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. (J Am Soc Mass Spectrom 2010, 21, 127-131) © 2010 American Society for Mass Spectrometry E lectrospray ionization is distinguished among other desorption/ionization sources for mass spectrometry by its ready generation of multiply charged molecules, the value of which was recognized early by Fenn's group: "This feature is very attractive in that it extends the effective mass range of any analyzer by a factor equal to the number of charges per ion. Moreover, because the ions have lower m/z values, they are generally easier to detect and weigh than are singly charged ions of the same mass [1]."After the utility of ESI-MS for protein analysis was demonstrated, multiple charging was rapidly extended to tandem mass spectrometry of large peptides and proteins [2,3]. Dissociation efficiency of large molecules is enhanced with increasing charge, allowing sequenceinformative product ions to be measured. Early ESI-MS studies investigated the potential of different solvents to increase charging [4]. However, useful enhancement of ESI multiple charging was not described clearly until Iavarone and Williams reported "supercharging" promoted by agents such as m-nitrobenzyl alcohol (m-NBA) [5,6]. For protein analyses, their work largely investigated solutions considered to be denaturing.Recently, we demonstrated that multiple charging of noncovalent protein complexes could be increased in ESI-MS when spectra are obtained from nondenaturing protein solutions containing up to 1% (vol/vol) m-NBA [7]. Increases in charge ranged from 8% for the 690 kDa 20S proteasome complex to 48% additional charge for zinc-bound 29 kDa carbonic anhydrase protein. These protein structures were cons...

Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based techniques by developing a multifunctional electrode with closed-loop temperature control, while minimizing overall system complexity, and addressing challenges associated with rapid evaporation. For the EWOD chip design, we discuss the performance of multifunctional surface electrodes for actuation, localized Joule heating, and thermistic temperature sensing. Furthermore, a hydrophilic pattern is formed in the multifunctional electrode to control the location of an evaporating droplet on the electrode. To demonstrate the capabilities and limitations of this technique, we performed three experiments and measured the results using MALDI-MS: (i) insulin disulfide reductions in DTT over a range of heater temperatures (22 to 70 °C) to show how reaction rates can be affected by thermal control, (ii) insulin disulfide reductions at 130 °C in DMSO to demonstrate a reaction in a high boiling point solvent, and (iii) tryptic digestions of cytochrome c at 22 and 40 °C to show that heated droplets can yield reasonably higher peptide sequence coverage than unheated droplets. Although they do not decouple the effects of changing temperatures and concentrations, these experiments verified that thermal cycling by EWOD electrodes accelerates reaction rates in liquid droplets in air.

Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottom-up techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MS ( n ) to protein analysis and show that ELDI is capable of multistage MS to MS (4) for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce "reactive-ELDI", which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.