title: The use of turbulent flow chromatography for rapid, on-line analysis of tryptic digests.
abstract: BACKGROUND
  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.
authors.affiliation: Viapath Analytics, Department of Clinical Biochemistry, King's College Hospital, Denmark Hill, London, SE5 9RS, UK.|Department of Cancer Studies, Leicester Royal Infirmary, University of Leicester, Leicester, LE2 7LX, UK.
authors.forename: L
authors.initials: L
authors.lastname: Couchman
authors.affiliation: Department of Cancer Studies, Leicester Royal Infirmary, University of Leicester, Leicester, LE2 7LX, UK.
authors.forename: D J L
authors.initials: DJ
authors.lastname: Jones
authors.affiliation: Department of Clinical Biochemistry, King's College Hospital, Denmark Hill, London, SE5 9RS, UK.
authors.forename: C F
authors.initials: CF
authors.lastname: Moniz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D012710:Serum Albumin, Bovine; D014357:Trypsin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D014357:Trypsin