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Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography–mass spectrometry

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Abstract

Peptide quantification by liquid chromatography–mass spectrometry (LC–MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC–MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI–MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.

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References

  1. Niwa M, Enomoto K, Yamashita K (1999) J Chromatogr B 729:245–253

    Article  CAS  Google Scholar 

  2. Zhang X, Nieforth K, Lang J-M, Rouzier-Panis R, Reynes J, Dorr A, Kolis S, Stiles MR, Kinchelow T, Patel IH (2002) Clin Pharmacol Ther 72:10–19

    Article  CAS  PubMed  Google Scholar 

  3. Prokai L, Zharikova AD, Janaky T, Prokai-Tatrai K (2000) Rapid Commun Mass Spectrom 14:2412–2418

    Article  CAS  PubMed  Google Scholar 

  4. Michels P, Tarnow J (2001) Anasthesiol Intensivmed Notfallmed Schmerzther 36:406–416

    Article  CAS  Google Scholar 

  5. Bestle MH, Olsen NV, Christensen P, Jensen BV, Bie P (1999) Am J Physiol 276:R684–R695

    CAS  PubMed  Google Scholar 

  6. Hupe-Sodmann K, McGregor GP, Bridenbaugh R, Göke R, Göke B, Thole H, Zimmermann B, Voigt K (1995) Regul Pept 58:149–156

    Article  CAS  PubMed  Google Scholar 

  7. Wilkinson IB, Affolter JT, de Haas SL, Pellegrini P, Boyd J, Winter MJ, Balment RJ, Webb DJ (2002) Cardiovasc Res 53:341–347

    Article  CAS  PubMed  Google Scholar 

  8. Douglas SA (2003) Curr Opin Pharmacol 3:159–167

    Article  CAS  PubMed  Google Scholar 

  9. Goltzman D (2002) Nat Rev Drug Discovery 1:784–796

    Article  CAS  Google Scholar 

  10. Oosterkamp AI, Gelpi E, Abian J (1998) J Mass Spectrom 33:976–983

    Article  CAS  Google Scholar 

  11. De Kock SS, Rodgers JP, Swanepoel BC (2001) Rapid Commun Mass Spectrom 15:1191–1197

    Article  PubMed  Google Scholar 

  12. Dalluge JJ (2002) Curr Protein Pept Sci 3:181–189

    CAS  PubMed  Google Scholar 

  13. Chace DH, Hillman SL, Millington DS, Kahler SG, Roe CR, Naylor EW (1995) Clin Chem 41:62–68

    CAS  Google Scholar 

  14. Chace DH, Hillman SL, Millington DS, Kahler SG, Adam BW, Levy HL (1996) Clin Chem 42:349–355

    Google Scholar 

  15. Chace DH, Hillman SL, van Hove JL, Naylor EW (1997) Clin Chem 43:2106–2113

    CAS  PubMed  Google Scholar 

  16. Xiao Z, Jiang X, Beckett ML, Wright Jr GL (2000) Protein Exp Purif 19:12–21

    Article  CAS  Google Scholar 

  17. Hajee CAJ, van Rhijn HA, Lasaroms JJP, Keukens HJ, de Jong J (2001) Analyst 126:1332–1338

    Article  CAS  PubMed  Google Scholar 

  18. Weckwerth W, Willmitzer L, Fiehn O (2000) Rapid Commun Mass Spectrom 14:1677–1681

    PubMed  Google Scholar 

  19. Barnidge DR, Dratz EA, Martin T, Bonilla LE, Moran LB, Lindall A (2003) Anal Chem 75:445–451

    Article  CAS  PubMed  Google Scholar 

  20. Zhu L, Tamvakopoulos C, Xie D, Dragovic J, Shen X, Fenyk-Melody JE, Schmidt K, Bagchi A, Griffin PR, Thornberry NA, Roy RS (2003) J Biol Chem 278:22418–22423

    Article  CAS  PubMed  Google Scholar 

  21. Powell MF, Grey H, Gaeta F, Sette A, Colon S (1992) J Pharm Sci 81:731–735

    CAS  PubMed  Google Scholar 

  22. Lee KC, Lee YJ, Song HM, Chun CJ, DeLuca PP (1992) Pharm Res 9:1521–1523

    Article  CAS  Google Scholar 

  23. Feighner JP, Ehrensing RH, Kastin AJ, Leonard BE, Sverdlov L, Nicolau G, Patel A, Hlavka J, Abajian H, Noble JF (2000) J Affect Disord 61:119–126

