<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>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/19338101 |
| Date | 20 April 2022 |
| Creators | Hsi-Chun Chao (12224828) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/INVESTIGATION_OF_MULTIPLE_CHARGING_PHENOMENON_AND_GAS-PHASE_ION_ION_REACTIONS_FOR_BIOLOGICAL_SYNTHETIC_POLYMERS_AND_GLYCOLIPIDS/19338101 |
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