Open Tubular Enzyme Reactors (OTERs) for On-line Protein Digestion in Liquid Chromatography Mass Spectrometry Proteomics

Abstract

Sample preparation in bottom-up proteomics consists of denaturation, reduction and alkylation of the proteins, before enzymatic digestion of the proteins into peptides. The rate limiting step is the enzymatic digestion, and digestion overnight is recommended. Hence, for faster analysis, the digestion time should be reduced. The main focus of this master thesis was the development of 20 μm inner diameter (ID) open tubular enzyme reactors (OTERs) based on 2-hydroxyethyl methacrylate-co-vinyl azlactone (HEMA-VDM) for on-line protein digestion of limited sample sizes in a nano liquid chromatography (LC) - mass spectrometry (MS) system. Monolithic solid phase extraction (SPE) pre-columns based on butyl methacrylate (BuMa) and polystyrene divinylbenzene (PS-DVB) were prepared in 50 μm ID capillaries and used for trapping of peptides generated by the OTER. These peptides were then separated using 10 μm ID PS-DVB porous layer open tubular (PLOT) columns. The developed OTER was prepared by polymerization using a polymerization mixture consisting of HEMA and VDM as monomer, 1-heptanol or 1-decanol as porogen, and 2,2´azobis(2-methylpropinonitrile) (AIBN) as initiator. Short OTERs were prepared with 1-decanol as porogen, while for longer OTERs, 1-heptanol was used. The sample was loaded onto the OTER with a loading buffer consisting of 50 mM NH4OAc pH 8.75 with 4 % acetonitrile (ACN). A trapping time of 4 min from the OTER to the SPE column was found to be optimal (for the short OTER) using the manual LC-MS-system (developed by Hanne K. Hustoft). The run-to-run retention time repeatability in this system, was 0.25-0.44 % in relative standard deviation (RSD %), and 300 attomoles of targeted recombinant progastrin-releasing peptide isoform 1 (ProGRP) could be detected. The developed long OTER, immobilized with Trypsin/endoproteinase Lys-C (T/L) gave sequence coverages (SQ %) up to 95 % of standard proteins. The optimal reactor temperature during digestion and the optimal digestion time was 37 ºC and 30 min, correspondingly. The OTER could be integrated in an automated LC-MS-system (Hanne K. Hustoft and set up by Tore Vehus), where the within and between digestion repeatability were satisfactory. About 1500 proteins were identified in a single analysis when injecting 1 μg of a human cell lysate sample using the OTER in the automated system.