Presentation Title

Derivatization of Peptide Carboxyl Groups for the Detection of Uncharged Peptides by Mass Spectrometry

Faculty Mentor

Markus Kalkum

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 40

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Major histocompatibility complex (MHC) I molecules are expressed on the surface of nuclear cells, and their role is to present antigen peptides to T cells, which recognize them as self or non-self. During hypertension, reactive oxygen species (ROS) are produced that oxidize fatty acids, leading to γ-ketoaldehydes, or isoketal formation. Isoketals covalently modify peptides and cause them to be recognized as non-self, activating T cells and eliciting an immune response that repeats this cycle. Identification of isoketal-peptides by mass spectrometry (MS) is challenging due to the elimination of the positive charge of the modified lysine and the neutral or negative charge of the ketal group. To enable peptide identification by MS, we used the quaternary amine cholamine to derivatize angiotensin II (Ang II) in order to determine the conditions under which carboxyl groups can be cholaminated. The first approach to cholamine modification involved reacting the peptide with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) and cholamine, but the modification was not present. Therefore, an alternative approach using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was implemented, and the cholamine modification was detected at m/z 1130.6. Because of the potential for cross linking between cholamine-peptide amino coupling, we propionylated potentially reactive amino groups. Between the single charged unmodified peptide and propionylated peptide, the m/z increased by 56 for the singly and by 28 for the double charged peptide. When the precursors were fragmented, the b series ions of the modified peptide showed the same m/z shifts. In conclusion, we established protocols for cholamine modification and EDC worked better than DMTMM as a coupling reagent. Propionylation may be necessary in order to sterically hinder reactive amino groups. Further experimentation will test the efficiency of cholamine coupling to isoketal-modified peptides.

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Nov 18th, 10:00 AM Nov 18th, 11:00 AM

Derivatization of Peptide Carboxyl Groups for the Detection of Uncharged Peptides by Mass Spectrometry

BSC-Ursa Minor 40

Major histocompatibility complex (MHC) I molecules are expressed on the surface of nuclear cells, and their role is to present antigen peptides to T cells, which recognize them as self or non-self. During hypertension, reactive oxygen species (ROS) are produced that oxidize fatty acids, leading to γ-ketoaldehydes, or isoketal formation. Isoketals covalently modify peptides and cause them to be recognized as non-self, activating T cells and eliciting an immune response that repeats this cycle. Identification of isoketal-peptides by mass spectrometry (MS) is challenging due to the elimination of the positive charge of the modified lysine and the neutral or negative charge of the ketal group. To enable peptide identification by MS, we used the quaternary amine cholamine to derivatize angiotensin II (Ang II) in order to determine the conditions under which carboxyl groups can be cholaminated. The first approach to cholamine modification involved reacting the peptide with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) and cholamine, but the modification was not present. Therefore, an alternative approach using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was implemented, and the cholamine modification was detected at m/z 1130.6. Because of the potential for cross linking between cholamine-peptide amino coupling, we propionylated potentially reactive amino groups. Between the single charged unmodified peptide and propionylated peptide, the m/z increased by 56 for the singly and by 28 for the double charged peptide. When the precursors were fragmented, the b series ions of the modified peptide showed the same m/z shifts. In conclusion, we established protocols for cholamine modification and EDC worked better than DMTMM as a coupling reagent. Propionylation may be necessary in order to sterically hinder reactive amino groups. Further experimentation will test the efficiency of cholamine coupling to isoketal-modified peptides.