The use of turbulent flow chromatography for rapid, on-line analysis of tryptic digests

• Published in: Rapid communications in mass spectrometry 2015-11, Vol.29 (22), p.2140-2146
• Main Authors: Couchman, L., Jones, D. J. L., Moniz, C. F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Chromatography; Chromatography, Liquid - instrumentation; Chromatography, Liquid - methods; Computational fluid dynamics; Equipment Design; Fluid flow; Mass Spectrometry - methods; Molecular Sequence Data; On-line systems; Peptide Fragments - analysis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptides; Recovery; Serum Albumin, Bovine; Trypsin - metabolism; Turbulence; Turbulent flow
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.7371

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.