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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 4.1The Purification of Proteins Is an Essential First Step in Understanding Their Function

An adage of biochemistry is, Never waste pure thoughts on an impure protein. Starting from pure proteins, we can determine amino acid sequences and evolutionary relationships between proteins in diverse organisms and we can investigate a protein's biochemical function. Moreover, crystals of the protein may be grown from pure protein, and from such crystals we can obtain x-ray data that will provide us with a picture of the protein's tertiary structure—the actual functional unit.

4.1.1. The Assay: How Do We Recognize the Protein That We Are Looking For?

Purification should yield a sample of protein containing only one type of molecule, the protein in which the biochemist is interested. This protein sample may be only a fraction of 1% of the starting material, whether that starting material consists of cells in culture or a particular organ from a plant or animal. How is the biochemist able to isolate a particular protein from a complex mixture of proteins?

The biochemist needs a test, called an assay, for some unique identifying property of the protein so that he or she can tell when the protein is present. Determining an effective assay is often difficult; but the more specific the assay, the more effective the purification. For enzymes, which are protein catalysts (Chapter 8), the assay is usually based on the reaction that the enzyme catalyzes in the cell. Consider the enzyme lactate dehydrogenase, an important player in the anaerobic generation of energy from glucose as well as in the synthesis of glucose from lactate. Lactate dehydrogenase carries out the following reaction:

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Nicotinamide adenine dinucleotide [reduced (NADH); Section 14.3.1] is distinguishable from the other components of the reaction by its ability to absorb light at 340 nm. Consequently, we can follow the progress of the reaction by examining how much light the reaction mixture absorbs at 340 nm in unit time—for instance, within 1 minute after the addition of the enzyme. Our assay for enzyme activity during the purification of lactate dehydrogenase is thus the increase in absorbance of light at 340 nm observed in 1 minute.

To be certain that our purification scheme is working, we need one additional piece of information—the amount of protein present in the mixture being assayed. There are various rapid and accurate means of determining protein concentration. With these two experimentally determined numbers—enzyme activity and protein concentration—we then calculate the specific activity, the ratio of enzyme activity to the amount of protein in the enzyme assay. The specific activity will rise as the purification proceeds and the protein mixture being assayed consists to a greater and greater extent of lactate dehydrogenase. In essence, the point of the purification is to maximize the specific activity.

4.1.2. Proteins Must Be Released from the Cell to Be Purified

Having found an assay and chosen a source of protein, we must now fractionate the cell into components and determine which component is enriched in the protein of interest. Such fractionation schemes are developed by trial and error, on the basis of previous experience. In the first step, a homogenate is formed by disrupting the cell membrane, and the mixture is fractionated by centrifugation, yielding a dense pellet of heavy material at the bottom of the centrifuge tube and a lighter supernatant above (Figure 4.1). The supernatant is again centrifuged at a greater force to yield yet another pellet and supernatant. The procedure, called differential centrifugation, yields several fractions of decreasing density, each still containing hundreds of different proteins, which are subsequently assayed for the activity being purified. Usually, one fraction will be enriched for such activity, and it then serves as the source of material to which more discriminating purification techniques are applied.

Figure 4.1. Differential Centrifugation.

Figure 4.1

Differential Centrifugation. Cells are disrupted in a homogenizer and the resulting mixture, called the homogenate, is centrifuged in a step-by-step fashion of increasing centrifugal force. The denser material will form a pellet at lower centrifugal force (more...)

4.1.3. Proteins Can Be Purified According to Solubility, Size, Charge, and Binding Affinity

Several thousand proteins have been purified in active form on the basis of such characteristics as solubility, size, charge, and specific binding affinity. Usually, protein mixtures are subjected to a series of separations, each based on a different property to yield a pure protein. At each step in the purification, the preparation is assayed and the protein concentration is determined. Substantial quantities of purified proteins, of the order of many milligrams, are needed to fully elucidate their three-dimensional structures and their mechanisms of action. Thus, the overall yield is an important feature of a purification scheme. A variety of purification techniques are available.

Salting Out

Most proteins are less soluble at high salt concentrations, an effect called salting out. The salt concentration at which a protein precipitates differs from one protein to another. Hence, salting out can be used to fractionate proteins. For example, 0.8 M ammonium sulfate precipitates fibrinogen, a blood-clotting protein, whereas a concentration of 2.4 M is needed to precipitate serum albumin. Salting out is also useful for concentrating dilute solutions of proteins, including active fractions obtained from other purification steps. Dialysis can be used to remove the salt if necessary.


