Abstract
Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH’s (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.
Similar content being viewed by others
References
Alfaro JF et al (2008) Chemo-enzymatic detection of protein isoaspartate using protein isoaspartate methyltransferase and hydrazine trapping. Anal Chem 80:3882–3889. doi:10.1021/ac800251q
Biastoff S, Teuber M, Zhou ZS, Dräger B (2006) Colorimetric activity measurement of a recombinant putrescine N-methyltransferase from datura stramonium. Planta Med 72:1136–1141. doi:10.1055/s-2006-947191
Böhme L, Bär JW, Hoffmann T, Manhart S, Ludwig H-H, Rosche F, Demuth H-U (2008) Isoaspartate residues dramatically influence substrate recognition and turnover by proteases. Biol Chem 389:1043–1053. doi:10.1515/bc.2008.123
Capasso S, Kirby AJ, Salvadori S, Sica F, Zagari A (1995) Kinetics and mechanism of the reversible isomerization of aspartic acid residues in tetrapeptides. J Chem Soc Perkin Trans 2:437–442. doi:10.1039/P29950000437
Chen Z-W, Bergman T, Östenson C-G, Efendic S, Mutt V, Jörnvall H (1997) Characterization of dopuin, a polypeptide with special residue distributions. Eur J Biochem 249:518–522. doi:10.1111/j.1432-1033.1997.t01-2-00518.x
Chen T et al (2010) Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferase 1 identified using phage display and biopanning. J Biol Chem 285:37281–37292. doi:10.1074/jbc.M110.157008
Chen WQ, Karnaukhova E, Lubec G (2013) The use of native gels for the concomitant determination of protein sequences and modifications by mass spectrometry with subsequent conformational and functional analysis of native proteins following electro-elution. Amino Acids 44:1381–1389. doi:10.1007/s00726-013-1477-1
Chumsae C, Gifford K, Lian W, Liu H, Radziejewski CH, Zhou ZS (2013) Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species. Anal Chem 85:11401–11409. doi:10.1021/ac402384y
Chumsae C et al (2014) Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody. Anal Chem 86:8932–8936. doi:10.1021/ac502179m
Clarke S (2003) Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair. Ageing Res Rev 2:263–285. doi:10.1016/S1568-1637(03)00011-4
Dai S, Ni W, Patananan AN, Clarke SG, Karger BL, Zhou ZS (2013) Integrated proteomic analysis of major isoaspartyl-containing proteins in the urine of wild type and protein l-isoaspartate O-methyltransferase-deficient mice. Anal Chem 85:2423–2430. doi:10.1021/ac303428h
Dashtiev M, Wäfler E, Röhling U, Gorshkov M, Hillenkamp F, Zenobi R (2007) Positive and negative analyte ion yield in matrix-assisted laser desorption/ionization. Int J Mass Spectrom 268:122–130. doi:10.1016/j.ijms.2007.07.001
Drapeau GR, Boily Y, Houmard J (1972) Purification and properties of an extracellular protease of Staphylococcus aureus. J Biol Chem 247:6720–6726
Du Y, Wang F, May K, Xu W, Liu H (2012) Determination of deamidation artifacts introduced by sample preparation using 18O-labeling and tandem mass spectrometry analysis. Anal Chem 84:6355–6360. doi:10.1021/ac3013362
Gráf L, Bajusz S, Patthy A, Barát E, Cseh G (1971) Revised amide location for porcine and human adrenocorticotropic hormone. Acta Biochim Biophys Acad Sci Hung 6:415–418
Gui S, Wooderchak-Donahue WL, Zang T, Chen D, Daly MP, Zhou ZS, Hevel JM (2013) Substrate-induced control of product formation by protein arginine methyltransferase 1. Biochemistry 52:199–209. doi:10.1021/bi301283t
Hao P, Ren Y, Datta A, Tam JP, Sze SK (2015) Evaluation of the effect of trypsin digestion buffers on artificial deamidation. J Proteome Res 14:1308–1314. doi:10.1021/pr500903b
Houmard J, Drapeau GR (1972) Staphylococcal protease: a proteolytic enzyme specific for glutamoyl bonds. Proc Natl Acad Sci USA 69:3506–3509. doi:10.1073/pnas.69.12.3506
Jiang H, Wu S-L, Karger BL, Hancock WS (2010) Characterization of the glycosylation occupancy and the active site in the follow-on protein therapeutic: TNK-tissue plasminogen activator. Anal Chem 82:6154–6162. doi:10.1021/ac100956x
Johnson BA, Aswad DW (1990) Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage. Biochemistry 29:4373–4380. doi:10.1021/bi00470a017
