SDS-PAGE is a variant of polyacrylamide gel electrophoresis, an analytical method in biochemistry for the separation of charged molecules in mixtures by their molecular masses in an electric field. It uses sodium dodecyl sulfate molecules to help isolate protein molecules. SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli, used as a method to separate proteins with molecular masses between 5 and 250 KDa; the publication describing it is the most cited paper by a single author, the second most cited overall. SDS-PAGE is an electrophoresis method; the medium is a polyacrylamide-based discontinuous gel. In addition, SDS is used. About 1.4 grams of SDS bind to a gram of protein, corresponding to one SDS molecule per two amino acids. SDS acts as a surfactant, covering the proteins' intrinsic charge and conferring them similar charge-to-mass ratios; the intrinsic charges of the proteins are negligible in comparison to the SDS loading, the positive charges are greatly reduced in the basic pH range of a separating gel.
Upon application of a constant electric field, the protein migrate towards the anode, each with a different speed, depending on its mass. This simple procedure allows precise protein separation by mass. SDS tends to form spherical micelles in aqueous solutions above a certain concentration called the critical micellar concentration. Above the critical micellar concentration of 7 to 10 millimolar in solutions, the SDS occurs as single molecules and as micelles, below the CMC SDS occurs only as monomers in aqueous solutions. At the critical micellar concentration, a micelle consists of about 62 SDS molecules. However, only SDS monomers bind to proteins via hydrophobic interactions, whereas the SDS micelles are anionic on the outside and do not adsorb any protein. SDS is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of protein structure. In SDS concentrations above 0.1 millimolar, the unfolding of proteins begins, above 1 mM, most proteins are denatured. Due to the strong denaturing effect of SDS and the subsequent dissociation of protein complexes, quaternary structures can not be determined with SDS.
Exceptions are e.g. proteins that were stabilised by covalent cross-linking and the SDS-resistant protein complexes, which are stable in the presence of SDS. To denature the SDS-resistant complexes a high activation energy is required, achieved by heating. SDS resistance is based on a metastability of the protein fold. Although the native folded, SDS-resistant protein does not have sufficient stability in the presence of SDS, the chemical equilibrium of denaturation at room temperature occurs slowly. Stable protein complexes are characterised not only by SDS resistance but by stability against proteases and an increased biological half-life. Alternatively, polyacrylamide gel electrophoresis can be performed with the cationic surfactants CTAB in a CTAB-PAGE, or 16-BAC in a BAC-PAGE; the SDS-PAGE method is composed of gel preparation, sample preparation, protein staining or western blotting and analysis of the generated banding pattern. When using different buffers in the gel, the gels are made up to one day prior to electrophoresis, so that the diffusion does not lead to a mixing of the buffers.
The gel is produced by radical polymerisation in a mold consisting of two sealed glass plates with spacers between the glass plates. In a typical mini-gel setting, the spacers have a thickness of 0.75 mm or 1.5 mm, which determines the loading capacity of the gel. For pouring the gel solution, the plates are clamped in a stand which temporarily seals the otherwise open underside of the glass plates with the two spacers. For the gel solution, acrylamide is mixed as gel-former, methylenebisacrylamide as a cross-linker, stacking or separating gel buffer, water and SDS. By adding the catalyst TEMED and the radical initiator ammonium persulfate the polymerisation is started; the solution is poured between the glass plates without creating bubbles. Depending on the amount of catalyst and radical starter and depending on the temperature, the polymerisation lasts between a quarter of an hour and several hours; the lower gel is poured first and covered with a few drops of a water-soluble alcohol, which eliminates bubbles from the meniscus and protects the gel solution of the radical scavenger oxygen.