    Article  CAS  PubMed  Google Scholar 

  24. Agerso H, Ynddal L, Sogaard B, Zdravkovic M (2001) J Clin Pharmacol 41:163–169

    Article  CAS  PubMed  Google Scholar 

  25. Wolf R, Rosche F, Hoffmann T, Demuth HU (2001) J Chromatogr B 926:21–27

    Article  CAS  Google Scholar 

  26. Chang D, Kolis SJ, Julian TF, Linderholm KH, Nachi R, Linder E, Lin PP, Lee JW, Bansal SK (2001) AAPS Pharm Sci 3:W4512

    Google Scholar 

  27. Krstulovic AM, Lee CR, Firmin S, Jacquet G, van Dau CN, Tessier D (2002) LC GC Europe 15:31–41

    CAS  Google Scholar 

  28. Baggerman G, Cerstiaens A, de Loof A, Schoofs L (2002) J Biol Chem 277:40368–40374

    Article  CAS  PubMed  Google Scholar 

  29. Heine G, Raida M, Forssmann WG (1997) J Chromatogr A 776:117–124

    Article  CAS  PubMed  Google Scholar 

  30. Dow RC, Bennie J, Fink G (1994) J Endocrinol 142:R1–R4

    CAS  PubMed  Google Scholar 

  31. Yang CS, Chou ST, Liu L, Tsai PJ, Kuo JS (1995) J Chromatogr B 674:23–30

    CAS  Google Scholar 

  32. Lawless MK, Hopkins S, Anwer MK (1998) J Chromatogr B 707:213–217

    Article  CAS  Google Scholar 

  33. Yang JZ, Bastian C, Moore RD, Stobaugh JF, Borchardt RT (2002) J Chromatogr B 780:269–281

    Article  CAS  Google Scholar 

  34. Nier V, Schultz I, Brenner B, Forssmann WG, Raida M (1999) FEBS Lett 461:246–252

    Article  CAS  PubMed  Google Scholar 

  35. Altria KD, Chen AB, Clohs L (2001) LC GC Europe 14:736–744

    CAS  Google Scholar 

  36. Craig AG, Hoeger CA, Miller CL, Goedken T, Rivier JE, Fischer WH (1994) Biol Mass Spectrom 23:519–528

    CAS  PubMed  Google Scholar 

  37. Richter R, Mägert HJ, Mifune H, Schulz-Knappe P, Forssmann WG (1998) Acta Anat 162:185–193

    Article  CAS  Google Scholar 

  38. Forssmann WG, Richter R, Meyer M (1998) Histochem Cell Biol 110:335–357

    Article  CAS  PubMed  Google Scholar 

  39. Kuhn M, Kulaksiz H, Adermann K, Rechkemmer G, Forssmann WG (1994) FEBS Lett 341:218–222

    Article  CAS  Google Scholar 

  40. Labeur C, Shepherd J, Rosseneu M (1990) Clin Chem 36:591–597

    CAS  PubMed  Google Scholar 

  41. Costello M, Baxter RC, Scott CD (1999) J Clin Endocrinol Metab 84:611–617

    CAS  PubMed  Google Scholar 

  42. Carstens J, Jensen KT, Ivarsen P, Rasmusen LM, Pedersen EB (1997) Clin Chem 43:638–643

    CAS  PubMed  Google Scholar 

  43. Meyer M, Armbruster FP, Maier I, Schwing J, Haller C, Missbichler A, Adermann K, Forssmann WG, Woloszczuk (1998) Clin Chem 44:2524–2529

    CAS  PubMed  Google Scholar 

  44. Shimizu M, Swanson P, Fukada H, Hara A, Dickhoff WW (2000) Gen Comp Endocrinol 119:26–36

    Article  CAS  PubMed  Google Scholar 

  45. Breier BH, Gallaher BW, Gluckman PD (1991) J Endocrinol 128:347–357

    CAS  Google Scholar 

  46. Gobom J, Kraeuter KO, Persson R, Stehen H, Roepstorff P, Ekman R (2000) Anal Chem 72:3320–3326

    Article  CAS  PubMed  Google Scholar 

  47. Desiderio DM, Wirth U, Lovelace JL, Fridland G, Umstot ES, Nguyen TMD, Schiller PW, Szeto HS, Clapp JF (2000) J Mass Spectrom 35:725–733

