Lecture 8 Chromatography in proteomics Affinity Ion Exchange Reversed-phase

1. Lecture 8 • Chromatography in proteomics • Affinity • Ion Exchange • Reversed-phase Oct 2010 SDMBT

2. Affinity Chromatography Principles of Affinity Chromatography • Affinity chromatography is based on biospecific binding • interactions between a ligand chemically bound (immobilised) to the • chromatographic packing and a target molecule in the sample. • Some examples of biospecific binding • anantigen to an antibody; • a substrate, inhibitor or cofactor to an enzyme; • a regulatory protein to a cell surface receptor; • etc. • The forces involved in the binding can be ionic or hydrophobic • interaction. • In affinity chromatography, one member of the ligand pair is • immobilized (i.e. covalently bonded/coupled) as a bonded phase. April 2006

3. Affinity Examples of biological interactions used in affinity chromatography April 2006

4. Affinity • a spacer arm or linker • to place some distance between the bound ligand • and the support matrix to improve protein accessibility to the ligand Immobilized ligand should only bind to one specific protein The immobilized ligand / support matrix combination should be highly selective stationary phase matrix • The support matrix (packing or base material) and • spacer arm (linker) themselves should have • minimalbinding interaction (nonspecific adsorption) • with any of the molecules in solution. April 2006

5. Affinity Properties of ligands • Ligand should have chemical properties that allow easy • immobilization to a matrix. • The ligandmust be able to form reversible complexes with the protein to be isolated or separated. • The complex formation equilibrium constant should be high enough for the formation of stable complexes or to give sufficient retardation. • It should be easy to dissociate the complex by a simple change in the medium, without irreversibly affecting the protein to be isolated or the ligand. April 2006

6. Affinity Types of ligands • Monospecific low molecular weight ligands • -- these ligands bind to a single or a very small number of proteins. • Group-specific low molecular weight ligands • -- the largest group of ligands, eg a wide variety of enzyme cofactors, • biomimetic dyes, boronic acid derivatives, and a number of amino • acids and vitamins. • The target proteins are most often enzymes and the most • thoroughly studied are the NAD+- and NADP+-dependent • dehydrogenases and kinases. April 2006

7. Affinity • Monospecific macromolecular ligands • It is through specific protein-protein interactions, • e.g. the binding of fibronectin to gelatin; • antithrombin to thrombin and heparin; • transferrin receptor to transferrin; • antibody to antigen; • etc. • Immunoadsorbents • It is through high specificity of antibodies. • Both antigens and antibodies can be used as affinity ligands. • The traditional immunoadsorbents are based on polyclonal antibodies. • The modern immunoadsorbents are based on monoclonal antibodies. April 2006

8. Affinity • Group-specific macromolecular ligands • This group includes several ligands that have found widespread • popularity; • e.g., lectins such as concanavalin (Con A) and lentil for glycoproteins; • protein A and protein G for antibody; • calmodulin for calcium-dependent enzymes; • etc. April 2006

9. Affinity Choice of spacer arm (linker) • Low molecular weight ligands might show poor function due • to low steric availability of the ligands. • The use of a spacer arm or linker can solve this problem. • Commonly used linkers are aliphatic, linear hydrocarbon chains • with two functional groups located at each end of the chain. • One of the groups (often a primary amine, -NH2) is attached to • the matrix, whereas the group at the other end (usually a • carboxyl, -COOH, or amino group, -NH2) is attached to the ligand. April 2006

10. 6 carbon chain – hydrophobic may attract hydrophobic proteins Stationary phase packing material – usually hydrophilic Affinity • The most common spacers are 6-aminohexanoic acid [H2N- • (CH2)6-COOH], hexamethylene diamine [H2N- (CH2)6-NH2], • and l,7-diamino-4-azaheptane (3,3-diaminodipropylamine). • A drawback with the hydrocarbon linkers, especially the longer ones, • is that they can give rise to unwanted nonspecific interactions – • hydrophobic interactions April 2006

11. Affinity Major affinity systems with pre-immobilized ligands Protein A / G affinity chromatography • Protein A / G, which is a specific protein originally extracted • from the surface of certain gram positive bacteria i.e. • staphylococcal and streptococcal, but now usually made • recombinantly, is immobilized onto eg Sepharose beads • These proteins selectively bind to a broad range of antibody • molecules, thus forming an affinity column for antibodies. • Proteins A and G differ in both their species and subclass • specificity for antibody binding (shown in Table 4-5.). April 2006

