Nanoscale LC-MS(n): technical design and applications to peptide and protein analysis

Abstract

We describe a nanoscale capillary column switching HPLC–ESI/MS⁽ⁿ⁾ system in detail. It is intended for routine operation with 50 μm ID packed, fused silica columns. The system accepts large injection volumes (typically 10 μL) and uses a trapping column for fast sample loading. Analytes are transferred and then separated on an analytical column at a flow rate of 100–125 nL/min. The design of the system focuses on both robustness for unattended operation and minimization of extracolumn dead volumes. Although the flow direction is controlled by switching valves, the analytes do not pass through any of these valves except the injection valve. A standard, high-pressure-mixing binary pump (not necessarily designed for nanoscale applications) is used, while the columns, splitter restrictors and electrospray emitters are home made. This provides maximum flexibility for a wide array of applications. Examples of protein and peptide identification at a concentration level of 100 amol/μL are given, including on-column derivatization. In addition, a minor modification of the setup provides the permanent option of using peak parking for interactive data acquisition (e. g. MS⁽ⁿ⁾ experiments during analyte elution).