More than half of proteins in humans are modified with carbohydrates in a process called glycosylation, yet this process remains poorly understood even though approximately 1% of the expressed human genome encodes biosynthetic machinery for glycosylation. Unlike genomics and proteomics where high throughput tools are now routinely used to generate hypothesis-directed research there is a lack of tools that can probe and profile the activity of enzyme mediating protein glycosylation in a similar manner. A promising analytic platform combines self-assembled monolayer surfaces and mass spectrometry to detect and quantitative enzymatic changes on diverse substrates, including proteins and carbohydrates. This approach is called Self-Assembled Monolayers coupled with Matrix-Assisted Desorption Ionization (SAMDI-MS) mass spectrometry and can be used to measure glycosyltransferase activity. In this dissertation, I explore the use of SAMDI-MS to measure glycosyltransferases activities on protein and carbohydrate substrates from complex samples such as cellular lysates. Appropriate details and background are discussed in Chapters 1 and 2 regarding glycosyltransferases and SAMDI-MS, respectively. I demonstrate the ability to detect endogenous activity from MDA-MB-231 breast cancer cell lysates. This work is discussed in Chapter 3. I also explore the capability of combining Cell-free Protein Synthesis technology with SAMDI-MS to detect and quantitate relative glycosylation on histidine-tagged protein substrates. I apply this method to study glycosyltransferase activity on a library of recombinant proteins by the activity of a cytoplasmic N-glycosyltransferase. This work is discussed in chapter 4. Additional information regarding this study and its particulars are included in the appendix. This dissertation demonstrates the potential of SAMDI-MS as an enabling profiling technology and details a rapid assay platform providing the throughput and means to rapidly evaluate hundreds of protein substrates without requiring cloning, utilization of antibodies, expression in living cells, or substantial quantities of material. The work presented addresses a technological gap in the measurement of glycosyltransferase activity relevant to efforts requiring large numbers of protein substrates and complement current efforts to design glycosylation sites on proteins of therapeutic interest.

Creator

• Techner, Jose-Marc

DOI

• https://doi.org/10.21985/n2-emf6-jg18

Subject

• Bioengineering
• Analytical chemistry
• Biochemistry

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:14598
• etdadmin_upload_661107

Keyword

• SAMDI
• High-throughput protein synthesis
• glycosylation
• self-assembled monolayers
• glycosyltransferase
• Mass spectrometry

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright