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Application and challenges in using LC–MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery

    Joanna Zheng

    * Author for correspondence:

    E-mail Address: joanna.zheng@bms.com

    Bioanalytical Research, Bristol-Myers Squibb, 311 Pennington Rocky Hill Rd, Pennington, NJ 08534, USA

    ,
    John Mehl

    Bioanalytical Research, Bristol-Myers Squibb, 311 Pennington Rocky Hill Rd, Pennington, NJ 08534, USA

    ,
    Yongxin Zhu

    Bioanalytical Research, Bristol-Myers Squibb, 311 Pennington Rocky Hill Rd, Pennington, NJ 08534, USA

    ,
    Baomin Xin

    Bioanalytical Research, Bristol-Myers Squibb, 311 Pennington Rocky Hill Rd, Pennington, NJ 08534, USA

    &
    Timothy Olah

    Bioanalytical Research, Bristol-Myers Squibb, 311 Pennington Rocky Hill Rd, Pennington, NJ 08534, USA

    Published Online:https://doi.org/10.4155/bio.14.36

    As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC–MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC–MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC–MS to support protein quantification in drug discovery.

    References

    • Dimitrov DS. Therapeutic proteins. Methods Mol. Biol.899,1–26 (2012).Crossref, Medline, CASGoogle Scholar
    • Li F, Fast D, Michael S. Absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC–MS. Bioanalysis3(21),2459–2480 (2011).Link, CASGoogle Scholar
    • Kuang B, King L, Wang HF. Therapeutic monoclonal antibody concentration monitoring: free or total? Bioanalysis2(6),1125–1140 (2010).Link, CASGoogle Scholar
    • Dubois M, Fenaille F, Clement G et al. Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum. Anal. Chem.80(5),1737–1745 (2008).Crossref, Medline, CASGoogle Scholar
    • Rauh M. LC–MS/MS for protein and peptide quantification in clinical chemistry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.884,59–67 (2012).CrossrefGoogle Scholar
    • Wang Y, Qu Y, Bellows CL, Ahn JS, Burkey JL, Taylor SW. Simultaneous quantification of davalintide, a novel amylin-mimetic peptide, and its active metabolite in beagle and rat plasma by online SPE and LC–MS/MS. Bioanalysis4(17),2141–2152 (2012).Link, CASGoogle Scholar
    • Ezan E, Bitsch F. Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis1(8),1375–1388 (2009).Link, CASGoogle Scholar
    • Hoofnagle AN, Wener MH. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J. Immunol. Methods347(1–2),3–11 (2009).Crossref, Medline, CASGoogle Scholar
    • Smith HW, Butterfield A, Sun D. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA. Regul. Toxicol. Pharmacol.49(3),230–237 (2007).Crossref, Medline, CASGoogle Scholar
    • 10  Wang YM, Fang L, Zhou L, Wang J, Ahn HY. A survey of applications of biological products for drug interference of immunogenicity assays. Pharm. Res.29(12),3384–3392 (2012).Crossref, Medline, CASGoogle Scholar
    • 11  Hart MH, de Vrieze H, Wouters D et al. Differential effect of drug interference in immunogenicity assays. J. Immunol. Methods372(1–2),196–203 (2011).Crossref, Medline, CASGoogle Scholar
    • 12  Wang SJ, Wu ST, Gokemeijer J et al. Attribution of the discrepancy between ELISA and LC–MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies. Anal. Bioanal. Chem.402(3),1229–1239 (2012).Crossref, Medline, CASGoogle Scholar
    • 13  Yang Z, Hayes M, Fang X, Daley MP, Ettenberg S, Tse FL. LC–MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup. Anal. Chem.79(24),9294–9301 (2007).Crossref, Medline, CASGoogle Scholar
    • 14  Heudi O, Barteau S, Zimmer D et al. Towards absolute quantification of therapeutic monoclonal antibody in serum by LC–MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry. Anal. Chem.80(11),4200–4207 (2008).Crossref, Medline, CASGoogle Scholar
