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.
Department of Chemistry and Biochemistry, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California, USA.
Shirley H
SH
Lomeli
Ivory X
IX
Peng
Sheng
S
Yin
Rachel R Ogorzalek
RR
Loo
Joseph A
JA
Loo
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