Ion Mobility Measurements of Nondenatured 12-150 kDa Proteins and Protein Multimers by Tandem Differential Mobility Analysis-Mass Spectrometry (DMA-MS)

The mobilities of electrosprayed proteins and protein multimers with molecular weights ranging from 12.4 kDa (cytochrome C monomers) to 154 kDa (nonspecific concanavalin A hexamers) were measured in dry air by a planar differential mobility analyzer (DMA) coupled to a time-of-flight mass spectrometer (TOF-MS). The DMA determines true mobility at atmospheric pressure, without perturbing ion structure from that delivered by the electrospray. A nondenaturing aqueous 20 mM triethylammonium formate buffer yields compact ions with low charge states, moderating polarization effects on ion mobility. Conversion of mobilities into cross-sections involves a reduction factor xi for the actual mobility relative to that associated with elastic specular collisions with smooth surfaces. xi is known to be 1.36 in air from Millikan's oil drop experiments. A similar enhancement effect ascribed to atomic-scale surface roughness has been found in numerical simulations. Adopting Millikan's value xi = 1.36 and assuming a spherical geometry yields a gas-phase protein density rho (p) = 0.949 +/- 0.053 g cm(-3) for all our protein data. This is substantially higher than the 0.67 g cm(-3) found in recent low-resolution DMA measurements of singly charged proteins. DMA-MS can distinguish nonspecific protein aggregates formed during the electrospray process from those formed preferentially in solution. The observed charge versus diameter relation is compatible with a protein charge reduction mechanism based on the evaporation of triethylammonium ions from electrosprayed drops.