High Throughput Screening of Disulfide-containing Proteins in a Complex Mixture

Overview

Journal Proteomics

Date 2013 Sep 14

PMID 24030959

Citations 9

Authors

Dong S Zhao

Zachery R Gregorich

Ying Ge

Affiliations

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Abstract

The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new LC/MS strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2-carboxyethyl)phosphine. LC/MS analysis of reduced and nonreduced protein mixtures quickly revealed disulfide-containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide-containing and disulfide-free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on-line LC/MS analysis), and reliable (no S-S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.

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References

Wu S, Jardine I, Hancock W, Karger B . A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron.... Rapid Commun Mass Spectrom. 2004; 18(19):2201-7. DOI: 10.1002/rcm.1609. View

Gorman J, Wallis T, Pitt J . Protein disulfide bond determination by mass spectrometry. Mass Spectrom Rev. 2002; 21(3):183-216. DOI: 10.1002/mas.10025. View

Xu G, Zhai H, Narayan M, McLafferty F, Scheraga H . Simultaneous characterization of the reductive unfolding pathways of RNase B isoforms by top-down mass spectrometry. Chem Biol. 2004; 11(4):517-24. DOI: 10.1016/j.chembiol.2004.03.020. View

Thevis M, Ogorzalek Loo R, Loo J . Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds. J Am Soc Mass Spectrom. 2003; 14(6):635-47. DOI: 10.1016/S1044-0305(03)00199-5. View

Peng Y, Chen X, Sato T, Rankin S, Tsuji R, Ge Y . Purification and high-resolution top-down mass spectrometric characterization of human salivary α-amylase. Anal Chem. 2012; 84(7):3339-46. DOI: 10.1021/ac300083y. View

Wu J, Watson J . A novel methodology for assignment of disulfide bond pairings in proteins. Protein Sci. 1997; 6(2):391-8. PMC: 2143640. DOI: 10.1002/pro.5560060215. View

Wedemeyer W, Welker E, Narayan M, Scheraga H . Disulfide bonds and protein folding. Biochemistry. 2000; 39(15):4207-16. DOI: 10.1021/bi992922o. View

Yen T, Joshi R, Yan H, Seto N, Palcic M, Macher B . Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. J Mass Spectrom. 2000; 35(8):990-1002. DOI: 10.1002/1096-9888(200008)35:8<990::AID-JMS27>3.0.CO;2-K. View

Zhang H, Ge Y . Comprehensive analysis of protein modifications by top-down mass spectrometry. Circ Cardiovasc Genet. 2011; 4(6):711. PMC: 3320739. DOI: 10.1161/CIRCGENETICS.110.957829. View

10.

Qin J, Chait B . Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry. Anal Chem. 1997; 69(19):4002-9. DOI: 10.1021/ac970489n. View

11.

Zhang J, Dong X, Hacker T, Ge Y . Deciphering modifications in swine cardiac troponin I by top-down high-resolution tandem mass spectrometry. J Am Soc Mass Spectrom. 2010; 21(6):940-8. PMC: 3056346. DOI: 10.1016/j.jasms.2010.02.005. View

12.

Zhang Y, Cui W, Zhang H, Dewald H, Chen H . Electrochemistry-assisted top-down characterization of disulfide-containing proteins. Anal Chem. 2012; 84(8):3838-42. PMC: 3661022. DOI: 10.1021/ac300106y. View

13.

Ge Y, Lawhorn B, ElNaggar M, Strauss E, Park J, Begley T . Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry. J Am Chem Soc. 2002; 124(4):672-8. DOI: 10.1021/ja011335z. View

14.

Loo J, Edmonds C, Udseth H, Smith R . Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra. Anal Chem. 1990; 62(7):693-8. DOI: 10.1021/ac00206a009. View

15.

Ge Y, Rybakova I, Xu Q, Moss R . Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state. Proc Natl Acad Sci U S A. 2009; 106(31):12658-63. PMC: 2722289. DOI: 10.1073/pnas.0813369106. View

16.

Kellie J, Tran J, Lee J, Ahlf D, Thomas H, Ntai I . The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput. Mol Biosyst. 2010; 6(9):1532-9. PMC: 3115741. DOI: 10.1039/c000896f. View