JOURNAL OF PROTEOMICS 75 (2012) 2275– 2296 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot Tutorial Amino acids: Chemistry, functionality and selected ☆ non-enzymatic post-translational modifications Rainer Bischoffa,⁎, Hartmut Schlüterb aUniversity of Groningen, Department of Pharmacy, Analytical Biochemistry, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands bInstitute of Clinical Chemistry, University Medicine Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany ARTICLE INFO ABSTRACT Article history: The ultimate goal of proteomics is determination of the exact chemical composition of Received 12 October 2011 protein species, including their complete amino acid sequence and the identification of Accepted 31 January 2012 each modified side chain, in every protein in a biological sample and their quantification. Available online 22 February 2012 We are still far from achieving this goal due to limitations in analytical methodology and data analysis but also due to the fact that we surely have not discovered all amino acid Keywords: modifications that occur in nature. To detect modified side chains and to discover new, Amino acid chemistry still unknown amino acid derivatives, an understanding of the chemistry of the reactive Post-translational modifications groups of amino acids is mandatory. This tutorial focuses on the chemistry of the amino Protein species acid side chains and addresses non-enzymatic modifications. By highlighting some exem- Thiol group plary reactions a glimpse of the huge diversity of modified amino acids provides the reader Deamidation with sufficient insight into amino acid chemistry to raise the awareness for unexpected side Carbonylation chain modifications. We further introduce the reader to a terminology, which enables the comprehensive description of the exact chemical composition of a protein species, includ- ing its full amino acid sequence and all modifications of its amino acid side chains. This Tu- torial is part of the International Proteomics Tutorial Programme (IPTP number 10). © 2012 Elsevier B.V. Open access under CC BY-NC-ND license. 1. Introduction hydrophilic to hydrophobic depending on the respective side chain R (Figs. 1–9). In 1806 Vauquelin and Robiquet iso- 1.1. Historical background lated the first amino acid as a substance from Asparagus sati- vus, which they named asparagine [2].Someyearslater Amino acids are the primary building blocks of proteins. Of Braconnot discovered glycine from an acid hydrolysate of the over 300 naturally occurring amino acids, 22 constitute gelatin [3]. Threonine was described by Rose et al. as an es- the monomer units of proteins [1], which are chemically sential food ingredient in 1935 [4] and selenocysteine and linked via peptide bonds in genetically predefined se- pyrrolysine, the last two of the 22 proteinogenic amino quences to constitute the backbone of all known proteins. acids to be discovered, were reported in 1986 [5] and 2002, Their properties range from acidic to basic and from respectively [6]. ☆ This Tutorial is part of the International Proteomics Tutorial Programme (IPTP number 10). Details can be found at: http://www. proteomicstutorials.org/. ⁎ Corresponding author. Tel.: +31 50 3633338. E-mail address: [email protected] (R. Bischoff). 1874-3919 © 2012 Elsevier B.V. Open access under CC BY-NC-ND license. doi:10.1016/j.jprot.2012.01.041 2276 JOURNAL OF PROTEOMICS 75 (2012) 2275– 2296 Amino acid structure COOH Carboxyl group H N A Amino group 2 C H General formula R Side-chain (R) - COOH 2.34 COO pH 7 + 9.60 H N 2 C H H3N C H B H H Glycine G Gly IP: 5.97 H: -0.4 75.032028 [u] Fig. 1 – (A) Chemical structure of the amino acid glycine with the side chain R = H. (B) Amino acids generally occur as zwitterions at physiological pH due to partial ionization of the carboxylic acid and the amino group. The formulas present the ionization state predominating at pH 7. The hydropathy index (H) can be used to predict the tendency of an amino acid to seek a hydrophobic (values >1) or an aqueous (values <1) environment. Ionization state, isoelectric points (IP), dissociation constant “ (pKa) values and hydropathy index from Lehninger Principles of Biochemistry. 2000. 