After the polymerisation of the separating gel, the alcohol is discarded and the residual alcohol is removed with filter paper. After addition of APS and TEMED to the stacking gel solution, it is poured on top of the solid separation gel. Afterwards, a suitable sample comb is inserted between the glass plates without creating bubbles; the sample comb is pulled out after polymerisation, leaving pockets for the sample application. For use of proteins for protein sequencing, the gels are prepared the day before electrophoresis to reduce reactions of unpolymerised acrylamide with cysteines in proteins. By using a gradient mixer, gradient gels with a gradient of acrylamide can be cast, which have a larger separation range of the molecular masses. Commercial gel systems use the buffer substance Bis-tris methane with a pH value between 6.4 and 7.2 both in the stacking gel and in the separating gel. These gels
Mass spectrometric immunoassay
Mass spectrometric immunoassay is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry; this method allows for "top down" and "bottom up" analysis. This This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay; this assay is capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer. There are various other immunoassy techniques that have been used such as radioimmunoassay and enzyme immunoassay; these techniques are sensitive however, there are many limitations to these methods.
For example, quantification for ELISA and EIA require several hours because the binding has to reach equilibrium. RIA's disadvantage is that you need radioactive particles which are universally known to be carcinogens; the creation of MSIA fulfilled the need to determine the presence of one or more antigens in a specimen as well as the quantification of those said species. This assay was patented in 2006 by Peter Williams and Jennifer Reeve Krone; the idea first came about with the development of ELISA and RIA. An earlier patent method suggested tagging antigens or antibodies with stable isotopes or long-lived radioactive elements, but limitations to both methods called for a better detection methods of proteins. The invention combines antigen-antibody binding with a mass spectrometer which aids in identifying qualitatively and quantifying analytes respectively. An early MSIA experiment was done on a venom laced human blood sample for the Antigen myotoxin; the experiment was successful in that the mass spectrum resulting from the analysis showed a distinct response for myotoxin at the molecular weight corresponding to 4,822 Da.
The m/z ratio at 5,242 Da is the molecular weight of the modified variant H-myotoxina, used as an internal reference species. The figure of the mass spectrum is shown below. An illustration of the MSIA procedure is depicted in the figure to the right. Analytes in a biological liquid sample are collected from solution by using a MSIA tip that contains a dervatized affinity frit. Biological samples contain various proteins that span a wide dynamic range so purification is needed to minimize the complex matrix and maximize mass spectrometry sensitivity; the MSIA tip serves as a place to purify these samples by immobilizing the analyte with high selectivity and specificity. Analytes are bound to the frit based on their affinities and all other nonspecific molecules are rinsed away; the specific targets are eluted on to a mass spectrometer target plate with a MALDI matrix. However, proteins may be digested prior to ms analysis. A MALDI-TOF-MS follows and targeted analytes are detected based on their m/z values.
This method is qualitative, but the addition of mass shifted variants of the analyte for use as an internal standard makes this method useful for quantitative analysis. Pipetor tips, which have been termed MSIA tips or affinity pipette tips play a key role in the process of detecting analytes within biological samples. MSIA tips contain porous solid support which has derivatized antigens or antibodies covalently attached. Different analytes have different affinity for the tips so it is necessary to derivatize MSIA tips based on the analyte of interest; the main use of these tips are to flow samples through and the analytes affinity for the bound antigen/antibody allows for the capture of analyte. Non bound compounds are rinsed out of the MSIA tips; the process can be simplified into 6 simple steps which Thermo termed the "work flow". Gather Sample Load Affinity Ligand Purify Target Analyte Elute Target Analyte Pre-MS Sampling Process MS AnalysisMany "work flows" are commercially available for purchase.
MSIA is a method that can be used as an assay for a variety of different molecules such as proteins, drugs and various pathogens found in biological fluids. MSIA has been applied to clinical samples and have been proven to be a unique assay for clinically relevant proteins. Assaying toxins and other pathogens are important to the environment as well as the human body. MSIA can be used for a range of environmental applications. An important application of mass spectrometric immunoassy is that it can be used as a rapid and accurate screening of apolipoproteins and mutations of them. Apolipoproteins represent a groups of proteins with many functions such as transport and clearance as well as enzyme activation. Recent studies have claimed that mutations in apopliproteins result in, or assist in the progression of various associated diseases including amyloidosis, amyloid cardiomyopathy, Alzheimer's disease, hypertriglyceridemic, lowered cholesterol and atherosclerosis to name a few. Nelson and colleagues did a study using MSIA to isolate apolipoproteins species.
