Molecular Weight Determination

All three of the major approaches to mass spectrometric determination of the molecular weights of intact proteins are available in the Medical University of South Carolina Mass Spectrometry Laboratory. These include FAB (the term as used here also includes cesium ion bombardment), electrospray ionization (ESI), and matrix assisted laser desorption ionization (MALDI). The JEOL HX110/HX110 tandem mass spectrometer is equipped with a high voltage (35 kV.) cesium gun and has a mass range of 14,500. Molecular weights of proteins in excess of 10,000 daltons have been successfully determined on this magnetic sector type instrument using FAB ionization. The resolution and mass accuracy of the JEOL instrument is such that the molecular weights can be determined to better than a single mass unit. In practice the protein is applied as a solution (on the order of 1 microliter) to the tip of a sample probe and mixed with a matrix liquid (e.g. glycerol, thioglycerol or nitrobenzyl alcohol). The probe is then inserted into the mass spectrometer and the sample bombarded with high energy cesium ions to sputter off intact (usually protonated) molecular ions of the protein in the sample. The ions are then accelerated into the mass spectrometer and the mass to charge ratio determined. Since one is usually observing singly charged ions, the result is a direct measurement of molecular weight. A pure sample is not necessary, and it is possible to measure molecular weights of multiple components of a mixture although competition effects can cause some components to be observed more easily than others. Buffer salts and detergents can impede the observation of sample components; optimal samples are those collected from a reversed phase HPLC separation.

Electrospray ionization permits measurement of much larger molecular weights by virtue of the fact that it produces multiply charged ions. Since mass spectrometers normally measure mass to charge ratio, increasing the number of charges on the ion effectively multiplies the mass range of the instrument. Mass measurement accuracy on the order of 0.01% is possible with this technique. In practice the sample is introduced into the ion source in a liquid stream containing an organic modifier and an acid delivered from a pump driven microliter syringe or as an HPLC effluent. A variety of acids and concentrations are used in ESI; a typical solvent system for discrete samples is 50% methanol in water containing 5% acetic acid. Buffer salts and ionic detergents must be avoided since they can dominate the ion current and prevent observation of the protein. Since electrospray ionization gives multiple peaks for each protein, mixtures can give very complex spectra. Algorithms have been developed to resolve the complex mixtures of multiply charged ion peaks into a spectrum with a true mass scale and one peak per component, but the technique works best with relatively pure samples.

Matrix assisted laser desorption (MALDI) mass spectrometry permits measurement of still larger molecular weights. In this technique, the sample is mixed with a UV absorbing matrix compound (commonly sinapinic acid) typically in a 30% aqueous acetonitrile solution, and 1-3 microliters of the solution is dried on the end of a sample probe or as a spot on a sample plate. The dried sample is then inserted into the mass spectrometer and the sample mixture exposed to the focussed beam from a pulsed UV laser. Absorption of the laser energy by the matrix results in ejection of intact molecular ions of the protein. The ions produced are typically singly charged, therefore the mass analyzer must be of a different type to measure very large masses. A time-of-flight analyzer is used to measure mass by measuring the time it takes for an ion, after electrostatic acceleration, to travel from the ion source to the detector. Heavier ions travel slower, hence, by measuring long times, the mass range of this type of analyzer is theoretically unlimited. Molecular weights as high as 160,000 have been measured on our home built instrument with mass measurement errors less than 1% (less than 0.1% for masses under 20,000). The newly installed (11/96) commercial instrument will provide higher performance. The MALDI technique is applicable to mixture analysis and is less affected than ESI by the presence of salts or ionic detergents. (However, some detergents are more troublesome than others; see the section below on MALDI and detergents.)

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We are located in room 305 of the Children's Research Institute Building at MUSC.

Biomolecular Mass Spectrometry Facility Department of Pharmacology Medical University of South Carolina 173 Ashley Avenue, CRI 305 Charleston, SC 29425	Telephone Numbers: 843-792-5849 (CRI 305) 843-792-2471 (department office) FAX: 843-792-2475