    • 15  Ji QC, Rodila R, El-Shourbagy TA. A sample preparation process for LC–MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody. J. Chromatogr. B847(2),133–141 (2007).Crossref, Medline, CASGoogle Scholar
    • 16  Ji QC, Rodila R, Gage EM, El-Shourbagy TA. A strategy of plasma protein quantitation by selective reaction monitoring of an intact protein. Anal. Chem.75(24),7008–7014 (2003).Crossref, Medline, CASGoogle Scholar
    • 17  Becher F, Pruvost A, Clement G, Tabet JC, Ezan E. Quantification of small therapeutic proteins in plasma by liquid chromatography–tandem mass spectrometry: application to an elastase inhibitor EPI-hNE4. Anal. Chem.78(7),2306–2313 (2006).Crossref, Medline, CASGoogle Scholar
    • 18  Yang Z, Ke J, Hayes M, Bryant M, Tse FL. A sensitive and high-throughput LC–MS/MS method for the quantification of PEGylated-interferon-alpha2a in human serum using monolithic C18 solid phase extraction for enrichment. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.877(18–19),1737–1742 (2009).Crossref, Medline, CASGoogle Scholar
    • 19  Hagman C, Ricke D, Ewert S, Bek S, Falchetto R, Bitsch F. Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography–tandem mass spectrometry. Anal. Chem.80(4),1290–1296 (2008).Crossref, Medline, CASGoogle Scholar
    • 20  Ewles M, Goodwin L. Bioanalytical approaches to analyzing peptides and proteins by LC–MS/MS. Bioanalysis3(12),1379–1397 (2011).Link, CASGoogle Scholar
    • 21  Mesmin C, Fenaille F, Ezan E, Becher F. MS-based approaches for studying the pharmacokinetics of protein drugs. Bioanalysis3(5),477–480 (2011).Link, CASGoogle Scholar
    • 22  Nowatzke WL, Rogers K, Wells E, Bowsher RR, Ray C, Unger S. Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs. Bioanalysis3(5),509–521 (2011).Link, CASGoogle Scholar
    • 23  van de Merbel NC, Bronsema KJ, Nemansky M. Protein quantification using LC–MS: can it make a difference? Bioanalysis4(17),2113–2116 (2012).Link, CASGoogle Scholar
    • 24  van den Broek I, Niessen WM, van Dongen WD. Bioanalytical LC–MS/MS of protein-based biopharmaceuticals. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.929,161–179 (2013).Crossref, Medline, CASGoogle Scholar
    • 25  Bischoff R, Bronsema KJ, van de Merbel NC. Analysis of biopharmaceutical proteins in biological matrices by LC–MS/MS I. Sample preparation. Trends Analyt. Chem.48(0),41–51 (2013).Crossref, CASGoogle Scholar
    • 26  Hopfgartner G, Lesur A, Varesio E. Analysis of biopharmaceutical proteins in biological matrices by LC–MS/MS II. LC–MS/MS analysis. Trends Analyt. Chem.48(0),52–61 (2013).Crossref, CASGoogle Scholar
    • 27  van den Broek I, Sparidans RW, Schellens JH, Beijnen JH. Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.872(1–2),1–22 (2008).Crossref, Medline, CASGoogle Scholar
    • 28  Stejskal K, Potesil D, Zdrahal Z. Suppression of peptide sample losses in autosampler vials. J. Proteome Res.12(6),3057–3062 (2013).Crossref, Medline, CASGoogle Scholar
    • 29  van Midwoud PM, Rieux L, Bischoff R, Verpoorte E, Niederlander HA. Improvement of recovery and repeatability in liquid chromatography–mass spectrometry analysis of peptides. J. Proteome Res.6(2),781–791 (2007).Crossref, Medline, CASGoogle Scholar
    • 30  Xu Y, Mehl JT, Bakhtiar R, Woolf EJ. Immunoaffinity purification using anti-PEG antibody followed by two-dimensional liquid chromatography–tandem mass spectrometry for the quantification of a PEGylated therapeutic peptide in human plasma. Anal. Chem.82(16),6877–6886 (2010).Crossref, Medline, CASGoogle Scholar
    • 31  Zhang H, Xin B, Caporuscio C, Olah TV. Bioanalytical strategies for developing highly sensitive liquid chromatography–tandem mass spectrometry based methods for the peptide GLP-1 agonists in support of discovery PK/PD studies. Rapid Commun. Mass Spectrom.25(22),3427–3435 (2011).Crossref, Medline, CASGoogle Scholar