3rd ed. Nelson DL, Cox MM. Worth Publishers. New York”. The exact monoisotopic mass is given in u (source: http://pubchem.ncbi.nlm.nih.gov). COO− 2.34 Amino Acids with Alkyl Residues + 9.69 H3N C H − 2.36 CH COO 3 + Alanine 9.68 H3N C H A Ala HC– CH3 IP: 6.01 CH H: 1.8 2 COO − 2.36 89.047678 [u] CH + 2 COO − 9.60 H N H 3 C Isoleucine H2C CH2 + 9.62 H N H 2.32 H 3 C CH I C Ile H2C + − C C IP: 6.02 N COO 1.99 H H 10.96 H H C CH H: 4.5 2 2 2 H2C CH2 131.094629 [u] Proline Valine Leucine P V L Pro Val Leu IP: 6.48 IP: 5.97 IP: 5.98 H: 1.6 H: 4.2 H: 3.8 115.063329 [u] 117.14634 [u] 131.094629 [u] Fig. 2 – Proteinogenic amino acids with alkyl residues. The formulas present the ionization state predominating at pH 7. The hydropathy index (H) can be used to predict the tendency of an amino acid to seek a hydrophobic (values >1) or an aqueous (values <1) environment. Ionization state, IP, pK values and hydropathy index from “Lehninger Principles of Biochemistry. 2000. 3rd ed. Nelson DL, Cox MM. Worth Publishers. New York”. The exact monoisotopic mass is given in u (source: http://pubchem.ncbi.nlm.nih.gov). JOURNAL OF PROTEOMICS 75 (2012) 2275– 2296 2277 Amino Acid with an Aryl Residue Amino Acids with Carboxy Residues COO − 1.83 COO − 1.88 COO − 2.19 + + + 9.13 H3N C H 9.60 9.67 H3N C H H3N C H CH2 CH2 CH2 − 3.65 COO CH2 COO − 4.25 Aspartic acid Phenylalanine D Glutamic acid F Asp E Phe IP: 2.77 Glu IP: 5.48 H: - 3.5 IP: 3.22 H: 2.8 133.037508 [u] 165.078979 [u] H: - 3.5 147.053158 [u] Fig. 3 – Proteinogenic amino acid with an aryl residue. The Fig. 5 – Proteinogenic amino acids with carboxy residues. formulas present the ionization state predominating at pH 7. The formulas present the ionization state predominating at The hydropathy index (H) can be used to predict the pH 7. The hydropathy index (H) can be used to predict the tendency of an amino acid to seek a hydrophobic (values >1) tendency of an amino acid to seek a hydrophobic (values >1) or an aqueous (values <1) environment. Ionization state, IP, or an aqueous (values <1) environment. Ionization state, IP, pK values and hydropathy index from “Lehninger Principles pK values and hydropathy index from “Lehninger Principles of Biochemistry. 2000. 3rd ed. Nelson DL, Cox MM. Worth of Biochemistry. 2000. 3rd ed. Nelson DL, Cox MM. Worth Publishers. New York”. Publishers. New York”. The exact monoisotopic mass is given in u (source: http:// The exact monoisotopic mass is given in u (source: http:// pubchem.ncbi.nlm.nih.gov). pubchem.ncbi.nlm.nih.gov). 2. The chemistry of amino acids at least one carboxylic acid group providing a negative charge if deprotonated. At physiological pH both charges are present. 2.1. General aspects With respect to the functional side chain groups amino acids can have basic properties like arginine (isoelectric point (IP): Amino acids are zwitterions because they have at least one 10.76; Fig. 7) or acidic like glutamic acid (IP: 3.22; Fig. 5). amino group yielding a positive charge when protonated and Amino groups are protonated if the pH is lower than their Amino Acids with Hydroxy Residues COO − 2.21 COO − 2.11 COO − 2.20 + + + 9.15 9.11 H3N C H 9.62 H3N C H H3N C H CH2OH H-C-OH CH2 Serine CH3 S Threonine Ser T IP: 5.68 Thr H: - 0.8 OH 10.07 IP: 5.87 105.042593 [u] H: - 0.7 Tyrosine 119.058243 [u] Y Tyr IP: 5.66 H: -1.3 181.073893 [u] Fig. 4 – Proteinogenic amino acids with hydroxy residues. The formulas present the ionization state predominating at pH 7. The hydropathy index (H) can be used to predict the tendency of an amino acid to seek a hydrophobic (values >1) or an aqueous (values <1) environment. Ionization state, IP, pK values and hydropathy index from “Lehninger Principles of Biochemistry. 2000. 3rd ed. Nelson DL, Cox MM. Worth Publishers. New York”. The exact monoisotopic mass is given in u (source: http://pubchem.ncbi.nlm.nih.gov). 2278 JOURNAL OF PROTEOMICS 75 (2012) 2275– 2296 Amino Acids with Amide and Indole Residues COO − 2.02 COO − 2.17 COO − 2.38 + + + 8.80 9.13 9.39 H3N C H H3N C H H3N C H CH2 CH2 CH2 C=O CH2 Indole C=O NH2 N H Asparagine NH2 Tryptophan N W Asn Glutamine Trp IP: 5.41 Q IP: 5.89 H: - 3.5 Gln H: - 0.9 132.053492 [u] IP: 5.65 H: - 3.5 204.089878 [u] 146.069142 [u] Fig. 6 – Proteinogenic amino acids with amide and indole residues. The formulas present the ionization state predominating at pH 7. The hydropathy index (H) can be used to predict the tendency of an amino acid to seek a hydrophobic (values >1) or an aqueous (values <1) environment. Ionization state, IP, pK values and hydropathy index from “Lehninger Principles of Biochemistry. 2000. 3rd ed. Nelson DL, Cox MM. Worth Publishers. New York”. The exact monoisotopic mass is given in u (source: http://pubchem.ncbi.nlm.nih.gov).