Johnson BA, Freitag NE, Aswad DW (1985) Protein carboxyl methyltransferase selectively modifies an atypical form of calmodulin. Evidence for methylation at deamidated asparagine residues. J Biol Chem 260:10913–10916
Klaene JJ, Ni W, Alfaro JF, Zhou ZS (2014) Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization. J Pharm Sci 103:3033–3042. doi:10.1002/jps.24074
Krokhin OV, Antonovici M, Ens W, Wilkins JA, Standing KG (2006) Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: consequences for MALDI and HPLC-MALDI analysis. Anal Chem 78:6645–6650. doi:10.1021/ac061017o
Lee J-C et al (2012) Protein L-isoaspartyl methyltransferase regulates p53 activity. Nat Commun 3:927. doi:10.1038/ncomms1933
Li X, Cournoyer JJ, Lin C, O’Connor PB (2008) Use of 18O labels to monitor deamidation during protein and peptide sample processing. J Am Soc Mass Spectrom 19:855–864. doi:10.1016/j.jasms.2008.02.011
Liu M, Cheetham J, Cauchon N, Ostovic J, Ni W, Ren D, Zhou ZS (2012) Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis. Anal Chem 84:1056–1062. doi:10.1021/ac202652z
Liu H, Wang F, Xu W, May K, Richardson D (2013a) Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis. Anal Biochem 432:16–22. doi:10.1016/j.ab.2012.09.024
Liu M, Zhang Z, Zang T, Spahr C, Cheetham J, Ren D, Zhou ZS (2013b) Discovery of undefined protein cross-linking chemistry: a comprehensive methodology utilizing 18O-labeling and mass spectrometry. Anal Chem 85:5900–5908. doi:10.1021/ac400666p
Liu M, Zhang Z, Cheetham J, Ren D, Zhou ZS (2014) Discovery and characterization of a photo-oxidative histidine-histidine cross-link in IgG1 antibody utilizing 18O-labeling and mass spectrometry. Anal Chem 86:4940–4948. doi:10.1021/ac500334k
Manning M, Chou D, Murphy B, Payne R, Katayama D (2010) Stability of protein pharmaceuticals: an update. Pharm Res 27:544–575. doi:10.1007/s11095-009-0045-6
Mosley SL, Bakke BA, Sadler JM, Sunkara NK, Dorgan KM, Zhou ZS, Seley-Radtke KL (2006) Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase. Bioorg Med Chem 14:7967–7971. doi:10.1016/j.bmc.2006.07.052
Ni W, Dai S, Karger BL, Zhou ZS (2010) Analysis of isoaspartic acid by selective proteolysis with Asp-N and electron transfer dissociation mass spectrometry. Anal Chem 82:7485–7491. doi:10.1021/ac101806e
Noguchi S (2010) Structural changes induced by the deamidation and isomerization of asparagine revealed by the crystal structure of Ustilago sphaerogena ribonuclease U2B. Biopolymers 93:1003–1010. doi:10.1002/bip.21514
O’Connor PB, Cournoyer JJ, Pitteri SJ, Chrisman PA, McLuckey SA (2006) Differentiation of aspartic and isoaspartic acids using electron transfer dissociation. J Am Soc Mass Spectrom 17:15–19. doi:10.1016/j.jasms.2005.08.019
Oliyai C, Borchardt R (1993) Chemical pathways of peptide degradation. IV. Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide. Pharm Res 10:95–102. doi:10.1023/a:1018981231468
Orrù S, Vitagliano L, Esposito L, Mazzarella L, Marino G, Ruoppolo M (2000) For the record: Effect of deamidation on folding of ribonuclease A. Protein Sci 9:2577–2582. doi:10.1110/ps.9.12.2577
Palmisano G, Melo-Braga MN, Engholm-Keller K, Parker BL, Larsen MR (2012) Chemical deamidation: a common pitfall in large-scale N-linked glycoproteomic mass spectrometry-based analyses. J Proteome Res 11:1949–1957. doi:10.1021/pr2011268
Paranandi MV, Guzzetta AW, Hancock WS, Aswad DW (1994) Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator. J Biol Chem 269:243–253
Patananan AN, Capri J, Whitelegge JP, Clarke SG (2014) Non-repair pathways for minimizing protein isoaspartyl damage in the yeast saccharomyces cerevisiae. J Biol Chem 289:16936–16953. doi:10.1074/jbc.M114.564385
Patel K, Borchardt R (1990) Chemical pathways of peptide degradation. II. Kinetics of deamidation of an asparaginyl residue in a model hexapeptide. Pharm Res 7:703–711. doi:10.1023/a:1015807303766
Perła-Kaján J, Jakubowski H (2012) Paraoxonase 1 and homocysteine metabolism. Amino Acids 43:1405–1417. doi:10.1007/s00726-012-1321-z