    • 32  Reubsaet JL, Beijnen JH, Bult A, van Maanen RJ, Marchal JA, Underberg WJ. Analytical techniques used to study the degradation of proteins and peptides: chemical instability. J. Pharm. Biomed. Anal.17(6–7),955–978 (1998).Crossref, Medline, CASGoogle Scholar
    • 33  Wolf R, Rosche F, Hoffmann T, Demuth HU. Immunoprecipitation and liquid chromatographic-mass spectrometric determination of the peptide glucose-dependent insulinotropic polypeptides GIP1–42 and GIP3–42 from human plasma samples. New sensitive method to analyze physiological concentrations of peptide hormones. J. Chromatogr. A926(1),21–27 (2001).Crossref, Medline, CASGoogle Scholar
    • 34  Fanciulli G, Azara E, Wood TD, Delitala G, Marchetti M. Liquid chromatography–mass spectrometry assay for quantification of gluten exorphin B5 in cerebrospinal fluid. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.852(1–2),485–490 (2007).Crossref, Medline, CASGoogle Scholar
    • 35  Wu ST, Ouyang Z, Olah TV, Jemal M. A strategy for liquid chromatography–tandem mass spectrometry based quantitation of PEGylated protein drugs in plasma using plasma protein precipitation with water-miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection. Rapid Commun. Mass Spectrom.25(2),281–290 (2011).Crossref, Medline, CASGoogle Scholar
    • 36  Olah T, Ranasinghe A, Zhang H et al. The development and implementation of LC–MS-based bioanalytical methods for the quantification of protein therapeutics in drug discovery. In: Characterization of Protein Therapeutics Using Mass Spectrometry. Chen G (Ed.). Springer, NY, USA, 95–115 (2013).Google Scholar
    • 37  Barr JR, Maggio VL, Patterson DG Jr et al. Isotope dilution–mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin. Chem.42(10),1676–1682 (1996).Crossref, Medline, CASGoogle Scholar
    • 38  Russell WK, Park ZY, Russell DH. Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry. Anal. Chem.73(11),2682–2685 (2001).Crossref, Medline, CASGoogle Scholar
    • 39  Norrgran J, Williams TL, Woolfitt AR, Solano MI, Pirkle JL, Barr JR. Optimization of digestion parameters for protein quantification. Anal. Biochem.393(1),48–55 (2009).Crossref, Medline, CASGoogle Scholar
    • 40  Proc JL, Kuzyk MA, Hardie DB et al. A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J. Proteome Res.9(10),5422–5437 (2010).Crossref, Medline, CASGoogle Scholar
    • 41  Havlis J, Thomas H, Sebela M, Shevchenko A. Fast-response proteomics by accelerated in-gel digestion of proteins. Anal. Chem.75(6),1300–1306 (2003).Crossref, Medline, CASGoogle Scholar
    • 42  Lesur A, Varesio E, Hopfgartner G. Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection. J. Chromatogr. A1217(1),57–64 (2010).Crossref, Medline, CASGoogle Scholar
    • 43  Halquist MS, Karnes HT. Quantification of Alefacept, an immunosuppressive fusion protein in human plasma using a protein analogue internal standard, trypsin cleaved signature peptides and liquid chromatography–tandem mass spectrometry. J. Chromatogr. B879(11–12),789–798 (2011).Crossref, Medline, CASGoogle Scholar
    • 44  Lu Q, Zheng X, McIntosh T et al. Development of different analysis platforms with LC–MS for pharmacokinetic studies of protein drugs. Anal. Chem.81(21),8715–8723 (2009).Crossref, Medline, CASGoogle Scholar
    • 45  Kehler J, Akella N, Citerone D, Szapacs M. Application of DBS for the quantitative assessment of a protein biologic using on-card digestion LC–MS/MS or immunoassay. Bioanalysis3(20),2283–2290 (2011).Link, CASGoogle Scholar
    • 46  Liu H, Manuilov AV, Chumsae C, Babineau ML, Tarcsa E. Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography–mass spectrometry. Anal. Biochem.414(1),147–153 (2011).Crossref, Medline, CASGoogle Scholar