There are many benefits to using a mass spectrometric immunoassay. Most the assay is fast and the data are reproducible, automated, they are sensitive and allows for absolute quantification. Analytes can be detected to low detection limits (as low
The ultracentrifuge is a centrifuge optimized for spinning a rotor at high speeds, capable of generating acceleration as high as 1 000 000 g. There are two kinds of the preparative and the analytical ultracentrifuge. Both classes of instruments find important uses in molecular biology and polymer science. In 1924 Theodor Svedberg built a centrifuge capable of generating 7,000 g, called it the ultracentrifuge, to juxtapose it with the Ultramicroscope, developed previously. In 1925-1926 Svedberg constructed a new ultracentrifuge that permitted fields up to 100,000 g. Modern ultracentrifuges are classified as allowing greater than 100,000 g. Svedberg won the Nobel Prize in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge; the vacuum ultracentrifuge was invented by Edward Greydon Pickels in the Physics Department at the University of Virginia. It was his contribution of the vacuum which allowed a reduction in friction generated at high speeds. Vacuum systems enabled the maintenance of constant temperature across the sample, eliminating convection currents that interfered with the interpretation of sedimentation results.
In 1946, Pickels cofounded Spinco to market analytical and preparative ultracentrifuges based on his design. Pickels considered his design to be too complicated for commercial use and developed a more operated, “foolproof” version, but with the enhanced design, sales of analytical centrifuges remained low, Spinco went bankrupt. The company survived by concentrating on sales of preparative ultracentrifuge models, which were becoming popular as workhorses in biomedical laboratories. In 1949, Spinco introduced the Model L, the first preparative ultracentrifuge to reach a maximum speed of 40,000 rpm. In 1954, Beckman Instruments, now Beckman Coulter, purchased the company, forming the basis of its Spinco centrifuge division. In an analytical ultracentrifuge, a sample being spun can be monitored in real time through an optical detection system, using ultraviolet light absorption and/or interference optical refractive index sensitive system; this allows the operator to observe the evolution of the sample concentration versus the axis of rotation profile as a result of the applied centrifugal field.
With modern instrumentation, these observations are electronically digitized and stored for further mathematical analysis. Two kinds of experiments are performed on these instruments: sedimentation velocity experiments and sedimentation equilibrium experiments. Sedimentation velocity experiments aim to interpret the entire time-course of sedimentation, report on the shape and molar mass of the dissolved macromolecules, as well as their size-distribution; the size resolution of this method scales with the square of the particle radii, by adjusting the rotor speed of the experiment size-ranges from 100 Da to 10 GDa can be covered. Sedimentation velocity experiments can be used to study reversible chemical equilibria between macromolecular species, by either monitoring the number and molar mass of macromolecular complexes, by gaining information about the complex composition from multi-signal analysis exploiting differences in each components spectroscopic signal, or by following the composition dependence of the sedimentation rates of the macromolecular system, as described in Gilbert-Jenkins theory.
Sedimentation equilibrium experiments are concerned only with the final steady-state of the experiment, where sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a time-independent concentration profile. Sedimentation equilibrium distributions in the centrifugal field are characterized by Boltzmann distributions; this experiment is insensitive to the shape of the macromolecule, directly reports on the molar mass of the macromolecules and, for chemically reacting mixtures, on chemical equilibrium constants. The kinds of information that can be obtained from an analytical ultracentrifuge include the gross shape of macromolecules, the conformational changes in macromolecules, size distributions of macromolecular samples. For macromolecules, such as proteins, that exist in chemical equilibrium with different non-covalent complexes, the number and subunit stoichiometry of the complexes and equilibrium constant constants can be studied. Analytical ultracentrifugation has seen a rise in use because of increased ease of analysis with modern computers and the development of software, including a National Institutes of Health supported software package, SedFit.
Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great range of experiments. Most rotors are designed to hold tubes that contain the samples. Swinging bucket rotors allow the tubes to hang on hinges so the tubes reorient to the horizontal as the rotor accelerates. Fixed angle rotors are made of a single block of material and hold the tubes in cavities bored at a predetermined angle. Zonal rotors are designed to contain a large volume of sample in a single central cavity rather than in tubes; some zonal rotors are capable of dynamic loading and unloading of samples while the rotor is spinning at high speed. Preparative rotors are used in biology for pelleting of fine particulate fractions, such as cellular organelles and viruses, they can be used for gradient separations, in which the tubes are filled from top to bottom with an increasing concentration of a dense substance in solution. Sucrose gradients are used for separation of cellular organelles. Gradients of caesium salts are used for separation of nucleic acids.
After the samp
Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. Their functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane, controlling the flow of ions across secretory and epithelial cells, regulating cell volume. Ion channels are present in the membranes of all excitable cells. Ion channels are one of the two classes of ionophoric proteins, the other being ion transporters; the study of ion channels involves biophysics, electrophysiology, pharmacology, while using techniques including voltage clamp, patch clamp, immunohistochemistry, X-ray crystallography, RT-PCR. Their classification as molecules is referred to as channelomics. There are two distinctive features of ion channels that differentiate them from other types of ion transporter proteins: The rate of ion transport through the channel is high. Ions pass through channels down their electrochemical gradient, a function of ion concentration and membrane potential, "downhill", without the input of metabolic energy.
Ion channels are located within the membrane of all excitable cells, of many intracellular organelles. They are described as narrow, water-filled tunnels that allow only ions of a certain size and/or charge to pass through; this characteristic is called selective permeability. The archetypal channel pore is just one or two atoms wide at its narrowest point and is selective for specific species of ion, such as sodium or potassium. However, some channels may be permeable to the passage of more than one type of ion sharing a common charge: positive or negative. Ions move through the segments of the channel pore in single file nearly as as the ions move through free solution. In many ion channels, passage through the pore is governed by a "gate", which may be opened or closed in response to chemical or electrical signals, temperature, or mechanical force. Ion channels are integral membrane proteins formed as assemblies of several individual proteins; such "multi-subunit" assemblies involve a circular arrangement of identical or homologous proteins packed around a water-filled pore through the plane of the membrane or lipid bilayer.
For most voltage-gated ion channels, the pore-forming subunit are called the α subunit, while the auxiliary subunits are denoted β, γ, so on. Because channels underlie the nerve impulse and because "transmitter-activated" channels mediate conduction across the synapses, channels are prominent components of the nervous system. Indeed, numerous toxins that organisms have evolved for shutting down the nervous systems of predators and prey work by modulating ion channel conductance and/or kinetics. In addition, ion channels are key components in a wide variety of biological processes that involve rapid changes in cells, such as cardiac and smooth muscle contraction, epithelial transport of nutrients and ions, T-cell activation and pancreatic beta-cell insulin release. In the search for new drugs, ion channels are a frequent target. There are over 300 types of ion channels just in the cells of the inner ear. Ion channels may be classified by the nature of their gating, the species of ions passing through those gates, the number of gates and localization of proteins.
Further heterogeneity of ion channels arises when channels with different constitutive subunits give rise to a specific kind of current. Absence or mutation of one or more of the contributing types of channel subunits can result in loss of function and underlie neurologic diseases. Ion channels may be classified by i.e. what opens and closes the channels. For example, voltage-gated ion channels open or close depending on the voltage gradient across the plasma membrane, while ligand-gated ion channels open or close depending on binding of ligands to the channel. Voltage-gated ion channels close in response to membrane potential. Voltage-gated sodium channels: This family contains at least 9 members and is responsible for action potential creation and propagation; the pore-forming α subunits are large and consist of four homologous repeat domains each comprising six transmembrane segments for a total of 24 transmembrane segments. The members of this family coassemble with auxiliary β subunits, each spanning the membrane once.
