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- Member of: Theses and Dissertations
There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity and limit of detection through matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis in Bruker’s Microflex LRF using polydimethylsiloxane (PDMS) reservoirs. In addition, initial imaging tests were performed on Bruker’s RapifleX MALDI Tissuetyper to determine the instrument’s imaging capabilities on proteins of interest through the use of a single layer “Christmas tree” microfluidic device, with the aim of applying a similar approach to future tissue samples. Data on 2µM insulin determined that a 95% laser power in the Microflex corresponded to 12-15% laser power in the RapifleX. Based on the experiments with insulin, the process of mixing insulin and saturated ɑ-Cyano-4-hydroxycinnamic acid (HCCA) matrix solvent in a 1:1 ratio using 10mM sodium phosphate buffer under area analysis is most optimized with a limit of detection value of 110 nM. With this information, the future aim is to apply this method to a double layer Christmas tree device in order to hopefully quantitatively analyze and image protein content in single or small cell populations.
I synthesized appropriate standards for secondary ion mass spectrometry (SIMS) measurements of 10Be–10B, necessary for accurate determination of the 10Be/9Be ratio. I then analyzed 32 CAIs for 10Be–10B as well as 6 CAIs for 26Al–26Mg and 5 CAIs for oxygen isotopes within this sample set using SIMS. Previous studies analyzed CAIs primarily from CV3 chondrites, which are known to have experienced thermal metamorphism and aqueous alteration. My work included a variety of CAIs (Type A, B, fine-grained, igneous) from CV3oxidized, CV3reduced, CO3, CR2, and CH/CB chondrites. Finally, after evaluating my data and literature data consistently, I statistically tested whether all CAIs belong to a single 10Be population. I find that the majority (~85%) of the normal (i.e., without large isotopic fractionations or anomalies), 26Al-bearing CAIs recorded a single value, 10Be/9Be = (7.0 ± 0.2) × 10-4. Although 6 CAIs recorded higher or lower values, these are plausibly explained by secondary alteration processes. The galaxy-wide average value of 10Be/9Be from GCR interactions 4.56 billion years ago is predicted to be <2 × 10-4; the value I measured is more than 3 times higher. Because GCRs trace supernovae and star formation, my results suggest a similarly enhanced star formation rate in the molecular cloud within ~1 kpc of the Sun, in the ~15 Ma prior to the Sun’s birth.
(LC-MS/MS) is used to identify and quantify peptides and proteins. LC-MS/MS produces mass spectra, which must be searched by one or more engines, which employ
algorithms to match spectra to theoretical spectra derived from a reference database.
These engines identify and characterize proteins and their component peptides. By
training a convolutional neural network on a dataset of over 6 million MS/MS spectra
derived from human proteins, we aim to create a tool that can quickly and effectively
identify spectra as peptides prior to database searching. This can significantly reduce search space and thus run time for database searches, thereby accelerating LCMS/MS-based proteomics data acquisition. Additionally, by training neural networks
on labels derived from the search results of three different database search engines, we
aim to examine and compare which features are best identified by individual search
engines, a neural network, or a combination of these.