    CAS  PubMed  Google Scholar 

  48. Kokko KP, Dix TA (2002) Anal Biochem 308:34–41

    Article  CAS  PubMed  Google Scholar 

  49. Kang MJ, Tholey A, Heinzle E (2001) Rapid Commun Mass Spectrom 15:1327–1333

    CAS  PubMed  Google Scholar 

  50. Powell KD, Ghaemmaghami S, Wang MZ, Ma L, Oas TG, Fitzgerald MC (2002) J Am Chem Soc 124:10256–10257

    Article  CAS  PubMed  Google Scholar 

  51. Schenone MM, Warder SE, Martin JA, Prorok M, Castellino FJ (2000) J Pept Res 56:438–445

    Article  CAS  PubMed  Google Scholar 

  52. Xiao Z, Adam BL, Cazares LH, Clements MA, Davis JW, Schellhammer PF, Dalmasso EA, Wright Jr GL (2001) Cancer Res 61:6029–6033

    CAS  PubMed  Google Scholar 

  53. Sharp ME, Marx SJ (1985) Clin Chim Acta 145:59–68

    Article  CAS  PubMed  Google Scholar 

  54. John H, Preissner KT, Forssmann WG, Ständker L (1999) Biochemistry 38:10217–10224

    Article  CAS  PubMed  Google Scholar 

  55. John H, Schulz S, Forssmann WG, Ständker L (2002) J Immunol Methods 268:233–237

    Article  CAS  PubMed  Google Scholar 

  56. Stegehuis DS, Tjaden UR, van den Beld CMB, van der Greef J (1991) J Chromatogr 549:185–193

    Article  CAS  PubMed  Google Scholar 

  57. Grove J, Fozard JR, Mamont PS (1981) J Chromatogr 223:409–416

    Article  CAS  PubMed  Google Scholar 

  58. Shen Z, Wang S, Bakhtiar R (2002) Rapid Commun Mass Spectrom 16:332–338

    Article  CAS  Google Scholar 

  59. Ramos L, Bakhtiar R, Majumdar T, Hayes M, Tse F (1999) Rapid Commun Mass Spectrom 13:2054–2062

    Article  CAS  PubMed  Google Scholar 

  60. Poot A, Beugeling T, van Aken WG, Bantjes A (1990) J Biomed Mater Res 24:1021–1036

    CAS  PubMed  Google Scholar 

  61. Lindberg BF, Andersson KE (1991) Pharmacol Toxicol 68:276–281

    CAS  PubMed  Google Scholar 

  62. Dagenais P, Desprez B, Albert J, Escher E (1994) Anal Biochem 222:149–155

    Article  CAS  PubMed  Google Scholar 

  63. Mizutani T (1980) J Pharm Sci 69:279–282

    CAS  PubMed  Google Scholar 

  64. Böcher M, Böldicke T, Kieß M, Bilitewski U (1997) J Immunol Methods 208:191–202

    Article  PubMed  Google Scholar 

  65. Hyenstrand P, Metcalf JS, Beattie KA, Codd GA (2001) Toxicon 39:589–594

    CAS  PubMed  Google Scholar 

  66. Hyenstrand P, Metcalf JS, Beattie KA, Codd GA (2001) Water Res 35:3508–3511

    Article  CAS  PubMed  Google Scholar 

  67. Song KH, An HM, Kim HJ, Ahn SH, Chung SJ, Shim CK (2002) J Chromatogr B 775:247–255

    Article  CAS  Google Scholar 

  68. Welinder BS, Sorensen HH, Hansen B (1987) J Chromatogr 408:191–199

    Article  CAS  PubMed  Google Scholar 

  69. Aguilar MI, Clayton DJ, Holt P, Kronina V, Boysen RI, Purcell AW, Hearn MTW (1998) Anal Chem 70:5010–5018

    Article  CAS  PubMed  Google Scholar 

  70. Woodhouse KA, Weitz JI, Brash JL (1994) J Biomed Mat Res 28:407–415

    CAS  Google Scholar 

  71. Raj PA, Johnsson M, Levine MJ, Nancollas GH (1992) 267:5968–5976

  72. Fouda H, Nocerini M, Schneider R, Gedutis C (1991) J Am Soc Mass Spectrom 2:164–167

    Article  CAS  Google Scholar 