12. Table 4-4. April 2006

13. Affinity • When both proteins will work, protein A is always recommended • because of the lower cost and harsher conditions which can be • used in cleaning and regeneration. • The most important type of antibody bound by protein G and not • normally by protein A is mouse IgG1 which is the most common • subclass of monoclonal antibodies. However, the addition of high • salt (2-3 M NaCl) with high pH (8-9) to the binding / wash buffer • will cause these antibodies to bind. April 2006

14. Affinity Immobilized metal affinity chromatography (IMAC) • A metal chelating group (typically imidodiacetate) is immobilized; • a multivalent transition metal ion (typically Cu2+>Ni2+>Zn2+ Co2+ • or Fe2+ in order of binding strength) is bound in such a way that one • or more coordination sites are available for interaction with proteins. • Certain surface amino acids (primarily histidines) bind specifically • to these free coordination sites. • The separation is based on the surface concentration of these • amino acids. April 2006

15. Affinity Elution Method Because ligand-ligand interaction complex mixture of hydrophobic, ionic forces – elution mechanisms may be complex • Elution can be done with any agent that disrupts the ligand- • ligand interaction. • The most common technique is to employ a shift to acidic pH • (usually to pH 2-4), which is used extensively for protein A/G • and for antibody ligand affinity methods. • Affinity elution is usually in the form of a step gradient. • For IMAC, a gradient elution in imidazole concentration is • normally used. Imidazole is the active functionality in histidine • which binds to the metal coordination site. Imidazole competes with protein to bind to the IMAC sepharose April 2006

16. Affinity - purification of protein complexes Tandem Afffinity Purification (TAP) A method to find out protein-protein interactions TAP tag consists of (i) calmodulin-binding peptide (ii) TEV protease cleavage site (iii) Protein A • DNA coding the TAP tag is inserted after the DNA for the protein of interest • Organism produces a recombinant protein with the TAP tag • The protein of interest is free to associate with other proteins • Cell is lysed and protein complex with TAP tag is released to bind to IgG sepharose • beads (IgG+protein A have specific attraction) • All other proteins are washed away • TEV protease used to cut off protein A • in TAP tag Oct 2010 SDMBT

17. Affinity - purification of protein complexes Tandem Afffinity Purification (TAP) • Residual protein binds to calmodulin beads through calmodulin binding peptide • Elute the protein with a buffer containing EGTA • EGTA chelates Ca2+ which is responsible for binding calmodulin to the calmodulin • binding peptide • Protein-protein interactions in complex are not disrupted so far. • Break up the complex by SDS and run SDS PAGE or trypsin digestion From: Is proteomics heading in the wrong direction? Lukas A. Huber, Nature Reviews Molecular Cell Biology 4, 74-80 (January 2003) Oct 2010 SDMBT http://www.bio.davidson.edu/Courses/Molbio/calmod/calmodulin.html

18. Affinity - purification of phosphopeptides by IMAC • Peptide mixture is methyl esterified with anhydrous • methanol and thionyl chloride (SOCl2) Acidic amino acids will also complex to Fe3+ in IMAC So esterification ensures only phosphorylated amino acids trapped Fe3+ • Methyl esterified peptide passed through Fe3+IMAC column • Traps only phosphorylated peptides • Phosphorylated peptides eluted with phosphate buffer – phosphate • competes to bind to Fe3+) Oct 2010 SDMBT

19. Ion-exchange chromatography (IEX) Fundamental Concepts • It separates proteins based on differences in their accessible • surface charges; • Charged proteins and other ions compete to bind to • the oppositely charged groups on an ion exchanger CATION-EXCHANGER attracts cations ANION-EXCHANGER attracts anions Anion-exchange resin Positive charge Attracts negative ions (eg anionic proteins) Protein accessible charge negative (-) anionic April 2006

20. IEX Cation-exchange resin Negative charge attracts positive ions (eg cationic proteins) Charged functional Group covalently bonded To resin/stationary phase bead Protein accessible charge positive (+) cationic Protein accessible charge positive (+) cationic Oct 2010 SDMBT

21. IEX • The interaction between small molecules and an ion • exchanger depends on the net charge and the ionic strength • of the medium – ionic strength, • When the concentration of competing ions is low, the ions • of interest bind to the ion exchanger, whereas when it is • high, they are desorbed; Ionic Strength i.e. concentration Na+ and Cl- Attraction between the protein and the ion-exchanger Na+ competes with the Cationic protein to bind to the ion-exchanger April 2006

