A new “best match” match model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GBapp) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that singly charged adducts were only observed for lower GB anions: HSO4-, I-, CF3COO-. Ions that have medium GBs (NO3-, Br-, H2PO4-) only form adducts having -2 charge states, whereas Cl- (higher GB) can form adducts having -3 charge states. Hydrogen bonds are the main interactions pertinent to the “Best Match” model, however, ion-ion interactions formed between peptides ([Glu]Fibrinopeptide B, Angiotensin I or [Asn1,Val5]-Angiotensin II) and low GB anions (ClO4- or HSO4-) have been established by CID-MS/MS. Evidence for ion-ion interactions comes especially from product ions formed during the first dissociation step, where, in addition to the expected loss of the anion or neutral acid, other product ions that require covalent bond cleavage (i.e., H2O or NH3 loss) are also observed. In this study, the “Best Match” model is further supported by the decomposition behavior of adducts formed when Na+/H+ exchange has occurred on peptides. Na+/H+ exchanges were found to occur preferentially at higher acidity sites. Without any Na+/H+ exchange, F- and CH3COO- can hardly form observable adducts with [Glu]Fibrinopeptide B. However, after multiple Na+/H+ exchanges, F- and CH3COO- do form stable adducts. This phenomenon can be rationalized by considering that Na+ cations serve to “block” the highly acidic sites, thereby forcing them to remain overall neutral. This leaves the less acidic protons available to match with higher GB anions. According to the "best match" model, high GB anions will match with high GBapp sites on the peptide, whereas low GB anions will match with low GBapp peptide sites. High charge states readily augment GBapp of the peptide (through-space effect). Na+/H+ exchanges substantially decrease GBapp by neutralizing charged sites, while slightly increasing intrinsic GBs by the inductive effect.

<p> Mass spectrometry (MS) is one of the most commonly used analytical techniques in bioanalytical analysis, allowing scientists to characterize molecules with very diverse chemical features. The advance in ionization strategies significantly improves the potential in using MS for that purpose, especially electrospray ionization (ESI) can generate ions directly from solution in ambient conditions, showing high flexibility in coupling with other techniques. Moreover, a hallmark of the ESI of large polymeric molecules is also its tendency to generate a distribution of charge states based on their chemical characteristics, allowing us to exploit the multiple charging phenomenon in various applications. </p> <p>This dissertation introduces the relationships between ESI and multiple charging phenomena with different proposed ionization models, and how condensed-phase and gas-phase approaches affect the multiple charging phenomenon. Moreover, multiply charged ions permit gas-phase ion/ion reactions to occur without neutralizing the ions. Therefore, various ion/ion reactions can be utilized for distinct analytical purposes. Objectively, this dissertation focuses on the investigation of the multiple charging phenomenon from ESI-MS, and the applications from taking the multiply charged ions to perform gas-phase ion chemistry in order to a) manipulate the charges of the targeted ions; b) invert the polarity of the targeted ions; c) and characterization of the ions from the gas-phase ion/ion reactions.</p> <p> The first work demonstrates how multiple components (i.e., complicated mixtures) lead to a highly congested spectrum of ions with overlapped m/z values, resulting from the multiple charging phenomenon after the ESI process. Utilizing ionic reactions can de-congest the spectra via manipulating the charges of the ions to separate the overlapped signals. A universal spectral pattern in the ESI mass spectra is observed while analyzing multiply-charged homopolymers. Various parameters, such as the charges of the ions, widths of polymer distributions, monomer mass, and cationizing agent masses, are investigated to show how they can affect the appearance of the unique patterns, which condense the information of the overall distribution of the homopolymers. Combined with gas-phase charge reduction (i.e., proton transfer reaction), we can characterize the size distribution of polydisperse homopolymer samples.</p> <p>Second, a novel type charge inversion ion/ion reaction summarizing the conversion of multiply charged protein ions to their opposite polarity and still holds multiple charges is reported. The reaction occurs via a single ion/ion collision with highly charged reagent ions, which we usually obtain from biological relevant polymers. Hyaluronic acid (HAs) anions and polyethylenimine (PEI) cations are used as the charge inversion reagents to react with protein ions. Remarkably, inversion of high absolute charge (up to 41) from the reaction is demonstrated. All mechanisms for ion/ion charge inversion involve low-energy ions proceeding via the formation of a long-lived complex. Factors that underlie the charge inversion of protein ions to the opposite polarity with high charge states in reaction with those reagent ions are hypothesized to include: (i) the relatively high charge densities of the HA anions and PEI cations that facilitate the extraction/donation of multiple protons from/to the protein leading to multiply charged protein anions/cations, (ii) the relatively high sum of absolute charges of the reactants that leads to high initial energies in the ion/ion complex, and (iii) the relatively high charge of the ion/ion complex following the multiple proton transfers that tends to destabilize the complex.</p> <p>Third, shotgun MS strategies coupled with different gas-phase ion chemistry and tandem MS to analyze glycolipids are demonstrated. Glycolipids contain both carbohydrates and lipids structure components that it is incredibly challenging to analyze with MS. Isomeric cerebrosides (n-HexCer) and glycosphingosines (n-HexSph), which hold isomerisms in diastereomeric sugar head groups (glucose and galactose), anomeric glycosidic linkages (alpha- or beta-), and isomeric amide-bonded monounsaturated fatty acyl chain (double bond location) are successfully differentiated by dissociating gas-phase ion/ion reaction products, the charge-inverted complex cations. Both relative and absolute quantification of the isomers is also achieved, and analytical performances are evaluated in terms of accuracy, precision, and inter-day precision, allowing us to perform mixture analysis. Porcine brains were used to demonstrate the ability to profile and quantify those isomers from biological extracts. Moreover, a parallel workflow is also proposed for gangliosides, which have more complicated structures among their glycan moiety. Metal cation transfer, proton transfer, and charge inversion reactions are utilized to manipulate the ion types to provide better structural information. The proposed workflow allows us to clean up the mass spectra by neutralizing interfering isobaric ions, differentiate isomeric gangliosides, and perform relative quantitation when the standards are available. The workflow also is used to obtain gangliosides profiles from biological matrices. Overall, work in this dissertation takes advantage of the multiple charging phenomenon and couples with gas-phase ion/ion reactions to achieve various analyses among a wide range of biological-related samples.</p>