Solid Phase Peptide Synthesis and Mass Spectrometry Analysis of Ac-WCGPCK, WCGPCK, and CAG
Format
Poster Presentation
Faculty Mentor Name
Jianhua Ren
Faculty Mentor Department
Chemistry
Graduate Student Mentor Name
Michael Browne
Graduate Student Mentor Department
Chemistry
Abstract/Artist Statement
Introduction:
The purpose of synthesizing peptides is to add these specific sequences to a peptide library, a growing assortment of amino acid sequences that will help aid in further understanding the protein, thioredoxin. We synthesized the peptides WCGPCK, an acetylated WCGPCK and CAG. The three peptides that were analyzed are going to be used to study the protein thioredoxin. This protein is essential for life as it has an important role in a lot of cellular processes such as photosynthesis. Thioredoxin is an antioxidant enzyme that can be found in nearly all living organisms and it plays a role in making the reduction of other proteins easier. The protein has an active disulfide site (CGPC) and synthesizing peptides that form disulfides may help us understand the protein itself better.
Method:
To get the desired peptides, we used solid phase peptide synthesis. We start with a rink amide resin to hold the first amino acid. Then, deprotection with 20% piperidine in DMF is done to get rid of the F-moc group on the Nitrogen of the resin. The same Fmoc group resides on the amino acid Nitrogen and the same process of deprotection is utilized. The next step is coupling of amino acids from C-Terminus to N-Terminus. The last step is cleaving the resin off the peptide with trifluoroacetic acid. If acetylating a peptide, we will do acetylation before cleavage. This adds an acetyl group on the N-terminus of the peptide. To purify our sample we add diethyl ether and centrifuge to precipitate the sample, we repeat this step one more time. After purification we dry the peptide with Nitrogen gas and lyophilize the sample overnight to dry it completely. After lyophilization we analyze the peptide using mass spectrum analysis.
Results:
Mass spectrometry analysis is done on the peptides to see if they were sequenced successfully. A full scan will show the peaks identifying the types of ions in the peptide. A peptide product ion scan shows if the peptide was sequenced in the correct order. It is compared to expected fragmentation values to check similarity.
Conclusion:
Peptide product ion scans of CAG, WCGPCK, Ac-WCGPCK show that observed values match expected values, therefore making the synthesis a success.
Location
Virtual
Start Date
25-4-2020 1:00 PM
End Date
25-4-2020 3:00 PM
Solid Phase Peptide Synthesis and Mass Spectrometry Analysis of Ac-WCGPCK, WCGPCK, and CAG
Virtual
Introduction:
The purpose of synthesizing peptides is to add these specific sequences to a peptide library, a growing assortment of amino acid sequences that will help aid in further understanding the protein, thioredoxin. We synthesized the peptides WCGPCK, an acetylated WCGPCK and CAG. The three peptides that were analyzed are going to be used to study the protein thioredoxin. This protein is essential for life as it has an important role in a lot of cellular processes such as photosynthesis. Thioredoxin is an antioxidant enzyme that can be found in nearly all living organisms and it plays a role in making the reduction of other proteins easier. The protein has an active disulfide site (CGPC) and synthesizing peptides that form disulfides may help us understand the protein itself better.
Method:
To get the desired peptides, we used solid phase peptide synthesis. We start with a rink amide resin to hold the first amino acid. Then, deprotection with 20% piperidine in DMF is done to get rid of the F-moc group on the Nitrogen of the resin. The same Fmoc group resides on the amino acid Nitrogen and the same process of deprotection is utilized. The next step is coupling of amino acids from C-Terminus to N-Terminus. The last step is cleaving the resin off the peptide with trifluoroacetic acid. If acetylating a peptide, we will do acetylation before cleavage. This adds an acetyl group on the N-terminus of the peptide. To purify our sample we add diethyl ether and centrifuge to precipitate the sample, we repeat this step one more time. After purification we dry the peptide with Nitrogen gas and lyophilize the sample overnight to dry it completely. After lyophilization we analyze the peptide using mass spectrum analysis.
Results:
Mass spectrometry analysis is done on the peptides to see if they were sequenced successfully. A full scan will show the peaks identifying the types of ions in the peptide. A peptide product ion scan shows if the peptide was sequenced in the correct order. It is compared to expected fragmentation values to check similarity.
Conclusion:
Peptide product ion scans of CAG, WCGPCK, Ac-WCGPCK show that observed values match expected values, therefore making the synthesis a success.