Proteins can be separated from small molecules by dialysis through a semipermeable membrane, such as a cellulose membrane with pores (Figure 4.2). Molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis bag, whereas smaller molecules and ions traverse the pores of such a membrane and emerge in the dialysate outside the bag. This technique is useful for removing a salt or other small molecule, but it will not distinguish between proteins effectively.

Figure 4.2. Dialysis.

Figure 4.2

Dialysis. Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium.

Gel-Filtration Chromatography

More discriminating separations on the basis of size can be achieved by the technique of gel-filtration chromatography (Figure 4.3). The sample is applied to the top of a column consisting of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose (which are carbohydrates) or polyacrylamide. Sephadex, Sepharose, and Bio-gel are commonly used commercial preparations of these beads, which are typically 100 μm (0.1 mm) in diameter. Small molecules can enter these beads, but large ones cannot. The result is that small molecules are distributed in the aqueous solution both inside the beads and between them, whereas large molecules are located only in the solution between the beads. Large molecules flow more rapidly through this column and emerge first because a smaller volume is accessible to them. Molecules that are of a size to occasionally enter a bead will flow from the column at an intermediate position, and small molecules, which take a longer, tortuous path, will exit last.

Figure 4.3. Gel Filtration Chromatography.

Figure 4.3

Gel Filtration Chromatography. A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones.

Ion-Exchange Chromatography

Proteins can be separated on the basis of their net charge by ion-exchange chromatography. If a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not (Figure 4.4). A positively charged protein bound to such a column can then be eluted (released) by increasing the concentration of sodium chloride or another salt in the eluting buffer because sodium ions compete with positively charged groups on the protein for binding to the column. Proteins that have a low density of net positive charge will tend to emerge first, followed by those having a higher charge density. Positively charged proteins (cationic proteins) can be separated on negatively charged carboxymethyl-cellulose (CM-cellulose) columns. Conversely, negatively charged proteins (anionic proteins) can be separated by chromatography on positively charged diethylaminoethyl-cellulose (DEAE-cellulose) columns.

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Figure 4.4. Ion-Exchange Chromatography.

Figure 4.4

Ion-Exchange Chromatography. This technique separates proteins mainly according to their net charge.

Affinity Chromatography

Affinity chromatography is another powerful and generally applicable means of purifying proteins. This technique takes advantage of the high affinity of many proteins for specific chemical groups. For example, the plant protein concanavalin A can be purified by passing a crude extract through a column of beads containing covalently attached glucose residues. Concanavalin A binds to such a column because it has affinity for glucose, whereas most other proteins do not. The bound concanavalin A can then be released from the column by adding a concentrated solution of glucose. The glucose in solution displaces the column-attached glucose residues from binding sites on concanavalin A (Figure 4.5). Affinity chromatography is a powerful means of isolating transcription factors, proteins that regulate gene expression by binding to specific DNA sequences. A protein mixture is percolated through a column containing specific DNA sequences attached to a matrix; proteins with a high affinity for the sequence will bind and be retained. In this instance, the transcription factor is released by washing with a solution containing a high concentration of salt. In general, affinity chromatography can be effectively used to isolate a protein that recognizes group X by (1) covalently attaching X or a derivative of it to a column, (2) adding a mixture of proteins to this column, which is then washed with buffer to remove unbound proteins, and (3) eluting the desired protein by adding a high concentration of a soluble form of X or altering the conditions to decrease binding affinity. Affinity chromatography is most effective when the interaction of the protein and the molecule that is used as the bait is highly specific.

Figure 4.5. Affinity Chromatography.

Figure 4.5

Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G).

High-Pressure Liquid Chromatography

The resolving power of all of the column techniques can be improved substantially through the use of a technique called high-pressure liquid chromatography (HPLC), which is an enhanced version of the column techniques already discussed. The column materials themselves are much more finely divided and, as a consequence, there are more interaction sites and thus greater resolving power. Because the column is made of finer material, pressure must be applied to the column to obtain adequate flow rates. The net result is high resolution as well as rapid separation (Figure 4.6).

Figure 4.6. High-Pressure Liquid Chromatography (HPLC).

