This is the first of four special supplements this year on Advances in Proteomics. The next will appear in the 29 April issue of Science.

Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

The emergence of genomics has opened the way to the next stage of research on the nature of life: proteomics. But for scientists trained to solve genomic problems, proteins mean trouble. Not only do they exist in huge numbers. Individually, proteins are more sophisticated than other biomolecules. Unlike DNA, which exists as linear strands, proteins take on three-dimensional shapes. An individual protein starts out as a peptide chain made up of 20 or so amino acids, and is then folded into a complex and fairly fragile three-dimensional structure. That physical structure ultimately determines any protein’s activity.

Those complex and changeable structures make it difficult for researchers to track down proteins’ activities. The difficulty applies particularly to low abundance proteins, which often get lost in clusters of more abundant proteins. “Low abundance proteins are often the most interesting, and they remain a problem,” says Darwin Asa, drug discovery development manager at ESA.

Multiple Technologies
Researchers who investigate all types of proteins must deal with another factor that differentiates proteomics from genomics. While structural genomics in the area of sequencing the human genome was generally approached with a single technology,” explains Achim Wehren, global marketing manager for biopharma at Tecan, “several technologies and methodologies are available for proteomics.”

Emerging tools and technologies have become key factors in facilitating several advances in proteomics. “One of the biggest achievements is the increased resolution of mass spectrometry,” says Donald Finley, product manager for recombinant protein expression at Sigma-Aldrich. “It enables us to see things we haven’t seen before.” Mark McDowall, strategic development manager for MS at Waters Corporation, extends that thought. “Prefractionation of proteins by multidimensional chromatography before mass spectrometry and multiplexing mass spectrometry analyses allow scientists to extract more information from a minimal amount of sample,” he says. Those approaches, adds Tom Wheat, principal scientist and manager in Waters’s life science application laboratory, “help to enrich the low abundance proteins so that we can use elegant software to extract information about these important proteins. There have been some very exciting things happening with the software for interpreting these analyses.”

Improved forms of liquid chromatography have also become significant tools in isolating and identifying proteins. “Methods such as multiplexed liquid chromatography and nano-high performance liquid chromatography coupled to electrospray or matrix-assisted laser desorption/ionization mass spectrometry [MALDI-MS] will definitely have an impact on proteomics research,” says Carsten Buhlmann, product manager for laboratory-on-a-chip assays at Agilent Technologies. Nanostream offers what it calls micro parallel liquid chromatography through its Veloce system, a microfluidic method that provides high throughput chromatographic separations.

Studies of proteomics don’t always demand new technology. “We often have to go back to the old methods, like 2-D gels and sometimes 1-D gel electrophoresis followed by mass spectrometry,” says Deb Chakravarti, director of proteomics and Beckman professor at the Keck Graduate Institute and editor-in-chief of Current Proteomics, published by Bentham Science Publishers. “We have to know the whole bag of tricks.” Wehren explains the outcome of such knowledge on proteomics. “Technology,” he says, “is driving the field.”

Unfamiliar Principles and Approaches
Emerging technologies often involve principles and approaches unfamiliar to scientists untrained in proteomics. “With the drive to come up with biological issues, people are less interested in studying the nuances of the chemistry,” explains George Lipscomb, product manager for protein expression and purification at Sigma-Aldrich. To ease those individuals into the protein world, suppliers increasingly emphasize user-friendliness. “The power of studying proteomics is bringing in a lot of people with diverse training and experience,” Finley says. “They need the absolute, simplest, most robust protocols available.” Why? “Nonproteomics people have to have an understanding of the results,” adds Chakravarti. “There’s a push for guidelines for experiments and interpretation of protein identification results.”

That puts the pressure on suppliers. “If your kit is not user-friendly,” says Shou Wong, manager of global R&D and business development at Merck KGaA which owns EMD Biosciences, “people won’t want to use it.” At the same time, vendors cannot afford to sell equipment that lacks appeal to proteomic specialists. “The trick here is to maximize ease of use but also to allow advanced users to have access to the advanced options,” Buhlmann explains.

Tecan sets out to create user friendliness by the breadth of its instruments’ appeal. “Once you have bought our liquid handling systems you usually have multiple applications,” Wehren says. “When your application repertoire changes, you can easily reconfigure your instrument.”

Two approaches help to familiarize newcomers to proteomics with the tools and technologies that facilitate research in the field. “Technical support is important,” Wong says. “Users can follow the instructions but still make simple mistakes; that’s where support comes in.” Wehren echoes that point. “We have been in the instrument market for 25 years,” he points out. “We have a very strong service organization.” In addition, Chakravarti says, “We’re finding more and more need for training workshops for proteomics. Many current curricula are predominantly oriented to recombinant DNA.”

One Key Factor
Newcomers to proteomics need to understand one key factor. “You can break proteomics into two types,” Wong explains. “You have the native proteins, from natural samples such as tissues, and recombinant proteins that scientists try to overexpress by biotechnological methods.”