    • 47  van den Broek I, Sparidans RW, Schellens JHM, Beijnen JH. Enzymatic digestion as a tool for the LC–MS/MS quantification of large peptides in biological matrices: measurement of chymotryptic fragments from the HIV-1 fusion inhibitor enfuvirtide and its metabolite M-20 in human plasma. J. Chromatogr. B854(1–2),245–259 (2007).Crossref, Medline, CASGoogle Scholar
    • 48  Ye H, Hill J, Kauffman J, Gryniewicz C, Han X. Detection of protein modifications and counterfeit protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry: studies of insulins. Anal. Biochem.379(2),182–191 (2008).Crossref, Medline, CASGoogle Scholar
    • 49  Kuzyk MA, Smith D, Yang J et al. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma. Mol. Cell. Proteomics8(8),1860–1877 (2009).Crossref, Medline, CASGoogle Scholar
    • 50  Hewavitharana AK, Herath HM, Shaw PN, Cabot PJ, Kebarle P. Effect of solvent and electrospray mass spectrometer parameters on the charge state distribution of peptides – a case study using liquid chromatography–mass spectrometry method development for beta-endorphin assay. Rapid Commun. Mass Spectrom.24(24),3510–3514 (2010).Crossref, Medline, CASGoogle Scholar
    • 51  Hahne H, Pachl F, Ruprecht B et al. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments. Nat. Methods10(10),989–991 (2013).Crossref, Medline, CASGoogle Scholar
    • 52  Shipkova P, Drexler DM, Langish R, Smalley J, Salyan ME, Sanders M. Application of ion trap technology to liquid chromatography–mass spectrometry quantitation of large peptides. Rapid Commun. Mass Spectrom.22(9),1359–1366 (2008).Crossref, Medline, CASGoogle Scholar
    • 53  Sherwood CA, Eastham A, Lee LW, Risler J, Vitek O, Martin DB. Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers. J. Proteome Res.8(9),4243–4251 (2009).Crossref, Medline, CASGoogle Scholar
    • 54  Paizs B, Suhai S. Fragmentation pathways of protonated peptides. Mass Spectrom. Rev.24(4),508–548 (2005).Crossref, Medline, CASGoogle Scholar
    • 55  Holstein CA, Gafken PR, Martin DB. Collision energy optimization of b- and y-ions for multiple reaction monitoring mass spectrometry. J. Proteome Res.10(1),231–240 (2010).Crossref, MedlineGoogle Scholar
    • 56  Neta P, Stein SE. Charge states of y ions in the collision-induced dissociation of doubly charged tryptic peptide ions. J. Am. Soc. Mass Spectrom.22(5),898–905 (2011).Crossref, Medline, CASGoogle Scholar
    • 57  Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics1(11),845–867 (2002).Crossref, Medline, CASGoogle Scholar
    • 58  Ouyang Z, Furlong M, Wu S et al. Pellet digestion: a simple and efficient sample preparation technique for LC–MS/MS quantification of large therapeutic proteins in plasma. Bioanalysis4(1),17–28 (2012).Link, CASGoogle Scholar
    • 59  Yuan L, Arnold ME, Aubry AF, Ji QC. Simple and efficient digestion of a monoclonal antibody in serum using pellet digestion: comparison with traditional digestion methods in LC–MS/MS bioanalysis. Bioanalysis4(24),2887–2896 (2012).Link, CASGoogle Scholar
    • 60  Duan X, Young R, Straubinger RM et al. A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome. J. Proteome Res.8(6),2838–2850 (2009).Crossref, Medline, CASGoogle Scholar
    • 61  Inoue K, Ikemura A, Tsuruta Y et al. Quantification of N-acetyl-seryl-aspartyl-lysyl-proline in hemodialysis patients administered angiotensin-converting enzyme inhibitors by stable isotope dilution liquid chromatography–tandem mass spectrometry. J. Pharm. Biomed. Anal.54(4),765–771 (2011).Crossref, Medline, CASGoogle Scholar
    • 62  ten Have S, Boulon S, Ahmad Y, Lamond AI. Mass spectrometry-based immuno-precipitation proteomics – the user’s guide. Proteomics11(6),1153–1159 (2011).Crossref, Medline, CASGoogle Scholar
    • 63  Ackermann BL, Berna MJ. Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers. Expert Rev. Proteomics4(2),175–186 (2007).Crossref, Medline, CASGoogle Scholar
    • 64  Dubois M, Becher F, Herbet A, Ezan E. Immuno-mass spectrometry assay of EPI-HNE4, a recombinant protein inhibitor of human elastase. Rapid Commun. Mass Spectrom.21(3),352–358 (2007).Crossref, Medline, CASGoogle Scholar
