Reversed‐Phase High Performance Liquid Chromatography of Proteins

Djuro Josic1, Spomenka Kovac2

1 Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospital and Brown University, Providence, Rhode Island, 2 Department of Chemistry, J. J. Strossmayer University, Osijek, Croatia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.7
DOI:  10.1002/0471140864.ps0807s61
Online Posting Date:  August, 2010
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Reversed‐phase HPLC (RP‐HPLC) is one of most important techniques for protein separations and the method of choice for peptide separation. RP‐HPLC has been applied on the nano, micro, and analytical scale, and has also been scaled up for preparative purifications, to large industrial scale. Because of its compatibility with mass spectrometry, RP‐HPLC is an indispensable tool in proteomic research. With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated at attomolar levels for further analysis. In addition, preparative RP‐HPLC is often used for large‐scale purification of proteins. This unit provides protocols for packing and testing a column, protein separation by use of gradient or step elution, desalting of protein solutions, and separation of enzymatic digests before mass spectrometric analyses. A protocol is also provided for cleaning, regenerating, and storing reversed‐phase chromatography columns. Curr. Protoc. Protein Sci. 61:8.7.1‐8.7.22. © 2010 by John Wiley & Sons, Inc.

Keywords: reversed‐phase HPLC; proteins; peptides; LC‐MS/MS

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Packing and Testing of Semi‐Preparative and Preparative RP‐HPLC Columns
  • Basic Protocol 2: Pilot Experiment to Choose a Matrix and Determine Conditions for Sample Application and Elution for RPC Separation of Proteins
  • Basic Protocol 3: Elution of Proteins from RPC Columns
  • Basic Protocol 4: Desalting of Proteins
  • Support Protocol 1: Regeneration, Cleaning, and Storage of RPC Columns
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Packing and Testing of Semi‐Preparative and Preparative RP‐HPLC Columns

  • Appropriate RPC support (silica‐ or polymer‐based) with a particle size between 15 and 90 µm
  • Packing solution: isopropanol or methanol
  • Sintered‐glass funnel (medium grade) and degasser and/or sonicator
  • Suction flask
  • Vacuum source
  • Chromatography column (e.g., 250 × 21.2–mm i.d.) with corresponding frits (Tosoh Bioscience or Knauer,
  • Adapter with tubing to connect the top of the column to a pump (Tosoh Bioscience, or Knauer,
  • Pump: for smaller columns, an analytical HPLC pump can be used; For semi‐preparative or preparative columns, the use of special pumps (e.g., Agilent, Knauer) is recommended

Basic Protocol 2: Pilot Experiment to Choose a Matrix and Determine Conditions for Sample Application and Elution for RPC Separation of Proteins

  • Reversed‐phase supports (e.g., large‐pore silica gel with C 4, C 8, or C 18 ligands; also see Table 8.7.1)
  • Starting solution (Eluent A), degassed: 0.1% (v/v) TFA in water or 0.1% (v/v) formic acid in water (∼1 liter will be needed)
  • Protein mixture to be purified (about 5 to 10 mg protein/ml), dissolved in Eluent A
  • Eluent B, degassed: acetonitrile containing 0.1 % TFA or formic acid (1 liter)
  • 1‐ml chromatography columns: several manufacturers, e.g., GE Healthcare and Tosoh Bioseparations, offer ready‐to‐use prepacked chromatographic columns with supports; the hardware (empty column, frits and column cartridge) can also be reused for packing and testing of new materials; disk‐shaped monolithic columns (BIA Separations, can be purchased and mounted in corresponding cartridges
  • Chromatographic system with pumps, gradient mixer, detector, and fraction collector (e.g., Agilent Technologies, Waters, Knauer,

Basic Protocol 3: Elution of Proteins from RPC Columns

  • Protein sample: adjust sample to 0.1% (v/v) trifluoroacetic acid (TFA); final sample pH should be <4.0
  • ZipTip (Millipore) C 4 or C 18 pipet tips with a standard bed volume of 0.6 µl for sample elution in 1 to 4 µl
  • Wetting solution: 100% acetonitrile
  • Equilibration/wash solution: 0.1% (v/v) TFA in HPLC‐ or MS‐grade H 2O
  • Elution solution: 0.1% (v/v) TFA in 50% (v/v) acetonitrile (or methanol); for fractionation of proteins prepare varying concentrations of acetonitrile in H 2O, e.g., 5%, 10%, 20%, 30%, and 50%) with or without 0.1% TFA
  • 10‐µl pipettor compatible with the ZipTips
NOTE: To achieve optimal sample uptake and delivery, set the pipettor to 10 µl and attach the tip containing RPC resin. Throughout the desalting procedure, depress and release the plunger slowly to ensure optimal movement of solution through the resin bed.NOTE: Because of their lower hydrophobicity, the pipet tips containing the C 4 resin are most applicable for proteins, while the tips containing the more hydrophobic C 18 resin are most suitable for low‐molecular‐weight proteins and peptides.