  73. Matuszewski BK, Constanzer ML, Chavez-Eng CM (1998) Anal Chem 70:882–889

    CAS  Google Scholar 

  74. Nelson MD, Dolan JW (2002) LC GC Europe 15:73–79

    Google Scholar 

  75. Niu PD, Perez-Sanchez J, Le Bail PY (1993) Fish Physiol Biochem 11:381–391

    CAS  Google Scholar 

  76. Niu PD, Le Bail PY (1993) J Exp Zool 265:627–636

    CAS  Google Scholar 

  77. Yamaguchi K, Takashima M, Uchimura T, Kobayashi S (2000) Biomed Chromatogr 14:77–81

    Article  CAS  PubMed  Google Scholar 

  78. Wilbert SM, Engrissei G, Yau EK, Grainger DJ, Tatalick L, Axworthy DB (2000) Anal Biochem 278:14–21

    Article  CAS  PubMed  Google Scholar 

  79. Marquez CD, Weintraub ST, Smith PC (1997) J Chromatogr B 694:21–30

    CAS  Google Scholar 

  80. Zimmer D, Pickard V, Czembor W, Müller C (1999) J Chromatogr A 854:23–35

    Article  CAS  PubMed  Google Scholar 

  81. Kirchner GI, Vidal C, Jacobsen W, Franzke A, Hallensleben K, Christians U, Sewing KF (1999) J Chromatogr B 721:285–294

    Article  CAS  Google Scholar 

  82. Kirchner GI, Vidal C, Winkler M, Mueller L, Jacobsen W, Franzke A, Sewing KF (1999) Ther Drug Monit 21:116–122

    Article  CAS  PubMed  Google Scholar 

  83. Deters M, Kirchner G, Resch K, Kaever V (2002) Clin Chem Lab Med 40:285–292

    CAS  PubMed  Google Scholar 

  84. Grigoriants OO, Tseng JL, Becklin RR, Desiderio DM (1997) J Chromatogr B 695:287–298

    Article  CAS  Google Scholar 

  85. Chan KK, Bakhtiar R, Jiang C (1997) Invest New Drug 15:195–206

    Article  CAS  Google Scholar 

  86. Garteiz DA, Madden T, Beck DE, Huie WR, McManus KT, Abbruzzese JL, Chen W, Newman RA (1998) Cancer Chemother Pharmacol 41:299–306

    Article  CAS  Google Scholar 

  87. Piot JM, Guillochon D, Thomas D (1988) Chromatographia 25:307–312

    CAS  Google Scholar 

  88. Walker V, Mills GA (2002) Ann Clin Biochem 39:464–477

    Article  CAS  PubMed  Google Scholar 

  89. Fenaille F, Tabet J-C, Guy PA (2002) Anal Chem 74:6298–6304

    Article  CAS  PubMed  Google Scholar 

  90. Ukena K, Koda A, Yamamoto K, Kobayashi T, Iwakoshi-Ukena E, Minakata H, Kikuyama S, Tsutsui K (2003) Endocrinol 144:3879–3884

    Article  CAS  Google Scholar 

  91. Wolf R, Hoffmann T, Rosche F, Demuth H-U (2003) J Chromatogr B, in press

  92. Shraiman BI, Siggia ED (2000) Nature 405:639–646

    Article  CAS  PubMed  Google Scholar 

  93. Jemal M, Qing Y, Whigan DB (1998) Rapid Commun Mass Spectrom 12:1389–1399

    Article  CAS  PubMed  Google Scholar 

  94. Ayrton J, Dear GJ, Leavens WJ, Mallett DN, Plumb RS (1998) J Chromatogr A 828:199–207

    CAS  PubMed  Google Scholar 

  95. Zimmer D, Pickard V, Czembor W, Müller C (1999) J Chromatogr A 854:23–35

    Article  CAS  PubMed  Google Scholar 

  96. Wagner K, Miliotis T, Marko-Varga G, Bischoff R, Unger KK (2002) Anal Chem 74:809–820

    Article  CAS  PubMed  Google Scholar 

  97. Hagestam ICH, Pinkerton TC (1985) Anal Chem 57:1757–1763

    CAS  Google Scholar 

  98. Machtejevas E, John H, Wagner K, Ständker L, Marko-Varga G, Forssmann WG, Bischoff R, Unger KK (2003) J Chromatogr, in press