    • 65  Palandra J, Finelli A, Zhu M, Masferrer J, Neubert H. Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC–MS/MS. Anal. Chem.85(11),5522–5529 (2013).Crossref, Medline, CASGoogle Scholar
    • 66  Huang L, Biolsi S, Bales KR, Kuchibhotla U. Impact of variable domain glycosylation on antibody clearance: an LC–MS characterization. Anal. Biochem.349(2),197–207 (2006).Crossref, Medline, CASGoogle Scholar
    • 67  Hall MP, Gegg C, Walker K et al. Ligand-binding mass spectrometry to study biotransformation of fusion protein drugs and guide immunoassay development: strategic approach and application to peptibodies targeting the thrombopoietin receptor. AAPS J.12(4),576–585 (2010).Crossref, Medline, CASGoogle Scholar
    • 68  Li H, Ortiz R, Tran L et al. General LC–MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies. Anal. Chem.84(3),1267–1273 (2012).Crossref, Medline, CASGoogle Scholar
    • 69  Li H, Ortiz R, Tran LT et al. Simultaneous analysis of multiple monoclonal antibody biotherapeutics by LC–MS/MS method in rat plasma following cassette-dosing. AAPS J.15(2),337–346 (2013).Crossref, Medline, CASGoogle Scholar
    • 70  Mirzaei H, Regnier F. Enhancing electrospray ionization efficiency of peptides by derivatization. Anal. Chem.78(12),4175–4183 (2006).Crossref, Medline, CASGoogle Scholar
    • 71  Ciccimaro E, Hanks SK, Yu KH, Blair IA. Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography–mass spectrometry. Anal. Chem.81(9),3304–3313 (2009).Crossref, Medline, CASGoogle Scholar
    • 72  Brun V, Dupuis A, Adrait A et al. Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol. Cell. Proteomics6(12),2139–2149 (2007).Crossref, Medline, CASGoogle Scholar
    • 73  Ocaña MF, Neubert H. An immunoaffinity liquid chromatography–tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum. Anal. Biochem.399(2),202–210 (2010).Crossref, Medline, CASGoogle Scholar
    • 74  Barnidge DR, Hall GD, Stocker JL, Muddiman DC. Evaluation of a cleavable stable isotope labeled synthetic peptide for absolute protein quantification using LC–MS/MS. J. Proteome Res.3(3),658–661 (2004).Crossref, Medline, CASGoogle Scholar
    • 75  Bronsema KJ, Bischoff R, van de Merbel NC. Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry. J. Chromatogr. B893–894(0),1–14 (2012).Crossref, MedlineGoogle Scholar
    • 76  Remily-Wood ER, Koomen JM. Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements. J. Mass Spectrom.47(2),188–194 (2012).Crossref, Medline, CASGoogle Scholar
    • 77  Buscher BAP, Gerritsen H, van Schöll I, Cnubben NHP, Brüll LP. Quantitative analysis of Tenecteplase in rat plasma samples using LC–MS/MS as an alternative for ELISA. J. Chromatogr. B852(1–2),631–634 (2007).Crossref, Medline, CASGoogle Scholar
    • 78  Marshall AG, Hendrickson CL. High-resolution mass spectrometers. Annu. Rev. Anal. Chem. (Palo Alto Calif.)1,579–599 (2008).Crossref, Medline, CASGoogle Scholar
    • 79  Wei H, Tymiak AA, Chen G. High-resolution MS for structural characterization of protein therapeutics: advances and future directions. Bioanalysis5(10),1299–1313 (2013).Link, CASGoogle Scholar
    • 80  Wong RL, Xin B, Olah T. Optimization of Exactive Orbitrap™ acquisition parameters for quantitative bioanalysis. Bioanalysis3(8),863–871 (2011).Link, CASGoogle Scholar
    • 81  Dillen L, Cools W, Vereyken L et al. Comparison of triple quadrupole and high-resolution TOF-MS for quantification of peptides. Bioanalysis4(5),565–579 (2012).Link, CASGoogle Scholar
    • 82  Ranasinghe A, Ramanathan R, Jemal M, D’Arienzo CJ, Humphreys WG, Olah TV. Integrated quantitative and qualitative workflow for in vivo bioanalytical support in drug discovery using hybrid Q-TOF-MS. Bioanalysis4(5),511–528 (2012).Link, CASGoogle Scholar