Figure 4.6

High-Pressure Liquid Chromatography (HPLC). Gel filtration by HPLC clearly defines the individual proteins because of its greater resolving power: (1) thyroglobulin (669 kd), (2) catalase (232 kd), (3) bovine serum albumin (67 kd), (4) ovalbumin (43 kd), (more...)

4.1.4. Proteins Can Be Separated by Gel Electrophoresis and Displayed

How can we tell whether a purification scheme is effective? One way is to ascertain that the specific activity rises with each purification step. Another is to visualize the effectiveness by displaying the proteins present at each step. The technique of electrophoresis makes the latter method possible.

Gel Electrophoresis

A molecule with a net charge will move in an electric field. This phenomenon, termed electrophoresis, offers a powerful means of separating proteins and other macromolecules, such as DNA and RNA. The velocity of migration (v) of a protein (or any molecule) in an electric field depends on the electric field strength (E), the net charge on the protein (z), and the frictional coefficient (f).

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The electric force Ez driving the charged molecule toward the oppositely charged electrode is opposed by the viscous drag fv arising from friction between the moving molecule and the medium. The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity (η) of the medium. For a sphere of radius r,

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Electrophoretic separations are nearly always carried out in gels (or on solid supports such as paper) because the gel serves as a molecular sieve that enhances separation (Figure 4.7). Molecules that are small compared with the pores in the gel readily move through the gel, whereas molecules much larger than the pores are almost immobile. Intermediate-size molecules move through the gel with various degrees of facility. Electrophoresis is performed in a thin, vertical slab of polyacrylamide. The direction of flow is from top to bottom. Polyacrylamide gels, formed by the polymerization of acrylamide and cross-linked by methylenebisacrylamide, are choice supporting media for electrophoresis because they are chemically inert and are readily formed (Figure 4.8). Electrophoresis is the opposite of gel filtration in that all of the molecules, regardless of size, are forced to move through the same matrix. The gel behaves as one bead of a gel-filtration column.

Figure 4.7. Polyacrylamide Gel Electrophoresis.

Figure 4.7

Polyacrylamide Gel Electrophoresis. (A) Gel electrophoresis apparatus. Typically, several samples undergo electrophoresis on one flat polyacrylamide gel. A microliter pipette is used to place solutions of proteins in the wells of the slab. A cover is (more...)

Figure 4.8. Formation of a Polyacrylamide Gel.

Figure 4.8

Formation of a Polyacrylamide Gel. A three-dimensional mesh is formed by co-polymerizing activated monomer (blue) and cross-linker (red).

Proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel under denaturing conditions. The mixture of proteins is first dissolved in a solution of sodium dodecyl sulfate (SDS), an anionic detergent that disrupts nearly all noncovalent interactions in native proteins. Mercaptoethanol (2-thioethanol) or dithiothreitol also is added to reduce disulfide bonds. Anions of SDS bind to main chains at a ratio of about one SDS anion for every two amino acid residues. This complex of SDS with a denatured protein has a large net negative charge that is roughly proportional to the mass of the protein. The negative charge acquired on binding SDS is usually much greater than the charge on the native protein; this native charge is thus rendered insignificant. The SDS-protein complexes are then subjected to electrophoresis. When the electrophoresis is complete, the proteins in the gel can be visualized by staining them with silver or a dye such as Coomassie blue, which reveals a series of bands (Figure 4.9). Radioactive labels can be detected by placing a sheet of x-ray film over the gel, a procedure called autoradiography.

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Figure 4.9. Staining of Proteins After Electrophoresis.

Figure 4.9

Staining of Proteins After Electrophoresis. Proteins subjected to electrophoresis on an SDS-polyacrylamide gel can be visualized by staining with Coomassie blue. [Courtesy of Kodak Scientific Imaging Systems.]

Small proteins move rapidly through the gel, whereas large proteins stay at the top, near the point of application of the mixture. The mobility of most polypeptide chains under these conditions is linearly proportional to the logarithm of their mass (Figure 4.10). Some carbohydrate-rich proteins and membrane proteins do not obey this empirical relation, however. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is rapid, sensitive, and capable of a high degree of resolution. As little as 0.1 μg (~2 pmol) of a protein gives a distinct band when stained with Coomassie blue, and even less (~0.02 μg) can be detected with a silver stain. Proteins that differ in mass by about 2% (e.g., 40 and 41 kd, arising from a difference of about 10 residues) can usually be distinguished.

