New reagents for increasing ESI multiple charging of proteins and protein complexes.
The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom. 2009, 20, 593-596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to "supercharge" proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging: benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation.
Lomeli
Shirley H
SH
Department of Chemistry and Biochemistry, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California, USA.
Peng
Ivory X
IX
Yin
Sheng
S
Loo
Rachel R Ogorzalek
RR
Loo
Joseph A
JA
D000818:Animals; D006736:Horses; D007202:Indicators and Reagents / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D055672:Static Electricity; D013876:Thiophenes / Q000737:chemistry
D007202:Indicators and Reagents; D009211:Myoglobin; D011506:Proteins; D013876:Thiophenes; C013693:sulfolane