Date of Graduation


Document Type


Degree Type



Eberly College of Arts and Sciences



Committee Chair

Stephen Valentine

Committee Member

Brian Popp

Committee Member

Glen Jackson

Committee Member

John Mertz

Committee Member

Jennifer Gallagher


Although the birth of native mass spectrometry (native MS) occurred only several years after the development of electrospray ionization (ESI), it is yet considered an emerging technique for MS-based studies. This stems in part from challenges associated with efficient ionization of biopolymers. Although steady gains in ion production for native structure characterization have been secured primarily with the use of nanoelectrospray ionization (nESI), under negative polarity, the ionization efficiency can be extensively reduced due to an effect called “corona discharge”. The corona discharge occurs at the tip of ESI emitters because of free electron acceleration in the high-field region produced during ESI. As a result, the stability of the spray decreases ultimately leading to ion signal loss. Many effective approaches have been introduced to suppress the discharge including instrumental modifications and methods development strategies. While these modifications have been efficacious, their ability to maintain the native state of biomolecules is still of concern. Moreover, several require major MS instrument modifications and can be costly. In this work, capillary vibrating sharp-edge spray ionization (cVSSI) is implemented for negative-ion, native-MS experiments. Early on, VSSI demonstrated an ability to behave as a soft ionization technique similar to ESI.

In the present study, a simple modification of cVSSI is introduced by coupling an electric field. During these experiments, the opportunity to suppress corona discharge and enhance ion production is observed. First, the enhancement in ion production and S/N levels is studied for various types of biomolecules including DNA oligonucleotides (Chapter 2). The intensity gains are compared with a commercial ion source (heated ESI or HESI), which uses nebulizing gas to suppress discharge. In general, 10 to 100-fold signal enhancement and 3 to 10-fold S/N gains are observed for DNA oligonucleotides when using field-assisted cVSSI. Moreover, the new method’s ability to preserve native conformations of higher-order DNAs and their complexes is demonstrated. In Chapter 3, the advantage of ion signal enhancement is utilized for tandem MS (MS/MS) and multistage tandem MS (MSn) experiments. High precursor ion intensities are considered to be important during MS/MS and MSn experiments because such signal is divided among the fragments at each fragmentation step. Due to the significantly higher precursor ion levels observed with field-assisted cVSSI, the fragment ions detected at the MS3 stage for several molecules have better defined peaks compared to commercial ESI. The relevance of ion intensity and S/N gains in obtaining high-quality fragment ion intensities is addressed. The results demonstrate that the precursor ion intensity gain plays a main role in producing informative, late-stage fragment ion mass spectra. Further, field-assisted cVSSI demonstrates higher sensitivity when compared to cESI and HESI. In the presence of organic solvent (10% and 20% methanol), the ion intensity increases for negatively-charged myoglobin with cVSSI and cESI. cVSSI shows an increase in total ion counts of up to 20% and exhibits a broader working voltage range. Overall, the research implies that the sensitivity boost afforded by cVSSI for MS/MS and MSn can be generalized to be ≥10 fold. In Chapter 4, a dual-tip cVSSI setup is implemented for in-droplet hydrogen/deuterium (HDX)-MS studies of oligonucleotides. Different folded conformers of DNA molecules are examined. The dramatically shortened labeling time provides the ability to distinguish different foldamers in solution; different reaction efficiencies are observed for single strand, duplex, and quadruplex conformers. In quadruplex conformers, polymorphic structures show greater deuterium incorporation in solution due in part to increased flexibility. Future work may focus on developing methods to guide VSSI-generated droplets into the MS inlet as well as investigating the potential of field-assisted cVSSI to examine other DNA, RNA, and protein conformation analyses. Overall, field-assisted cVSSI is a simple, robust, and cost-effective ionization approach with significant advantages for native-MS analysis.

Embargo Reason

Publication Pending