Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses

This study explores the potential of a novel electrospray based method, ter med gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent bio complexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions w ere formed by nano electrospray ionization (ESI) in the 'cone jet mode'. Th e multiple charged state of the monodisperse droplets/ions generated was re duced by means of bipolar ionized air (generated by an alpha -particle sour ce) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophore tic mobility in air. Finally, the ions are detected using a standard conden sation particle counter. Data were expressed as electrophoretic mobility di ameters by applying the Millikan equation. The measured electrophoretic mob ility diameters, or Millikan diameters, of 32 well-defined proteins were pl otted against their molecular weights in the range 3.5 to 1920 kDa and exhi bited an excellent squared correlation coefficient (r(2) = 0.999). This fin ding allowed the exact molecular weight determination of large (glyco)prote ins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the bin ding stoichiometry of the intact complex and the complex stability as a fun ction of pH, for example, can be derived. Examples of specific protein comp lexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecif ic complexes. A discussion of current and future applications of charge-red uced nano ESI GEMMA, such as chemical reaction monitoring (reduction proces s of immunglobulin G) or size determination of an intact virus, a supramole cular complex, and monitoring of partial dissociation of a human rhinovirus es, is provided. Copyright (C) 2001 John Wiley & Sons, Ltd.