Selective Derivation of Protein Carboxylate Esters: Development of a New Detection and Quantification Method Laura A. Gillies Department of Chemistry, Washington State University, Pullman, WA 99164 Spring 2004 Senior Thesis Advisor: Zhouhui Sunny Zhou ---..... Honors Thesis ************************* PASS WITH DISTINCTION \. TO THE UNIVERSITY HONORS COLLEGE: As thesis advisor for Lo..u (~ G,'I/ies, I have read this paper and find it satisfactory. ~jlj#-- /vf 4it .. (.'" 2. ~, 2. 00't Date Selective Derivation of Protein Carboxylate Esters: Development of a New Detection and Quantification Method Laura A. Gillies Department of Chemistry, Washington State University, Pullman, WA 99164 Spring 2004 Senior Thesis Advisor: Zhouhui Sunny Zhou Precis Post-translational modifications of a protein can affect the structural stability or biological activity of a protein. One such modification is the esterification of protein carboxylic acids. Formation of protein carboxylate esters is involved in protein damage repair and signaling transduction. Current methods for detecting protein carboxylate esters are cumbersome and only semi-quantitative. The ability to detect and quantify these modifications using a proteomics approach is an important step to aid in further study of these modifications. Here we show a method being developed for facile detection and precise quantification of protein carboxylate esters. In the first step of the method, esters react with hydrazine to fonn a hydrazide. We utilized several UV active model esters to optimize the conditions for this reaction. These compounds allow for quick and facile screening of reaction conditions using UV-visible spectrometry or high performance liquid chromatography. Against common notions, we found this reaction gave high yields of the hydrazide. Furthermore, under these conditions proteins tested up to 147 IlM remained stable. From the studies of model carboxylic esters, a 2nd-order kinetic dependence on hydrazine concentration was found. The stability of several randomly selected proteins in 1.0 M hydrazine was investigated; all of these proteins remained intact even after prolonged incubation up to 10 days. The second step is to detect and quantify the hydrazide intermediate. For precise detection it is important only the hydrazide formed in step one is derivatized, and not other amines in the sample. Possible derivatizing agents include UV active or fluorescent compounds, or compounds which can be easily detected by mass spectrometry. First, we tried derivatizing the hydrazide with an aldehyde tag to form a hydrazone. Several aldehyde tags were used to successfully derivatize model hydrazides. 2 Using L-Iysine we were able to show selectivity of the aldehyde tag for reaction with hydrazide. However the hydrazones formed were found to be unstable under the acidic conditions commonly used for mass spectrometry analysis of peptides. For facile detection and application for protein research it is essential our method is suitable for analysis by mass spectrometry. Therefore, as an alternative derivatizing agent we are testing a dansyl containing tag. Derivation with the dansyl tag will allow mass spectrometry analysis under acidic conditions, and has been shown selective for hydrazide. We will next use ~-delta sleep inducing peptide to further optimize the method conditions. When complete, our method will allow for facile and precise detection and quantification of protein carboxylate ester in biological samples. Future applications include further study of protein repair processes, signaling transduction, and discovery of new biological functions of protein carboxylate esters. 3 Table of Contents Selective Derivation of Protein Carboxylate Esters: Development of a New Detection and Quantification Method Precis 2 List of Figures and Schemes 5 Acknowledgements 6 Abbreviations 6 Introduction 7 Materials and Methods 10 Results and Discussion 14 Conclusion 27 References 28 4 List of Figures and Schemes Scheme 1: PIMT repair cycle 8 Scheme 2: Proposed method 9 Scheme 3: Rxn of 4-nitrophenoxy acylal with hydrazine 14 Figure 1: UV spectra of4-nitrophenoxy acylal with hydrazine 16 Figure 2: Rate is dependent on hydrazine concentration 16 Scheme 4: Rxn of Ac-Tyr-OMe with hydrazine 17 Figure 3: Ester converts to hydrazide in 82% yield 18 Scheme 5: [3-DSIP reaction 19 Figure 4: Acetic hydrazide with 4-nitrobenzaldehyde fonn hydrazone 20 Scheme 6: Rxn of4-nitrobenzaldehyde with acetic hydrazide 21 Figure 5: Hydrazone fonnation rate is pH dependent 21 Scheme 7: Rxn of Ac-Tyr-Hyd with 3-hydroxy-4-nitrobenzaldehyde 23 Figure 6: Aldehyde and hydrazone peaks co-elute 23 Scheme 8: Rxn of Lysine with aldehyde and Ac-Tyr-Hyd 24 Scheme 9: Reduction of hydrazone using pyridine borane 26 Scheme 