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, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) 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 electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins 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 binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.
Bacher
G
G
Institute for Analytical Chemistry, University of Vienna, Währinger Str. 38, A-1090 Vienna, Austria.
Szymanski
W W
WW
Kaufman
S L
SL
Zöllner
P
P
Blaas
D
D
Allmaier
G
G
D004586:Electrophoresis; D006023:Glycoproteins / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012229:Rhinovirus / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014780:Viruses / Q000737:chemistry*
D006023:Glycoproteins; D007074:Immunoglobulin G; D010455:Peptides; D011506:Proteins