To understand proteins’ makeup, scientists must first isolate and separate intact proteins from cells or tissue. The processes involve several steps, each of which can cause the degradation or loss of the proteins of interest if carried out incorrectly. Avoiding such problems is particularly critical in the case of low abundance proteins. So is choosing the right approach. “There are perhaps up to a dozen principles of protein separation available,” says Wheat of Waters.

As the first step in isolating a protein, scientists must disrupt the cells or tissue and extract the protein fraction of interest. Disruption generally requires a strong denaturing solution. Often based on detergents such as CHAPS, such solutions also include urea for rapid inactivation of any enzymes that might degrade the target proteins. Researchers often add protease inhibitor cocktails that prevent the proteins from degrading in crude cellular extracts that contain many active proteases. Several suppliers that specialize in protein chemistry, including BD Biosciences Clontech, EMD Biosciences, and Pierce Biotechnology, provide chemicals and reagents of this type.

Many of these companies have also developed kits that provide researchers with relatively simple tools for isolating proteins without developing their own protocols from scratch. “For recombinant proteins, EMD Biosciences offers everything for protein expression and purification,” Wong says. “EMD Biosciences has ligation independent cloning which allows you to clone genes in an orientation dependent fashion such that the insert can go into your vector in only one orientation. Since this process doesn’t use ligase, its efficiency is very high; the whole process takes less than five minutes at room temperature with an efficiency of 99 percent or better.” For native proteins from tissues and cell cultures, EMD Biosciences has developed efficient user-friendly extraction kits to purify nuclear proteins (the NucBuster Protein Extraction Kit), total proteins from bacteria, yeast and mammalian cells and tissues (ProteoExtract Complete Proteome Extraction Kits), and partial and subcellular proteins from mammalian cells (the ProteoExtract Partial Proteome Extraction and ProteoExtract Subcellular Proteome Extraction Kit).

Physical and Chemical Properties
Once they have isolated a mixture of proteins from a cell, researchers can separate the individual proteins according to physical properties such as their size, shape, and charge, or such chemical characteristics as hydrophobicity and affinity for other molecules. Affinity chromatography is a popular method of separating a specific protein or family of proteins. This method takes advantage of the fact that some proteins bind to specific substrates or to antibodies generated specifically for that purpose. When available, antibodies raised against a protein offer highly specific tools for separating or purifying one protein from a mixture for further analysis.

Antibodies generated to a specific protein can be used to “fish” for a unique protein in a cellular extract. Chromatography media packed in columns secure the antibodies to a solid support. This allows protein-containing samples, buffers, and other solutions to be run through a column, capturing the protein of interest, followed by elution of the protein-antibody complexes. This elution is accomplished by changing solvent conditions in the column, which diminishes the strength of the protein-ligand interaction.

Several companies, including IBA GmbH, EMD Biosciences, Promega, and Sigma-Aldrich, have developed novel protein purification systems based on the insertion of small peptide sequences into a specific protein that can enable isolation of the protein using a molecule with a strong affinity to the peptide sequence. Many researchers use a HIS type of tag for inexpensive purification of recombinant proteins. EMD Biosciences offers a line of His-tag affinity products from vectors that contain a wide range of tags to purification resins and kits. Sigma-Aldrich’s HIS-Select family of products purifies histidine-containing fusion proteins with high selectivity. The company also offers its FLAG system, designed for highly sensitive detection of recombinant protein. “It adds a small octopeptide tag to a protein,” Finley explains. “The tag tends to end up on the outside of a protein instead of being folded in. It’s one of our most popular products in the recombinant protein arena.”

Purification and detection tools for many other fusion proteins are also available. For example, Sigma-Aldrich offers agarose-based affinity resins for purification of c-Myc, HA, and Maltose Binding Protein fusion proteins.

Two Types of Gel
Scientists developed two-dimensional (2-D) gel electrophoresis specifically to separate mixtures of proteins. The systems consist of two types of gel electrophoresis; the first dimension is based on isoelectric focusing (IEF), while the second uses a denaturing polyacrylamide gel matrix.

The first gel (or dimension) traditionally incorporates ampholytes that form a pH gradient in the gel. This gradient helps to separate the proteins based on the isoelectric point of each protein. Having separated a mixture of proteins in this first dimension, the scientist places the separated proteins on a vertical gel electrophoresis unit. This second dimension is usually a denaturing sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) – a relatively routine tool found in most laboratories.

Companies such as Bio-Rad Laboratories, GE Healthcare, and Invitrogen offer complete systems with the units, power supplies, and accessories required to perform protein separations. These and other companies also produce precast polyacrylamide gels and/or IEF strips that provide highly consistent results and allow scientists to use the systems without having to master the casting of polyacrylamide gels, which can be technically difficult.