Basic Protocol 4: Desalting of Proteins

  • Eluent A: 0.1% (v/v) TFA in H 2O
  • Eluent B: 0.1% (v/v) TFA in acetonitrile
  • 0.5 to 1.0 M NaOH
  • Concentrated acetic acid
  • Storage solvent: 70% methanol, 70% to 80% acetonitrile, or 70% to 80% isopropanol
  • Used RPC column
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  •   FigureFigure 8.7.1 Analytical RP‐HPLC of crude mixture of proteins from rabbit skeletal muscle: tropomyosin (Tm) and components of troponin (Tn) complex, comprising troponin T (TnT), troponin I (TnI), and troponin C (TnC). (A) Analytical RP‐HPLC. Column, Zorbax SB300 C8 (150 × 4.6 mm I.D., 5 µm particle size, 300 Å pore size; Agilent Technologies). Conditions: linear A‐B gradient (1% B/min starting from 25% acetonitrile) at a flow rate of 1 ml/min. Eluent A, water containing 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. (B) Preparative RP‐HPLC (optimized after scaling‐up experiments). Column, µBondapack C8 packing, 5 µm particle size, 300 Å pore size, from Waters, in column of dimensions 280 × 50 mm I.D. Eluent A, water containing 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. Sample load, 5700 mg in 440 ml water containing 0.05% TFA (∼13 mg protein/ml); following sample loading at 22 ml/min, a 10‐min isocratic hold (at constant solvent concentration) with water containing 0.05% TFA, followed by linear A‐B gradient (1.7% acetonitrile/min) up to 25% acetonitrile, then 0.1% acetonitrile/min up to 35% acetonitrile and, finally, 0.5% acetonitrile/min up to 55% acetonitrile. The positions of the individual components identified following fraction analysis are denoted in histograms. Reprinted from Mant and Hodges (), with permission
  •   FigureFigure 8.7.2 Most common column ligands used in reversed‐phase HPLC.
  •   FigureFigure 8.7.3 Structure and surface chemistry of supports for reversed‐phase HPLC. (A) Schematic presentation of surface chemistry of an end‐capped reversed‐phase C8 silica‐based support (reprinted from Engelhardt et al., , with permission). (B) Structure of a polystyrene‐based reversed‐phase HPLC support (Courtesy of GE Healthcare)
  •   FigureFigure 8.7.4 Structure of HPLC monolithic supports. (A) Silica‐based monolith (reprinted with permission from Luo, et al., . (B) Polymer‐based monolith (reprinted with permission from Lee, et al., )
  •   FigureFigure 8.7.5 Column test for a monolithic, polymer‐based, disk‐shaped column. Separation of a mixture of standard proteins. Separation conditions: column, polystyrene‐divinylbenzene CIM disk, 12 mm I.D. × 3 mm bed height, column volume 0.34 ml; Eluent A, 20% acetonitrile containing 0.15% TFA; Eluent B, 70% acetonitrile containing 0.15% TFA; gradient, 100% Eluent A for 6 sec, followed by 0 to 100% Eluent B over 90 sec; isocratic run for 10 sec at 100% B, followed by column equilibration (100% Eluent A for 20 sec), flow rate 3 ml/min, back pressure 3 bar, detection at 280 nm, room temperature. Sample: test protein solution dissolved in water for HPLC, injection volume, 20 µl. Peaks (according to the elution order): impurity from ribonuclease A, ribonuclease A (1.5 mg/ml), cytochrome C (0.5 mg/ml), bovine serum albumin (2.5 mg/ml), ovalbumin (3.0 mg/ml), and impurity from ovalbumin. Courtesy of Drs. A. Strancar and M. Barut, of BIASeparations.
  •   FigureFigure 8.7.6 Isolation of recombinant protein TM 1‐99 (mol. wt. 12,837 Da) from crude sample mixture by use of an optimized gradient. Target protein (peak P) is eluted in the middle of the gradient as a single peak, and it is separated from less hydrophobic impurities that elute earlier (front part of the chromatogram) and some more hydrophobic components that elute later. Conditions: linear gradient (1% Eluent B/min) at a flow rate of 0.3 ml/min. Eluent A: 0.05% TFA in water, Eluent B: 0.05% TFA in acetonitrile, temperature: 25°C, column: Zorbax 300SB‐C8 (150 mm × 2.1 mm I.D.; 3.5 µm particle size, 300 Ån pore size), from Agilent Technologies. Reprinted with permission from Mills et al. ()


