HPLC-MS : Chapter Three

Chapter three

Electrospray Ionization with Low-Energy Collisionally

Activated Dissociation Tandem Mass Spectrometry of

Complex Lipids: Structural Characterization and

Mechanisms of Fragmentation

Fong-Fu Hsu and John Turk

Mass Spectrometry Resource, Division of Endocrinology, Diabetes, Metabolism, and Lipid

Research, Department of Internal Medicine, Washington University School of Medicine, St.

Louis, MO 63110

Introduction

The development of the desorption technique of fast atom bombardment (FAB) combined with mass spectrometry (MS) has permitted direct analyses of intact complex lipid molecules (1–4). One of the earliest applications of the technique to achieve structural characterization of complex lipids was the analysis of long-chain fatty acids by using tandem magnetic sector/high-energy collisionally activated dissociation (CAD) (5,6). These studies led to the discovery of charge-remote fragmentation (CRF), which yields ions informative for locating the position(s) of double bonds within the fatty acyl chains (7–10). The product ions from FAB-generated molecular ions of phospholipids, following CAD with high- or low-energy, could also identify the head groups and the positions of fatty acid substituents on the glycerol backbones. Many of the structurally informative ions generated by CAD are thought to derive from CRF processes (1–10). Although FAB is powerful and, with MS/MS, can yield rich structural information, its sensitivity is limited. As a result, tremendous effort is required to obtain sufficient quantities of biological specimens to yield informative FAB mass spectra (11,12). However, the recent introduction of electrospray ionization (ESI) MS (13,14) to analyze intact complex lipids has further advanced the research in this field to a new level (15,16). ESI-MS analyses of phospholipids are 2 to 3 orders of magnitude more sensitive than those achieved by FAB/MS (17–19). The superb sensitivity and ease of continuous sample introduction with ESI, coupled with tandem MS, provide opportunities to explore the structures and fragmentation processes of complex lipids in greater detail. The ions generated by ESI have also minimized some of the problems associated with FAB/MS, such as the complication of spectra with matrix ions.