Protocol

Preparation and Use of an Integrated Microcapillary HPLC Column and ESI Device for Proteomic Analysis

This protocol was adapted from "The Use of Mass Spectrometry in Proteomics," Chapter 8, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Implementation of separation techniques in miniaturized formats on-line with high-performance mass spectrometers and the development of miniaturized sprayers as electrospray ionization (ESI) ion sources have reduced the amount of peptide required for complete and routine sequence characterization from several picomoles to a few femtomoles and below. Arguably, much of this gain in sensitivity is due to the combination of concentration-dependent type ionization devices such as ESI and on-line capillary separation devices of very small internal diameter (I.D.). This can be primarily attributed to a reduced mass flow rate of solvents and other background constituents into the ESI source, which allows for greater sample ionization efficiency. For reasons of robustness, most microcapillary HPLC (µLC) work is done with 75- or 100-µm I.D. capillary columns that clog less frequently than 50-µm I.D. capillary columns. The following protocol describes the preparation of an integrated C₁₈-packed capillary column-ESI microspray device. In this protocol, a polyimide capillary tip tapered to ~5 µm is used to hold the C₁₈-derivatized particles in place.