Figure 4.10. Electrophoresis Can Determine Mass.

Figure 4.10

Electrophoresis Can Determine Mass. The electrophoretic mobility of many proteins in SDS-polyacrylamide gels is inversely proportional to the logarithm of their mass. [After K. Weber and M. Osborn, The Proteins, vol. 1, 3d ed. (Academic Press, 1975), (more...)

We can examine the efficacy of our purification scheme by analyzing a part of each fraction by SDS-PAGE. The initial fractions will display dozens to hundreds of proteins. As the purification progresses, the number of bands will diminish, and the prominence of one of the bands should increase. This band will correspond to the protein of interest.

Isoelectric Focusing

Proteins can also be separated electrophoretically on the basis of their relative contents of acidic and basic residues. The isoelectric point (pl) of a protein is the pH at which its net charge is zero. At this pH, its electrophoretic mobility is zero because z in equation 1 is equal to zero. For example, the pI of cytochrome c, a highly basic electron-transport protein, is 10.6, whereas that of serum albumin, an acidic protein in blood, is 4.8. Suppose that a mixture of proteins undergoes electrophoresis in a pH gradient in a gel in the absence of SDS. Each protein will move until it reaches a position in the gel at which the pH is equal to the pI of the protein. This method of separating proteins according to their isoelectric point is called isoelectric focusing. The pH gradient in the gel is formed first by subjecting a mixture of polyampholytes (small multicharged polymers) having many pI values to electrophoresis. Isoelectric focusing can readily resolve proteins that differ in pI by as little as 0.01, which means that proteins differing by one net charge can be separated (Figure 4.11).

Figure 4.11. The Principle of Isoelectric Focusing.

Figure 4.11

The Principle of Isoelectric Focusing. A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (more...)

Two-Dimensional Electrophoresis

Isoelectric focusing can be combined with SDS-PAGE to obtain very high resolution separations. A single sample is first subjected to isoelectric focusing. This single-lane gel is then placed horizontally on top of an SDS-polyacrylamide slab. The proteins are thus spread across the top of the polyacrylamide gel according to how far they migrated during isoelectric focusing. They then undergo electrophoresis again in a perpendicular direction (vertically) to yield a twodimensional pattern of spots. In such a gel, proteins have been separated in the horizontal direction on the basis of isoelectric point and in the vertical direction on the basis of mass. It is remarkable that more than a thousand different proteins in the bacterium Escherichia coli can be resolved in a single experiment by two-dimensional electrophoresis (Figure 4.12).

Figure 4.12. Two-Dimensional Gel Electrophoresis.

Figure 4.12

Two-Dimensional Gel Electrophoresis. (A) A protein sample is initially fractionated in one dimension by isoelectric focusing as described in Figure 4.11. The isoelectric focusing gel is then attached to an SDS-polyacrylamide gel, and electrophoresis is (more...)

Proteins isolated from cells under different physiological conditions can be subjected to two-dimensional electrophoresis, followed by an examination of the intensity of the signals. In this way, particular proteins can be seen to increase or decrease in concentration in response to the physiological state. How can we tell what protein is being regulated? A former drawback to the power of the two-dimensional gel is that, although many proteins are displayed, they are not identified. It is now possible to identify proteins by coupling two-dimensional gel electrophoresis with mass spectrometric techniques. We will consider these techniques when we examine how the mass of a protein is determined (Section 4.1.7).

4.1.5. A Protein Purification Scheme Can Be Quantitatively Evaluated

To determine the success of a protein purification scheme, we monitor the procedure at each step by determining specific activity and by performing an SDS-PAGE analysis. Consider the results for the purification of a fictitious protein, summarized in Table 4.1 and Figure 4.13. At each step, the following parameters are measured:

Table 4.1. Quantification of a purification protocol for a fictitious protein.

Table 4.1

Quantification of a purification protocol for a fictitious protein.

Figure 4.13. Electrophoretic Analysis of a Protein Purification.

Figure 4.13

Electrophoretic Analysis of a Protein Purification. The purification scheme in Table 4.1 was analyzed by SDS-PAGE. Each lane contained 50 μg of sample. The effectiveness of the purification can be seen as the band for the protein of interest becomes (more...)