  99. Naidong W, Chen YL, Shou W, Jiang X (2001) J Pharm Biomed Anal 26:753–767

    Article  CAS  PubMed  Google Scholar 

  100. Geng X, Regnier FE (1984) J Chromatogr 296:15–30

    Article  CAS  PubMed  Google Scholar 

  101. Huang X, Zhang S, Schultz GA, Henion J (2002) Anal Chem 74:2336–2344

    Article  CAS  PubMed  Google Scholar 

  102. Schmidt A, Karas M, Dülcks T (2003) J Am Soc Mass Spectrom 14:492–500

    Article  CAS  Google Scholar 

  103. Licklider LJ, Thoreen CC, Peng J, Gygi SP (2002) Anal Chem 74:3076–3083

    Article  CAS  PubMed  Google Scholar 

  104. Ihling C, Berger K, Höfliger MM, Führer D, Beck-Sickinger AG, Sinz A (2003) Rapid Commun Mass Spectrom 17:1240–1246

    Article  CAS  PubMed  Google Scholar 

  105. Varesio E, Rudaz S, Krause K-H, Veuthey J-L (2002) J Chromatogr A 974:135–142

    Article  CAS  PubMed  Google Scholar 

  106. Schellenberg KH, Linder M, Groeppelin A, Erni F (1987) J Chromatogr 394:239–251

    Article  CAS  PubMed  Google Scholar 

  107. Rashed MS (2001) J Chromatogr B 758:27–48

    Article  CAS  Google Scholar 

  108. Yamashita M, Fenn JB (1984) J Phys Chem 88:4451–4459

    CAS  Google Scholar 

  109. Whitehouse CM, Dreyer RN, Yamashita M, Fenn JB (1985) Anal Chem 57:675–679

    CAS  PubMed  Google Scholar 

  110. Fenn JB (2002) J Biomol Technol 13:101–118

    Google Scholar 

  111. Schulz-Knappe P, Forssmann K, Herbst F, Hock D, Pipkorn R, Forssmann WG (1988) Klin Wochenschr 66:752–759

    CAS  PubMed  Google Scholar 

  112. Hunt DF, Yates III JR, Shabanowitz J, Winston S, Hauer CR (1986) Proc Natl Acad Sci USA 83:6233–6237

    PubMed  Google Scholar 

  113. John H, Forssmann W-G (2001) Rapid Commun Mass Spectrom 15:1222–1228

    Article  CAS  PubMed  Google Scholar 

  114. Wan H, Desiderio DM (2003) Rapid Commun Mass Spectrom 17:538–546

    Article  CAS  PubMed  Google Scholar 

  115. Bobin S, Popot MA, Bonnaire Y, Tabet JC (2001) Analyst 126:1996–2001

    Article  CAS  PubMed  Google Scholar 

  116. Wan H, Umstot ES, Szeto HH, Schiller PW, Desiderio DM (2003) J Chromatogr B, in press

  117. Blackstock WP, Weir MP (1999) Trends Biotechnol 17:121–127

    CAS  PubMed  Google Scholar 

  118. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Nat Biotechnol 17:994–999

    Google Scholar 

  119. Dethy JM, Ackermann BL, Delatour C, Henion JD, Schultz GA (2003) Anal Chem 75:805–811

    Article  CAS  PubMed  Google Scholar 

  120. Daughaday WH, Mariz IK, Blethen SL (1980) J Clin Endocrinol Metab 51:781–788

    CAS  PubMed  Google Scholar 

  121. Muddlman DC, Gusev AI, Proctor A, Hercules DM, Venkataramanan R, Diven W (1994) Anal Chem 66:2362–2368

    CAS  PubMed  Google Scholar 

  122. Hermann K, Ring J, Phillips MI (1990) J Chromatogr Sci 28:524–528

    CAS  PubMed  Google Scholar 

  123. Marquez CD, Lee ML, Weintraub ST, Smith PC (1997) J Chromatogr B 700:9–21

    CAS  Google Scholar 

  124. Drummer C, Fiedler F, Bub A, Kleefeld D, Dimitriades E, Gerzer R, Forssmann WG (1993) Pflügers Arch 423:372–377

    Google Scholar 

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Acknowledgment

We thank Jutta Barras-Akhnoukh, Kathleen Listemann, and Stefanie Schulz from IPF PharmaCeuticals GmbH for their excellent technical assistance in peptide analysis.

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Correspondence to Harald John.

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John, H., Walden, M., Schäfer, S. et al. Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography–mass spectrometry. Anal Bioanal Chem 378, 883–897 (2004). https://doi.org/10.1007/s00216-003-2298-y

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