Both α and β subunits are extensively glycosylated. Voltage-gated calcium channels: This family contains 10 members, though these members are known to coassemble with α2δ, β, γ subunits; these channels play an important role in both linking muscle excitation with contraction as well as neuronal excitation with transmitter release. The α subunits have an overall structural resemblance to those of the sodium channels and are large. Cation channels of sperm: This small family of channels referred to as Catsper channels, is related to the two-pore channels and distantly related to TRP channels. Voltage-gated potassium channels: This family contains 40 members, which are further divided into 12 subfamilies; these channels are known for their role in repolarizing the cell membrane following action potentials. The α subunits have six transmembrane segments, homologous to a single domain of the sodium channels. Correspondingly, they assemble; some transient receptor potential channels: This group of channels referred to as
Nuclear magnetic resonance spectroscopy of proteins
Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, nucleic acids, their complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, by Ad Bax, Marius Clore, Angela Gronenborn at the NIH, among others. Structure determination by NMR spectroscopy consists of several phases, each using a separate set of specialized techniques; the sample is prepared, measurements are made, interpretive approaches are applied, a structure is calculated and validated. NMR involves the quantum mechanical properties of the central core of the atom; these properties depend on the local molecular environment, their measurement provides a map of how the atoms are linked chemically, how close they are in space, how they move with respect to each other. These properties are fundamentally the same as those used in the more familiar magnetic resonance imaging, but the molecular applications use a somewhat different approach, appropriate to the change of scale from millimeters to nanometers, a factor of a million.
This change of scale requires much higher sensitivity of detection and stability for long term measurement. In contrast to MRI, structural biology studies do not directly generate an image, but rely on complex computer calculations to generate three-dimensional molecular models. Most samples are examined in a solution in water, but methods are being developed to work with solid samples. Data collection relies on placing the sample inside a powerful magnet, sending radio frequency signals through the sample, measuring the absorption of those signals. Depending on the environment of atoms within the protein, the nuclei of individual atoms will absorb different frequencies of radio signals. Furthermore, the absorption signals of different nuclei may be perturbed by adjacent nuclei; this information can be used to determine the distance between nuclei. These distances in turn can be used to determine the overall structure of the protein. A typical study might involve how two proteins interact with each other with a view to developing small molecules that can be used to probe the normal biology of the interaction or to provide possible leads for pharmaceutical use.
The interacting pair of proteins may have been identified by studies of human genetics, indicating the interaction can be disrupted by unfavorable mutations, or they may play a key role in the normal biology of a "model" organism like the fruit fly, the worm C. elegans, or mice. To prepare a sample, methods of molecular biology are used to make quantities by bacterial fermentation; this permits changing the isotopic composition of the molecule, desirable because the isotopes behave differently and provide methods for identifying overlapping NMR signals. Protein nuclear magnetic resonance is performed on aqueous samples of purified protein; the sample consists of between 300 and 600 microlitres with a protein concentration in the range 0.1 – 3 millimolar. The source of the protein can be either natural or produced in a production system using recombinant DNA techniques through genetic engineering. Recombinantly expressed proteins are easier to produce in sufficient quantity, this method makes isotopic labeling possible.
The purified protein is dissolved in a buffer solution and adjusted to the desired solvent conditions. The NMR sample is prepared in a thin-walled glass tube. Protein NMR utilizes multidimensional nuclear magnetic resonance experiments to obtain information about the protein. Ideally, each distinct nucleus in the molecule experiences a distinct electronic environment and thus has a distinct chemical shift by which it can be recognized. However, in large molecules such as proteins the number of resonances can be several thousand and a one-dimensional spectrum has incidental overlaps. Therefore, multidimensional experiments that correlate the frequencies of distinct nuclei are performed; the additional dimensions decrease the chance of overlap and have a larger information content, since they correlate signals from nuclei within a specific part of the molecule. Magnetization is transferred into the sample using pulses of electromagnetic energy and between nuclei using delays. Pulse sequences allow the experimenter to investigate and select specific types of connections between nuclei.
