UCDHSC Anesthesiology Research - Clinical Nutrition Research Unit

Using Liquid Chromatography AND NANO-LC/MS in Proteomics Research

Electrospray ionization is a method of generating a very fine aerosol by electrostatic charging. This is accomplished by passing a liquid through a nozzle and electrically charging the liquid with a very high voltage (around 5000 volts). The charged liquid in the nozzle becomes unstable as it is forced to hold more and more charge and it blows apart into a cloud of tiny, highly charged droplets. These tiny droplets are less than 10 µm in diameter, and rapidly shrink as solvent molecules evaporate from their surface, often aided by a heated drying gas. As the droplets desolvate and shrink, the distance between electrical charges in the droplet dramatically decreases. When the electrical charge density reaches a critical state, the like charges repel each other and the droplet violently blows apart again.

The electrospray process has profoundly affected the field of mass spectrometry because it generates a distribution of multiply-charged ions, allowing structural analysis of large biomolecules such as peptides, proteins and DNA (which normally would be outside of the mass range of most mass spectrometers). In addition, electrospray ionization is directly compatible with liquid chromatography methods.

The most common electrospray apparatus pumps liquid through a pointed hollow metal tube, such as a syringe needle. A high-voltage power supply is used to generate a potential difference between the outlet of the tube and a nearby plate (counter-electrode). Either the tube or plate (entrance to the mass spectrometer) is commonly held at ground potential to charge the liquid. When the power supply is turned on and adjusted for the proper voltage, the liquid being pumped through the tube transforms into a fine continuous mist of droplets that fly rapidly toward the counter-electrode.

Commercial ESI mass spectrometers typically utilize flow rates ranging from10 µL/min to 1 mL/min. Because of the large volume of liquid exiting the tube, aerosol formation is usually assisted by pneumatic nebulization and/or by thermal heating in the effort to obtain a stable spray. This requirement is especially pronounced for highly aqueous liquids.

When the flow rate is reduced to nanoliters per minute (termed nanospray), droplet formation occurs readily, requiring only the applied voltage to generate spray. Spray is much more stable and signal is improved for aqueous mobile phases such as the 0.1% formic acid used in HPLC of peptides. Since electrospray ionization is a concentration-sensitive technique, lowering the flow rate effectively increases the analyte concentration exiting the tube. If the aperture of the tube is also decreased, the concentration effect increases more. If a nanobore LC column (75 um ID as compared with 2.1 or 4.6 mm ID used in standard electrospray) is interfaced to the system, even more analyte concentration can be achieved. Signal intensities for nanospray are often 2-3 orders of magnitude higher than those obtained using standard electrospray.

Sample Liquid Chromatography Experiments An excellent resource for nanospray information can be found at www.newobjective.com.

The base peak chromatogram plots the signal intensity of the most abundant ion in each mass spectrum during the time course of a chromatographic separation. This chromatogram is from an in gel digest of a 2D gel spot from a rat kidney preparation. The MS spectrum at a retention time of 26.4 minutes is displayed below.

The intensity of peptide ions is plotted vs. mass-to-charge ratio in the MS spectrum. Here we see the singly-charged ion at 1118.7 and the doubly-charged ion at 560. The doubly-charged ion was selected for fragmentation and the resultant product ion (MS/MS) spectrum is shown below.

The intensity of peptide fragment ions is plotted vs. mass-to-charge ratio in the MS/MS spectrum. The distance between the peaks is related to the amino acid mass, therefore computer algorithms can be used to determine the amino acid sequence of the peptide based upon its fragmentation pattern.

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