10: Hydrazide derivatization with dansyl chloride 26 5 Acknowledgements Many people contributed to the completion of this project through their support, feedback, and helpful discussions. I would like to thank Joshua Alfaro, Katy Ritter, Shahrzad Mansouri, and my other lab mates for their technical support and project feedback. I am thankful for the support and encouragement provided by Ralph Yount, my family and friends. Thank you to the College of Sciences Undergraduate Mini-Grant, College of Phannacy Summer Undergraduate Research Fellowship, and Hennan Frasch Foundation (541-HF02 to Z.S.Z.) for their financial support. Finally, I would like to thank my mentor Professor Sunny Zhou for his support, patience, and mentoring on this project. I have gained a priceless amount of knowledge about what it means to be a scientist, experimental methods, and technical writing. His drive and enthusiasm encourage and inspire me to do more than I often think is possible. Abbreviations Ac-Tyr-Hydrazide, N-acetyl-L-tyrosine hydrazide; Ac-Tyr-OMe, N-acetyl-L-tyrosine methyl ester; AdoMet, S-adenosyl-L-methionine; ~-DSIP, ~-delta sleep inducing peptide; DMF, dimethyl fonnamide; DMSO, dimethyl sulfoxide; HEPES, N-(2­ hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; HPLC, high perfonnance liquid chromatography; LuxS, S-ribosylhomocysteinease; PIMT, protein isoaspartyl methyltransferase; TFA, trifluoric acetic acid. 6 Introduction Protein post translational modifications can affect the biological activity and structural stability of a protein. [1,2] One such modification is the esterification of protein carboxylic acid groups. For instance, aspartyl and asparginyl groups on aged proteins can deaminate, racemize, and isomerize.[3] This leads to fonnation of carboxylic acid containing D and L-isoaspartyl and D and L-isoasparginyl residues. These damaged proteins are repaired by the enzyme protein isoaspartyl methyldtransferase (PIMT, Ee 2.1.1.77). PIMT converts the damaged residue from a carboxylic acid to methyl ester through transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the damaged residue.[4,5] The newly fonned methyl ester can cyclize with the protein backbone to fonn a succinimide intennediate. Depending on which bond is broken, succinimide can hydrolyze back to the natural L-aspartyl residue. This cycle allows for repair of age­ damaged proteins (Scheme 1). 7 o o ~lOH ~NH2 r-""'" N~( NH""0, r-""'" N~( NH""o, H 0 H 0 L-asparagine L-aspartate ~ oY / N""""'­ r-"""'N ~"­ H 0 L-succinimide 0/ ~o ,JlNH""" ~NH""" PIMT r-""'" N H • Ayo AdoMet r-""'" N~O'CH3 H 0 H 0 L-isoaspartate L-isoaspartate methyl ester SCHEME 1: Repair cycle for age-damaged proteins. From the succinic intermediate damaged proteins can, depending on what bond is broken, return to a normal residue, or continue in the repair cycle. Formation of carboxylate ester occurs during the repair stage of the cycle. Further study of protein carboxylate esters requires precise detection and quantification. Current methods for detection of these esters are cumbersome and often biologically incompatible.[6] One common method uses radio labeled AdoMet to transfer a radioactive methyl group to the protein carboxylic acid. Another current method for protein carboxylate ester quantification measures the methanol formed during the conversion of carboxylic acid to carboxylate ester. Other methods require lengthy and cumbersome high performance liquid chromatography (HPLC) teclmiques. Here we show a method for facile 8 detection and precise quantification of protein carboxylate esters under biologically compatible conditions (Scheme 2.) First, as shown in Scheme 2, the reaction of hydrazine with esters results in formation of a hydrazide intennediate. This reaction has been suggested, but not reported in the literature.[7] Utilizing model compounds we optimized conditions of this reaction in aqueous hydrazine at pH 8.0. Second, the hydrazide intennediate is derivatized with a selective tag, thus allowing the original carboxylate ester to be precisely detected and quantified. Derivatizations of hydrazides with aldehydes have been previously reported. [8­ lO] In addition, as an alternative derivatizing agent we will use dansyl chloride to derivatize hydrazide. STEP 1 H ~r~CH3 + H2N-NH2 • ~r~NH2 Hydrazine Y Ester Hydrazide Acid STEP 2 Hr§) H H o O~N /~ ~N'~ 2-a H N Hydrazone °r 'NH2 .-vvv"" 'VJVVV Hydrazide 2·b s~ o NONH 'N-S /; ~ /\ N/ H " os/; frVVVJ\ "'"""""" 0 ­ CI-S \ r o ~ /; Dansyl Hydrazide Dansyl Chloride SCHEME 2: In Step I, esters react with hydrazine to fonn a hydrazide. In Step 2, hydrazide is derivatized with a selective tag. Possible tags include aldehyde compounds, resulting in fonnation of a hydrazone product (2-a), or dansyl chloride (2-b.) 9 Methods