The array of nuclear magnetic resonance experiments used on proteins fall in two main categories — one where magnetization is transferred through the chemical bonds, one where the transfer is through space, irrespective of the bonding structure. The first category is used to assign the different chemical shifts to a specific nucleus, the second is used to generate the distance restraints used in the structure calculation, in the assignment with unlabelled protein. Depending on the concentration of the sample, on the magnetic field of the spectrometer, on the type of experiment, a single multidimensional nuclear magnetic resonance experiment on a protein sample may take hours or several days to obtain suitable signal-to-noise ratio through signal averaging, to allow for sufficient evolution of magnetization transfer through the various dimensions of the experiment. Other things being equal, higher-dimensional experiments will take longer than lower-dimensional experiments; the first experiment to be measured with an isot
Enzymes are macromolecular biological catalysts. Enzymes accelerate chemical reactions; the molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into different molecules known as products. All metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps; the study of enzymes is called enzymology and a new field of pseudoenzyme analysis has grown up, recognising that during evolution, some enzymes have lost the ability to carry out biological catalysis, reflected in their amino acid sequences and unusual'pseudocatalytic' properties. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are proteins; the latter are called ribozymes. Enzymes' specificity comes from their unique three-dimensional structures. Like all catalysts, enzymes increase the reaction rate by lowering its activation energy; some enzymes can make their conversion of substrate to product occur many millions of times faster.
An extreme example is orotidine 5'-phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, activators are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly outside its optimal temperature and pH, many enzymes are denatured when exposed to excessive heat, losing their structure and catalytic properties; some enzymes are used commercially, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.
By the late 17th and early 18th centuries, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by which these occurred had not been identified. French chemist Anselme Payen was the first to discover an enzyme, diastase, in 1833. A few decades when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that this fermentation was caused by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms, he wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."In 1877, German physiologist Wilhelm Kühne first used the term enzyme, which comes from Greek ἔνζυμον, "leavened" or "in yeast", to describe this process. The word enzyme was used to refer to nonliving substances such as pepsin, the word ferment was used to refer to chemical activity produced by living organisms.
Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of experiments at the University of Berlin, he found that sugar was fermented by yeast extracts when there were no living yeast cells in the mixture, he named the enzyme that brought about the fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in Chemistry for "his discovery of cell-free fermentation". Following Buchner's example, enzymes are named according to the reaction they carry out: the suffix -ase is combined with the name of the substrate or to the type of reaction; the biochemical identity of enzymes was still unknown in the early 1900s. Many scientists observed that enzymatic activity was associated with proteins, but others argued that proteins were carriers for the true enzymes and that proteins per se were incapable of catalysis. In 1926, James B. Sumner crystallized it; the conclusion that pure proteins can be enzymes was definitively demonstrated by John Howard Northrop and Wendell Meredith Stanley, who worked on the digestive enzymes pepsin and chymotrypsin.
These three scientists were awarded the 1946 Nobel Prize in Chemistry. The discovery that enzymes could be crystallized allowed their structures to be solved by x-ray crystallography; this was first done for lysozyme, an enzyme found in tears and egg whites that digests the coating of some bacteria. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail. An enzyme's name is derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are alcohol dehydrogenase and DNA polymerase. Different enzymes that catalyze the same chemical reaction are called isozymes; the International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers. The first number broadly classifies the enzyme based on its mechanism; the top-level classification is: EC 1, Oxidoreductases: catalyze oxidation/reducti
Electrospray ionization is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. ESI is different from other ionization processes since it may produce multiple-charged ions extending the mass range of the analyser to accommodate the kDa-MDa orders of magnitude observed in proteins and their associated polypeptide fragments. Mass spectrometry using ESI is called electrospray ionization mass spectrometry or, less electrospray mass spectrometry. ESI is a so-called'soft ionization' technique, since there is little fragmentation; this can be advantageous in the sense that the molecular ion is always observed, however little structural information can be gained from the simple mass spectrum obtained. This disadvantage can be overcome by coupling ESI with tandem mass spectrometry.