    • 83  Zhu M, Ma L, Zhang D et al. Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data. Drug Metab. Dispos.34(10),1722–1733 (2006).Crossref, Medline, CASGoogle Scholar
    • 84  Plumb RS, Johnson KA, Rainville P et al. UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Commun. Mass Spectrom.20(13),1989–1994 (2006).Crossref, Medline, CASGoogle Scholar
    • 85  Zhang H, Zhang D, Ray K, Zhu M. Mass defect filter technique and its applications to drug metabolite identification by high- resolution mass spectrometry. J. Mass Spectrom.44(7),999–1016 (2009).Crossref, Medline, CASGoogle Scholar
    • 86  Ruan Q, Ji QC, Arnold ME, Humphreys WG, Zhu M. Strategy and its implications of protein bioanalysis utilizing high-resolution mass spectrometric detection of intact protein. Anal. Chem.83(23),8937–8944 (2011).Crossref, Medline, CASGoogle Scholar
    • 87  Ramagiri S, Garofolo F. Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis4(5),529–540 (2012).Link, CASGoogle Scholar
    • 88  Plumb RS, Fujimoto G, Mather J et al. Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS. Bioanalysis4(5),605–615 (2012).Link, CASGoogle Scholar
    • 89  Duan X, Abuqayyas L, Dai L, Balthasar JP, Qu J. High-throughput method development for sensitive, accurate, and reproducible quantification of therapeutic monoclonal antibodies in tissues using orthogonal array optimization and nano liquid chromatography/selected reaction monitoring mass spectrometry. Anal. Chem.84(10),4373–4382 (2012).Crossref, Medline, CASGoogle Scholar
    • 90  Cao J, Gonzalez-Covarrubias V, Straubinger RM et al. A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC–MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver. Anal. Chem.82(7),2680–2689 (2010).Crossref, Medline, CASGoogle Scholar
    • 91  Gil J, Cabrales A, Reyes O et al. Development and validation of a bioanalytical LC–MS method for the quantification of GHRP-6 in human plasma. J. Pharm. Biomed. Anal.60(0),19–25 (2012).Crossref, Medline, CASGoogle Scholar
    • 92  Whiteaker JR, Zhao L, Anderson L, Paulovich AG. An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers. Mol. Cell. Proteomics9(1),184–196 (2010).Crossref, Medline, CASGoogle Scholar
    • 93  Halfinger B, Sarg B, Amann A, Hammerer-Lercher A, Lindner HH. Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method. Electrophoresis32(13),1706–1714 (2011).Crossref, Medline, CASGoogle Scholar
    • 94  Thomas A, Schanzer W, Delahaut P, Thevis M. Immunoaffinity purification of peptide hormones prior to liquid chromatography–mass spectrometry in doping controls. Methods56(2),230–235 (2012).Crossref, Medline, CASGoogle Scholar
    • 95  Huang EC, Henion JD. LC–MS and LC–MS/MS determination of protein tryptic digests. J. Am. Soc. Mass Spectrom.1(2),158–165 (1990).Crossref, Medline, CASGoogle Scholar
    • 96  Duan X, Weinstock-Guttman B, Wang H et al. Ultrasensitive quantification of serum vitamin D metabolites using selective solid-phase extraction coupled to microflow liquid chromatography and isotope-dilution mass spectrometry. Anal. Chem.82(6),2488–2497 (2010).Crossref, Medline, CASGoogle Scholar
    • 97  Christianson CC, Johnson CJ, Needham SR. The advantages of microflow LC–MS/MS compared with conventional HPLC–MS/MS for the analysis of methotrexate from human plasma. Bioanalysis5(11),1387–1396 (2013).Link, CASGoogle Scholar
    • 98  Ramanathan R, Raghavan N, Comezoglu SN, Humphreys WG. A low flow ionization technique to integrate quantitative and qualitative small molecule bioanalysis. Int. J. Mass Spectrom.301(1–3),127–135 (2011).Crossref, CASGoogle Scholar
    • 99  Murphy RE, Kinhikar AG, Shields MJ et al. Combined use of immunoassay and two-dimensional liquid chromatography–mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples. J. Pharm. Biomed. Anal.53(3),221–227 (2010).Crossref, Medline, CASGoogle Scholar