turbulent flow chromatography systems was greater than 30% for all remaining peptides (N = 17), and exceeded 50% for 12 of the 18 peptides studied. There was a broad correlation between the hydrophobicity factor and the observed recovery.
Conclusions: This study suggests that turbulent flow chromatography offers a rapid, on-line alternative to solid-phase extraction for the analysis of peptide digests by LC/MS. A wide range of column chemistries are available, and the technique can be further optimised for analyses which are targetted to specific peptides. As with turbulent flow chromatography for small-molecule workflows, this approach may be ideally suited to high-throughput applications, such as those which are emerging from within clinical laboratories.
Copyright © 2015 John Wiley & Sons, Ltd.