Another important advantage of ESI is that solution-phase information can be retained into the gas-phase. The electrospray ionization technique was first reported by Masamichi Yamashita and John Fenn in 1984; the development of electrospray ionization for the analysis of biological macromolecules was rewarded with the attribution of the Nobel Prize in Chemistry to John Bennett Fenn in 2002. One of the original instruments used by Dr. Fenn is on display at the Science History Institute in Philadelphia, Pennsylvania. In 1882, Lord Rayleigh theoretically estimated the maximum amount of charge a liquid droplet could carry before throwing out fine jets of liquid; this is now known as the Rayleigh limit. In 1914, John Zeleny published work on the behaviour of fluid droplets at the end of glass capillaries and presented evidence for different electrospray modes. Wilson and Taylor and Nolan investigated electrospray in the 1920s and Macky in 1931; the electrospray cone was described by Sir Geoffrey Ingram Taylor.
The first use of electrospray ionization with mass spectrometry was reported by Malcolm Dole in 1968. John Bennett Fenn was awarded the 2002 Nobel Prize in Chemistry for the development of electrospray ionization mass spectrometry in the late 1980s; the liquid containing the analyte of interest is dispersed into a fine aerosol. Because the ion formation involves extensive solvent evaporation, the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds. To decrease the initial droplet size, compounds that increase the conductivity are customarily added to the solution; these species act to provide a source of protons to facilitate the ionization process. Large-flow electrosprays can benefit from nebulization of a heated inert gas such as nitrogen or carbon dioxide in addition to the high temperature of the ESI source; the aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary carrying a potential difference of 3000V, which can be heated to aid further solvent evaporation from the charged droplets.
The solvent evaporates from a charged droplet until it becomes unstable upon reaching its Rayleigh limit. At this point, the droplet deforms as the electrostatic repulsion of like charges, in an ever-decreasing droplet size, becomes more powerful than the surface tension holding the droplet together. At this point the droplet undergoes Coulomb fission, whereby the original droplet'explodes' creating many smaller, more stable droplets; the new droplets undergo desolvation and subsequently further Coulomb fissions. During the fission, the droplet loses a small percentage of its mass along with a large percentage of its charge. There are two major theories that explain the final production of gas-phase ions: the ion evaporation model and the charge residue model; the IEM suggests that as the droplet reaches a certain radius the field strength at the surface of the droplet becomes large enough to assist the field desorption of solvated ions. The CRM suggests that electrospray droplets undergo evaporation and fission cycles leading progeny droplets that contain on average one analyte ion or less.
The gas-phase ions form after the remaining solvent molecules evaporate, leaving the analyte with the charges that the droplet carried. A large body of evidence shows either directly or indirectly that small ions are liberated into the gas phase through the ion evaporation mechanism, while larger ions form by charged residue mechanism A third model invoking combined charged residue-field emission has been proposed. Another model called; the ions observed by mass spectrometry may be quasimolecular ions created by the addition of a hydrogen cation and denoted +, or of another cation such as sodium ion, +, or the removal of a hydrogen nucleus, −. Multiply charged ions such as n+ are observed. For large macromolecules, there can be many charge states, resulting in a characteristic charge state envelope. All these are even-electron ion species: electrons are not added or removed, unlike in some other ionization sources; the analytes are sometimes involved in electrochemical processes, leading to shifts of the corresponding peaks in the mass spectrum.
This effect is demonstrated in the direct ionization of noble metals such as copper and gold using electrospray. The electro