22. IEX Principle of ion-exchange chromatography. Species with several positive charges (A3+) are adsorbed to the column; those with few charges move slowly, whereas those with zero net charge or a net negative charge pass through the column unretained ie they are not well separated April 2006

23. IEX • The interaction between a protein and an ion exchanger depends on • -- the net charge; • -- the ionic strength of the mobile phase; • -- the surface charge distribution of the protein; • -- pH; • -- the nature of particular ions in the solvent; • -- additives e.g. organic solvents • -- properties of the ion exchanger. • The more highly charged a protein is, the more strongly it will • bind to a given, oppositely charged ion exchanger. • More highly charged ion exchangers, usually bind proteins more • effectively than weakly charged exchangers. April 2006

24. IEX The pH parameter • pH determines charge on both the protein and the ion • exchanger, therefore it is one of the most important • parameters in determining protein binding. • At pH values far away from the pI, proteins bind strongly • and in practice do not desorb at low ionic strength. • Near to its pI, the net charge of a protein is less and • consequently it binds less strongly. • An ion exchanger is normally used in conditions where its • charges will not be significantly changed (titrated) • by small shifts in pH. April 2006

25. IEX Influence of Ions • The proteins compete with other ions in the mobile phase • buffer/solvent to bind to the charged groups on the ion exchanger; • If concentration of competing ions low, proteins preferably • bound through interactions between several charged groups • on the proteins and oppositely charged groups on the ion • exchanger. • If concentrations of competing ions high, the proteins • will start to be displaced from the ion exchanger. The most • weakly bound are displaced and eluted from the column • first. April 2006

26. IEX April 2006

27. IEX The stationary phase: ion exchangers • The stationary phase in ion-exchange (usually known • as an ion-exchange resin) is made of 2 components • packing or base material (section 3.3.2) • functional group or bonded phase (see below) Functional Groups - Functional groups are bonded covalently/ permamently to the packing material. April 2006

28. IEX eg Cation Exchange resin– the bound functional group has negative charge- the counterion associated with the functional group is positive (cation)- the counterion can be easily displaced by other cations since not permamently bound to resin particle – exchange cations • Summary: • , Cation exchange column separates cations (positive charge) • Anion exchange column separates anions (negative charge) April 2006

29. IEX • Anion or cation exchange functional groups can be classified as • either “weak” or “strong”; • Strong ion exchange resins - charge of resin is independent • of the pH of the mobile phase.. April 2006

30. IEX • Weak ion exchange groups - may gain or lose electrical charge as the • pH of the mobile phase changes. – can be used as over limited pH range * Note: the terms “strong” or “weak” do not refer to the strength of the binding but only to the effect of pH on the charge of the functional groups. April 2006

31. IEX • All cation exchangers have a limiting pH below which they • cannot be used. As a rule of thumb, the pKa is suggested as • the lower limit. • Weak anion exchangers have an upper pH limit for their use. • For the quaternary amines, there is no upper limit as they will • not lose the charge whatever the pH. April 2006

32. IEX Table 3-1. Functional groups used in ion exchangers April 2006

33. IEX The mobile phase: buffers and salts Buffer pH and concentration • Normally the concentration of buffer salts during protein • adsorption is low, around 0.01 to 0.05 M. • Criteria for choosing buffer: • (i) The buffer should have a high capacity, preferably with • the pKa of the buffering species less than 0.5 units • from the working pH; • (ii) The buffering species should not interact with the ion • exchanger. • For an anion exchanger, a positive buffering ion, such as Tris • (pKa 8.2), is often used and usually with Cl- as the counterion. • For a cation exchanger, a negatively charged buffering ion is • recommended, e.g., phosphate, acetate, and the counterions • are mostly Na+ and K+. April 2006

34. IEX • A nonbuffering salt, such as NaCl, is usually added to the buffer • to elute proteins from an ion exchanger • Elution methods may also include changes in pH along with (or • instead of) ionic strength increases. Because the pH can affect the charge of the sample molecules as well as the charge of the bonded phase (i.e. weak ion exchange media). Changes in pH can thus be used to weaken or eliminate charge-charge interactions, thereby causing elution. April 2006

35. IEX Experimental planning and preparation Choosing an ion exchange column • Use anion or cation exchange column. • It depends on the charge characteristics and the effect • of pH on stability and solubility of both the target • molecule itself and the other molecules in the sample. (ii) To maximize binding strength, select an operating pH range that is either above or below the pI of the target, based on where the biomolecule is most stable and soluble. If pH=pI of protein, protein Neutral (uncharged) will not bind to IEX resin April 2006