  • Total protein. The quantity of protein present in a fraction is obtained by determining the protein concentration of a part of each fraction and multiplying by the fraction's total volume.
  • Total activity. The enzyme activity for the fraction is obtained by measuring the enzyme activity in the volume of fraction used in the assay and multiplying by the fraction's total volume.
  • Specific activity. This parameter is obtained by dividing total activity by total protein.
  • Yield. This parameter is a measure of the activity retained after each purification step as a percentage of the activity in the crude extract. The amount of activity in the initial extract is taken to be 100%.
  • Purification level. This parameter is a measure of the increase in purity and is obtained by dividing the specific activity, calculated after each purification step, by the specific activity of the initial extract.

As we see in Table 4.1, the first purification step, salt fractionation, leads to an increase in purity of only 3-fold, but we recover nearly all the target protein in the original extract, given that the yield is 92%. After dialysis to lower the high concentration of salt remaining from the salt fractionation, the fraction is passed through an ion-exchange column. The purification now increases to 9-fold compared with the original extract, whereas the yield falls to 77%. Molecular exclusion chromatography brings the level of purification to 100-fold, but the yield is now at 50%. The final step is affinity chromatography with the use of a ligand specific for the target enzyme. This step, the most powerful of these purification procedures, results in a purification level of 3000-fold, while lowering the yield to 35%. The SDS-PAGE in Figure 4.13 shows that, if we load a constant amount of protein onto each lane after each step, the number of bands decreases in proportion to the level of purification, and the amount of protein of interest increases as a proportion of the total protein present.

A good purification scheme takes into account both purification levels and yield. A high degree of purification and a poor yield leave little protein with which to experiment. A high yield with low purification leaves many contaminants (proteins other than the one of interest) in the fraction and complicates the interpretation of experiments.

4.1.6. Ultracentrifugation Is Valuable for Separating Biomolecules and Determining Their Masses

We have already seen that centrifugation is a powerful and generally applicable method for separating a crude mixture of cell components, but it is also useful for separating and analyzing biomolecules themselves. With this technique, we can determine such parameters as mass and density, learn something about the shape of a molecule, and investigate the interactions between molecules. To deduce these properties from the centrifugation data, we need a mathematical description of how a particle behaves in a centrifugal force.

A particle will move through a liquid medium when subjected to a centrifugal force. A convenient means of quantifying the rate of movement is to calculate the sedimentation coefficient, s, of a particle by using the following equation:

Image ch4e3.jpg
where m is the mass of the particle, ν― is the partial specific volume (the reciprocal of the particle density), ρ is the density of the medium and f is the frictional coefficient (a measure of the shape of the particle). The (1 - ρ) term is the buoyant force exerted by liquid medium.

Sedimentation coefficients are usually expressed in Svedberg units (S), equal to 10-13 s. The smaller the S value, the slower a molecule moves in a centrifugal field. The S values for a number of biomolecules and cellular components are listed in Table 4.2 and Figure 4.14.

Table 4.2. S values and molecular weights of sample proteins.

Table 4.2

S values and molecular weights of sample proteins.

Figure 4.14. Density and Sedimentation Coefficients of Cellular Components.

Figure 4.14

Density and Sedimentation Coefficients of Cellular Components. [After L. J. Kleinsmith and V. M. Kish, Principles of Cell and Molecular Biology, 2d ed. (Harper Collins, 1995), p. 138.]

Several important conclusions can be drawn from the preceding equation:


The sedimentation velocity of a particle depends in part on its mass. A more massive particle sediments more rapidly than does a less massive particle of the same shape and density.


Shape, too, influences the sedimentation velocity because it affects the viscous drag. The frictional coefficient f of a compact particle is smaller than that of an extended particle of the same mass. Hence, elongated particles sediment more slowly than do spherical ones of the same mass.


A dense particle moves more rapidly than does a less dense one because the opposing buoyant force (1 - ρ) is smaller for the denser particle.


The sedimentation velocity also depends on the density of the solution. (ρ). Particles sink when ρ < 1, float when ρ > 1, and do not move when ρ = 1.