To identify the proteins separated by 2-D gel electrophoresis, scientists typically use mass spectrometry (MS). “We recently introduced the MALDI micro MX system to identify proteins excised from 2-D gels,” reports McDowall of Waters. “The novel thing is that it’s multiplexed. It performs MS-MS on all the peptide components at the same time in parallel. You “burn” less sample and you no longer need to serially select each peptide precursor ion for fragmentation, so that it’s very easy to use compared with traditional PSD.”

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Another separation approach, high performance liquid chromatography (HPLC), refers to the separation of molecules under high pressure in a stainless steel column filled with a solid matrix. Companies that specialize in HPLC include Agilent Technologies, Grace Vydac, Phenomenex, and Waters.

Attractive Forces
HPLC vendors use the attractive forces between molecules that carry oppositely charged groups in two different ways. Ion pairing primarily concerns rather small molecules, while ion exchange chromatography (IEC) can separate almost any type of charged molecule, from large proteins to small nucleotides and amino acids. In IEC, charged particles in the form of a solid matrix bind reversibly to proteins or other molecules. The bound molecules are then detached by increasing the salt concentration or by altering the pH of the mobile phase. Researchers frequently use ion exchangers that contain diethyl aminoethyl or carboxymethyl groups to study proteins and peptides under widely varying conditions.

Waters has taken the process a stage further with its nanoACQUITY Ultra Performance Liquid Chromatography System. “It’s a new workhorse for proteomics to prepare and introduce samples for mass spectrometry,” Wheat explains. “We’ve developed new packing materials based on much smaller particles that provide greater chromatographic resolution, peak capacity, MS sensitivity, and speed.”

Agilent Technologies, meanwhile, plans to launch a new HPLC chip later this year. “It will offer maximum sensitivity and minimum sample size,” Buhlmann says. “It will integrate sample handling, sample separation, and sample spraying into the mass spectrometer on a chip device that is reusable and very easy to run.”

Having separated proteins in an HPLC column, scientists must then detect them. While mass spectrometry remains the most popular approach, electrochemical detectors also provide highly sensitive and selective detection in HPLC, capable of quantifying picogram to femtogram levels of proteins in a sample. “When an electrochemical detector operates, it oxidizes a compound,” ESA’s Asa says. “Our electrochemical detectors are 100 percent efficient in their oxidation of compounds going through the cell. That makes the analysis much easier at the other end.”

ESA also offers what it calls the Corona CCA detector. “It’s probably one of the easiest HPLC detectors in existence,” Asa claims. “You just have to hook it up to a nitrogen source, plug it into your HPLC, and turn it on. And it delivers in gaps left by other HPLC detectors.”

Automation and Microfluidics
Like practitioners in other fields of life science, proteomics scientists increasingly rely on automation and robotics to pursue their research. Tecan’s Cellerity system, designed for use in protein production among other applications, illustrates the value of automation. “It’s a self-contained system for automated cell culturing that handles all the tasks necessary to grow, maintain, harvest, seed, and plate cell lines without user intervention for several days,” Wehren says. “We see increasing demands for this system.”

Several companies are now developing microfluidic devices and systems that can help to automate the handling processes involved in protein isolation and purification. “Microfluidics can integrate multiple manual steps in the workflow into integrated chip devices like the HPLC chip systems, giving high ease of use and good standardization,” explains Buhlmann. “If you’re looking for compliance, it’s important to have validated analytic methods.”

Last November, Agilent launched an automated lab-on-a-chip platform that permits automatic sizing and quantitation of thousands of protein samples from microtiter plates running overnight. “You can analyze up to 4,480 samples in an unattended way,” Buhlmann explains. “You feed the machine with all the chips and samples, and in the morning you can see the results in your database.”

Agilent has also developed the 2100 Bioanalyzer System that uses Caliper’s LabChip devices to analyze proteins and other molecules. “The system gives high resolution and high reproducibility,” Buhlmann says. “And it’s much faster than the traditional SDS-PAGE approach. You can get digital data for 10 samples in half an hour rather than four hours.”

Successfully unraveling the function and structure of proteins clearly begins with obtaining a target protein with its native characteristics still intact. Manufacturers who specialize in protein chemistry and proteomics research continue to improve the tools used in this field. Those tools have already led to significant advances. “In proteomics research, I see a lot of progress in the area of biomarkers,” Chakravarti of the Keck Graduate Institute says. “There’s a special focus on early diagnosis. There’s also interest in the localization of proteins within the cell.” Adds Sigma-Aldrich’s Lipscomb: “Proteomics has much more to do with human disease than genomics, in terms of biomarkers for cancer, for example.”

Chakravarti cites Current Proteomics to illustrate the promise enabled by new tools for proteomics. “We’ve completed our first year and have published 26 review articles in four issues,” he says. “The wide areas covered by these articles give you an idea of how diverse the field is.”

Peter Gwynne (pgwynne767{at}aol.com) is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner
(gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.