Perła-Kaján J, Twardowski T, Jakubowski H (2007) Mechanisms of homocysteine toxicity in humans. Amino Acids 32:561–572. doi:10.1007/s00726-006-0432-9
Pompach P et al (2009) Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds. J Mass Spectrom 44:1571–1578. doi:10.1002/jms.1609
Potter SM, Henzel WJ, Aswad DW (1993) In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV. Protein Sci 2:1648–1663. doi:10.1002/pro.5560021011
Radkiewicz JL, Zipse H, Clarke S, Houk KN (2001) Neighboring side chain effects on asparaginyl and aspartyl degradation: an Ab initio study of the relationship between peptide conformation and backbone NH acidity. J Am Chem Soc 123:3499–3506. doi:10.1021/ja0026814
Reissner KJ, Aswad DW (2003) Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals? Cell Mol Life Sci 60:1281–1295. doi:10.1007/s00018-003-2287-5
Robinson NE, Robinson AB (2001) Prediction of protein deamidation rates from primary and three-dimensional structure. Proc Natl Acad Sci USA 98:4367–4372. doi:10.1073/pnas.071066498
Robinson NE, Robinson ZW, Robinson BR, Robinson AL, Robinson JA, Robinson ML, Robinson AB (2004) Structure-dependent nonenzymatic deamidation of glutaminyl and asparaginyl pentapeptides. J Pept Res 63:426–436. doi:10.1111/j.1399-3011.2004.00151.x
Salzano AM, Renzone G, Scaloni A, Torreggiani A, Ferreri C, Chatgilialoglu C (2011) Human serum albumin modifications associated with reductive radical stress. Mol BioSyst 7:889–898. doi:10.1039/c0mb00223b
Tomlinson AJ, Johnson KL, Lam-Holt J, Mays DC, Lipsky JJ, Naylor S (1997) Inhibition of human mitochondrial aldehyde dehydrogenase by the disulfiram metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide: Structural characterization of the enzyme adduct by HPLC-tandem mass spectrometry. Biochem Pharmacol 54:1253–1260. doi:10.1016/S0006-2952(97)00359-6
Tyler-Cross R, Schirch V (1991) Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. J Biol Chem 266:22549–22556
Wan W, Zhao G, Al-Saad K, Siems WF, Zhou ZS (2004) Rapid screening for S-adenosylmethionine-dependent methylation products by enzyme-transferred isotope patterns analysis. Rapid Commun Mass Spectrom 18:319–324. doi:10.1002/rcm.1335
Wang Z, Rejtar T, Zhou ZS, Karger BL (2010) Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by MS. Rapid Commun Mass Spectrom 24:267–275. doi:10.1002/rcm.4383
Winter D, Pipkorn R, Lehmann WD (2009) Separation of peptide isomers and conformers by ultra performance liquid chromatography. J Sep Sci 32:1111–1119. doi:10.1002/jssc.200800691
Yao X, Freas A, Ramirez J, Demirev PA, Fenselau C (2001) Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal Chem 73:2836–2842. doi:10.1021/ac001404c
Yao X, Afonso C, Fenselau C (2003) Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates. J Proteome Res 2:147–152. doi:10.1021/pr025572s
Yu X, Warme C, Lee D, Zhang J, Zhong W (2013) Characterization of a low-level unknown isomeric degradation product using an integrated online-offline top-down tandem mass spectrometry platform. Anal Chem 85:8964–8967. doi:10.1021/ac401911n
Zang T, Dai S, Chen D, Lee BW, Liu S, Karger BL, Zhou ZS (2009) Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes. Anal Chem 81:9065–9071. doi:10.1021/ac9017132
Zhou ZS, Peariso K, Penner-Hahn JE, Matthews RG (1999) Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli. Biochemistry 38:15915–15926. doi:10.1021/bi992062b
Acknowledgments
The work is supported by the National Institutes of Health (Grant GM101396 to Z. S. Z.). We thank Sam Burns, Kalli Catcott, Mike Pablo, Clair Yu, Derek Lin and Wanlu Qu for the helpful discussion and critical reading. This is contribution number 1057 from the Barnett Institute.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Handling Editor: P. R. Jungblut.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Liu, S., Moulton, K.R., Auclair, J.R. et al. Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5. Amino Acids 48, 1059–1067 (2016). https://doi.org/10.1007/s00726-015-2166-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-015-2166-z