A technique called zonal, band, or most commonly gradient centrifugation can be used to separate proteins with different sedimentation coefficients. The first step is to form a density gradient in a centrifuge tube. Differing proportions of a low-density solution (such as 5% sucrose) and a high-density solution (such as 20% sucrose) are mixed to create a linear gradient of sucrose concentration ranging from 20% at the bottom of the tube to 5% at the top (Figure 4.15). The role of the gradient is to prevent connective flow. A small volume of a solution containing the mixture of proteins to be separated is placed on top of the density gradient. When the rotor is spun, proteins move through the gradient and separate according to their sedimentation coefficients. The time and speed of the centrifugation is determined empirically. The separated bands, or zones, of protein can be harvested by making a hole in the bottom of the tube and collecting drops. The drops can be measured for protein content and catalytic activity or another functional property. This sedimentation-velocity technique readily separates proteins differing in sedimentation coefficient by a factor of two or more.

Figure 4.15. Zonal Centrifugation.

Figure 4.15

Zonal Centrifugation. The steps are as follows: (A) form a density gradient, (B) layer the sample on top of the gradient, (C) place the tube in a swinging-bucket rotor and centrifuge it, and (D) collect the samples. [After D. Freifelder, Physical Biochemistry, (more...)

The mass of a protein can be directly determined by sedimentation equilibrium, in which a sample is centrifuged at relatively low speed so that sedimentation is counterbalanced by diffusion. The sedimentation-equilibrium technique for determining mass is very accurate and can be applied under nondenaturing conditions in which the native quaternary structure of multimeric proteins is preserved. In contrast, SDS-polyacrylamide gel electrophoresis (Section 4.1.4) provides an estimate of the mass of dissociated polypeptide chains under denaturing conditions. Note that, if we know the mass of the dissociated components of a multimeric protein as determined by SDS-polyacrylamide analysis and the mass of the intact multimeric protein as determined by sedimentation equilibrium analysis, we can determine how many copies of each polypeptide chain is present in the multimeric protein.

4.1.7. The Mass of a Protein Can Be Precisely Determined by Mass Spectrometry

Mass spectrometry has been an established analytical technique in organic chemistry for many years. Until recently, however, the very low volatility of proteins made mass spectrometry useless for the investigation of these molecules. This difficulty has been circumvented by the introduction of techniques for effectively dispersing proteins and other macromolecules into the gas phase. These methods are called matrix-assisted laser desorption-ionization (MALDI) and electrospray spectrometry. We will focus on MALDI spectrometry. In this technique, protein ions are generated and then accelerated through an electrical field (Figure 4.16). They travel through the flight tube, with the smallest traveling fastest and arriving at the detector first. Thus, the time of flight (TOF) in the electrical field is a measure of the mass (or, more precisely, the mass/charge ratio). Tiny amounts of biomolecules, as small as a few picomoles (pmol) to femtomoles (fmol), can be analyzed in this manner. A MALDI-TOF mass spectrum for a mixture of the proteins insulin and β-lactoglobulin is shown in Figure 4.17. The masses determined by MALDI-TOF are 5733.9 and 18,364, respectively, compared with calculated values of 5733.5 and 18,388. MALDI-TOF is indeed an accurate means of determining protein mass.

Figure 4.16. MALDI-TOF Mass Spectrometry.

Figure 4.16

MALDI-TOF Mass Spectrometry. (1) The protein sample, embedded in an appropriate matrix, is ionized by the application of a laser beam. (2) An electrical field accelerates the ions formed through the flight tube toward the detector. (3) The lightest ions (more...)

Figure 4.17. MALDI-TOF Mass Spectrum of Insulin and β -lactoglobulin.

Figure 4.17

MALDI-TOF Mass Spectrum of Insulin and β -lactoglobulin. A mixture of 5 pmol each of insulin (I) and β-lactoglobulin (L) was ionized by MALDI, which produces predominately singly charged molecular ions from peptides and proteins (I + H (more...)

Mass spectrometry has permitted the development of peptide mass fingerprinting. This technique for identifying peptides has greatly enhanced the utility of two-dimensional gels. Two-dimensional electrophoresis is performed as described in Section 4.1.4. The sample of interest is extracted and cleaved specifically by chemical or enzymatic means. The masses of the protein fragments are then determined with the use of mass spectrometry. Finally, the peptide masses, or fingerprint, are matched against the fingerprint found in databases of proteins that have been “electronically cleaved” by a computer simulating the same fragmentation technique used for the experimental sample. This technique has provided some outstanding results. For example, of 150 yeast proteins analyzed with the use of two-dimensional gels, peptide mass fingerprinting unambiguously identified 80%. Mass spectrometry has provided name tags for many of the proteins in twodimensional gels.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
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