<p>Electrospray ionization-tandem
mass spectrometry (ESI-MS/MS) has been established as a powerful tool for
qualitative and quantitative analysis of biomolecules. However, mass
spectrometric analysis of biomolecules is often limited by poor ionization
efficiency of analyte for sensitive detection and limited fragmentation for structural
characterization. Over the years, various solution phase as well as gas-phase
derivatization techniques, have been coupled with MS to increase the ionization
efficiency and facilitate the formation
of structural informative fragment ions. The research presented in this dissertation falls into
two major parts; focusing on method development and application of radical
chemistry for enhanced biomolecule analysis on an ESI-MS/MS platform. In the
first part, a method of rapid charge tagging of neutral lipids (e.g. sterols,
glycerides) with a thiol radical-based charge tag is developed, followed by
comprehensive analysis via ESI-MS/MS without the use of a chromatographic
separation (shotgun lipidomics). This charge tagging is performed in an easily
constructible fused silica capillary-based
microflow photo-reactor which is relatively low in cost and requires no
instrument modifications. This method significantly enhances the ionization efficiency of the neutral lipids for
sensitive MS detection (pM range). This method can be applied to the small volume of biological complex samples (e.g.
1 µL plasma) and doesn’t require extensive sample pretreatment procedure
(analysis time of 2 min vs. traditional >60 min on GC-MS and HPLC-MS
systems). Furthermore, the derivatized neutral lipids can also be fragmented
via soft collision-induced dissociation to obtain fatty acyl chain composition
of the neutral lipids (sterol esters, diacylglycerols, triacylglycerols, etc.) for structural characterization. This can
especially be useful for determination
for fatty acyl compositional isomers in neutral lipids for analysis related to
biomarker detection. The characteristic fragmentation pattern of tagged neutral
lipids has also been utilized for quantitation of lipids from biological
mixture samples. Initial application of
this method has shown alteration in the concentration of diacylglycerol lipid
species in clinical samples of Type 2 Diabetes Mellitus patients, suggesting
the potential of understanding the biological roles of such lipids in insulin
resistance. </p>
<p>In the second part, a unique approach of radical-induced disulfide bond cleavage in
peptides and proteins is demonstrated. Using 254 nm UV emission, acetone was
used as a photoinitiator to initiate secondary radical formation i.e.
hydroxyalkyl radical, from alcohol co-solvents used for electrospray. These
radicals can then be used to efficiently cleave the disulfide bonds (R-S-S-R)
in peptide/proteins to give reduced reaction products (RSH) at the cleavage
site. Upon soft collision-induced
dissociation, the reduced product gave abundant <i>b-</i> and <i>y-</i> type fragment
ions for complete or enhanced sequence coverage as compared to intact disulfide-linked peptides and proteins. With
the use of a simple microflow photo-reactor, this radical based approach can
also be coupled with infusion ESI-MS/MS for a rapid online-based peptide and protein
analysis. The yield for disulfide bond reduction was almost 100% within less
than 5 s of UV irradiation. Furthermore, by adjusting the UV irradiance time,
different degrees of partial reduction could be achieved, which greatly
facilitated the disulfide linkage mapping in peptides and proteins with
multiple disulfide bonds. This method has been incorporated with both bottom-up
and top-down approach for protein analysis for unraveling the molecular
complexity, quantifying and deep sequencing of disulfide-linked proteins.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/7988045 |
| Date | 10 June 2019 |
| Creators | Sarju Adhikari (5929454) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/RADICAL_CHEMISTRY_AND_MASS_SPECTROMETRY_FOR_ENHANCED_BIOMOLECULE_ANALYSIS/7988045 |
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