36. IEX (iii) The ion exchanger is then chosen by the following rule: -- If pH of medium > pI, the protein molecule is negative. Try anion exchange column first. -- If pH of medium < pI the protein molecule is positive. Try cation exchange column first. April 2006

37. (ie pI>7) (ie pI < 7) IEX pH 7 pH < 7 Many other biomolecules have a solubility and stability pH range that encompasses their pI, so that either anion or cation exchange can be used. April 2006

38. IEX • Use a weak or strong ion exchange functional group. -- For most biomolecules and pH ranges, either strong or weak ion exchange media may be used; -- As a starting point for method development, use strong ion exchange media, since they operate over a broader pH range and equilibrate more easily than weak ion exchange media. Need to soak weak ion exchange media with buffer containing a counterion Eg cation exchanger – H+, Na+, K+ April 2006

39. IEX -- In extreme pH conditions (pH>10 for anion exchange and pH<3-5 for cation exchange), weak ion exchange media lose most of their charge, and thus bind molecules very weakly or not at all; -- In addition, weak ion exchange media can take much longer to equilibrate because the column itself has a significant buffering capacity. If media have no charge – then cannot attract ions - cannot be ion exchange April 2006

40. Reversed-phase RP -Reversed-phase chromatography – stationary phase made of porous silica beads modified by long hydrophobic alkyl C18H37 (C18) chains - Stationary phase is hydrophobic and attracts hydrophobic molecules • Many different silica base materials available • e.g. Zorbax, Poroshell etc – different pore • sizes and particle sizes available Oct 2010 SDMBT

41. Reversed-phase RP • Typical mobile phases are water/methanol • or water/acetonitrile mixtures - Typically samples are eluted by a gradient of increasing non-polar solvent (methanol or acetonitrile) concentration. e.g. start with 2% increasing to 80% acetonitrile - Usually buffered or acidified e.g. 0.1% trifluoroacetic acid, or formic acid (volatile acid) – if sample is for LC-MS The more polar the compound (has more OH groups, C=O etc), shorter retention time.The less polar the compound (has more C-H, C-C bonds),longer retention time. Oct 2010 SDMBT

42. Reversed-phase RP -SH more polar than disulfide linked peptides Tryptic digest was separated on a ZORBAX 300SB-C18 column before (bottom panel) and after (top panel) reduction with TCEP - Tris(2-carboxyethyl)phosphine hydrochloride. Major peaks, which disappeared following reduction are indicated by T1 to T3. The peptide constituents of the complexes are shown in Figure 11.11.2. T2* and T3* indicate incomplete tryptic cleavages of T2 and T3, respectively. Single peptides that appear as a result of reduction are indicated in the top panel by their residue numbers. From Determination of Disulfide‐Bond Linkages in Proteins Hsin‐Yao Tang, David W. Speicher, Current Protocols in Protein Science, 2004 Oct 2010 SDMBT

43. From Determination of Disulfide‐Bond Linkages in Proteins Hsin‐Yao Tang, David W. Speicher, Current Protocols in Protein Science, 2004 Oct 2010 SDMBT

44. MudPIT approach to proteomics Column is a SCX (strong cation exchanger) followed by a reverse phase column Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry Andrew J. Link, Jennifer L. Jennings, Michael P. Washburn, Current Protocols in Protein Science 2004 Oct 2010 SDMBT

45. MudPIT approach to proteomics Cell lysate Digest with trypsin Load samples on SCX with e.g. 5% acetonitrile 0.1% formic acid Elute with increasing % acetonitrile – uncharged peptides separated by polarity Change to higher ionic strength buffer e.g. add some 5% acetonitrile, 0.1 % formic acid 500 mM ammonium acetate to mobile phase to elute low charge peptides Elute with increasing % acetonitrile – low charge peptides separated by polarity Change to even higher ionic strength buffer to elute more highly charged peptides Oct 2010 SDMBT

46. MudPIT approach to proteomics All compounds eluting from the RP-HPLC ionised straightaway by electrospray ionisation MS captures the molecular weight of peptides eluting at a particular time Programme MS to select the top 4-5 peptides to fragment further MS/MS spectra – can tell the amino sequence of part of peptide – identify protein Oct 2010 SDMBT

47. MudPIT vs 2-D Gels Oct 2010 SDMBT

48. MudPIT vs 2-D Gels Oct 2010 SDMBT