Literature Cited

   Aguilar, M.‐E. and Hearn, M.T.W. 1996. High‐resolution reversed‐phase high‐performance liquid chromatography of peptides and proteins. Methods Enzymol. 270:3‐26.
   Chen, H. and Horvath, C.S. 1995. High‐speed high‐performance liquid chromatography of peptides and proteins. J. Chromatogr. A 705:3‐20.
   Coffman, J.L., Kramarczyk, J.F., and Kelley, B.D. 2008. High‐throughput screening of chromatographic separations: I. Method development and column modeling. Biotech. Bioeng. 100:605‐618.
   Dolan, J.W. 2002. Temperature selectivity in reversed‐phase high performance liquid chromatography. J. Chromatogr. A 965:195‐205.
   Engelhardt, H., Blay, C., and Saar, J. 2005. Reversed‐phase chromatography: The mystery of surface silanols. Chromatographia 62:S19‐S29.
   Eschelbach, J.W. and Jorgenson, J.W., 2006. Improved protein recovery in reversed‐phase liquid chromatography by the use of ultrahigh pressure. Anal. Chem. 78:1697‐1706.
   Hancock, W.S., Chloupek, R.S., Kirkland, J.J., and Snyder, L.R. 1994. Temperature as a variable in reversed‐phase high‐performance liquid chromatographic separations of peptide and protein samples: I. Optimizing the separation of a growth hormone tryptic digest. J. Chromatogr. A 686:31‐43.
   Hearn, M.T.W. 1984. Reversed‐phase high performance liquid chromatography. Methods Enzymol. 104:190‐212.
   Josic, D.J. and Clifton, J.G. 2007. Use of monolithic supports in proteomics technology. J. Chromatogr. A 1144:2‐13.
   Josic, D.J. and Strancar, A. 1997. Application of membranes and compact, porous units for separation of biopolymers. Ind. Eng. Chem. Res. 38:333‐342.
   Josic, D.J., Reutter, W., and Molnar, I. 1982. HPLC of membrane bound proteins. In Practical Aspects of Modern HPLC (I. Molnar, ed.) pp. 109‐121. Walter de Gruyter, Berlin, New York.
   Lee, D., Svec, F., and Fréchet, J.M. 2004. Photopolymerized monolithic capillary columns for rapid micro high‐performance liquid chromatographic separation of proteins. J. Chromatogr. A 1051:53‐61
   Luo, Q., Shen, Y., Hixson, K.K., Zhao, R., Yang, F., Moore, R.J., Mottaz, H.M., and Smith, R.D. 2005. Preparation of 20 µm‐i.d. silica‐based monolithic columns and their performance for proteomics analyses. Anal. Chem. 77:5028‐5035
   MacNair, J., Lewis, K.C., and Jorgenson, J.W. 1997. Ultrahigh‐pressure reversed‐phase liquid chromatography in packed capillary columns. Anal. Chem. 69:983‐989.
   Mant, C.T. and Hodges, R.S. 2002. Reversed‐phase liquid chromatography of proteins from rabbit skeletal troponin, a multi‐protein complex. J. Chromatogr. A 972:101‐114.
   Martosella, J., Zolotarjova, N., Liu, H., Moyer, S.C., Perkins, P.D., and Boyes, B.E. 2006. High recovery HPLC separation of lipid rafts for membrane proteome analysis. J. Proteome Res. 5:1301‐1312.
   McNay, J. and Fernandez, E.J. 1999. How does a protein unfold on a reversed‐phase liquid chromatography surface? J. Chromatogr. A 849:135‐148.
   Mills, J.B., Mant, C.T., and Hodges, R.S. 2006. One step purification of a recombinant protein from a whole cell extract by reversed‐phase high performance liquid chromatography. J. Chromatogr. A 1133:248‐253.
   Molnar, I. 2005. Searching for robust HPLC methods: Csaba Horváth and the solvophobic theory. Chromatographia 62:S7‐S17.
   Porath, J., Sundberg, L., Fornstedt, N., and Olsson, I. 1973. Salting‐out in amphiphilic gels as a new approach to hydrophilic adsorption. Nature 245:465‐466.
   Shi, Y., Xiang, R., Horvath, C.S., and Wilkins, J.A. 2004. Role of chromatographic techniques in proteomic analysis. J. Chromatogr. A 1053:27‐36.
   Sinanoglu, O. 1980. The solvophobic theory for the prediction of molecular conformations and biopolymer bindings in solution with recent direct experimental tests. Int. J. Quant. Chem. 18:381‐392.
   Svec, F. and Huber, C.G. 2006. Monolithic materials: Promises, challenges, achievements. Anal. Chem. 78:2101‐2107.
   Wagner, K., Miliotis, T., Marko‐Varga, G., Bischoff, R., and Unger, K.K. 2002. An automated on‐line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal. Chem. 74:809‐820.
   Wolters, D.A., Washburn, M.P., and Yates, J.R. III 2001. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73:5683‐5690.
   Yang, Y., Boysen, R.I., Harris, S.J., and Hearn, M.T.W. 2009. Peptide mapping with mobile phases of intermediate pH value using capillary reversed‐phase high‐performance liquid chromatography/electrospray ionisation tandem mass spectrometry. J. Chromatogr. A 1216:3767‐3773.
Key References
   Aguilar and Hearn, 1996. See above.
  Describes both theoretical and practical aspects of analytical and preparative reversed‐phase chromatography and includes a number of references to theory as well as practical aspects of this method in the literature.
   Shi et al., 2004. See above.
  An excellent review about the use of chromatographic techniques, especially reversed‐phase HPLC in proteomics technology.
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