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Slow Evolution for Protein Detection, Characterization
Old tools will likely remain a mainstay for the foreseeable future, but new approaches aim to get data faster, reproducibly, and with smaller samples
Lori Valigra
Valigra is a freelance writer
based in Cambridge, Mass.
A Texas cow whose tissue tested positive for mad cow disease in June turned some time-honored protein tests into household words. Consumers and the cattle industry alike debated whether the US Department of Agriculture (USDA) had performed rigorous enough tests on the animal, which initially was deemed negative for bovine spongiform encephalopathy (BSE).
But it wasn't biosensors or other cutting-edge technologies that confirmed the deposits of abnormal prions in a postmortem tissue sample. Instead, it was a positive result
About 12 proteins comprise up to 96% of the protein mass in plasma. (Source: Plasma Proteome Institute and Beckman Coulter)
from an old standby, the Western blot, that prompted the USDA to dig beyond earlier inconclusive and negative results from enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry (IHC) tests (see sidebar on "Anatomy of BSE Detection"). The USDA and The Veterinary Laboratories Agency, a world reference lab for BSE in Weybridge, UK, conducted additional tests and concurred the sample was positive. The Texas case led the USDA to change its BSE protocol. All inconclusive ELISA tests now will be followed by both an IHC and Western blot rather than just the IHC alone, says Jim Rogers, a spokesman for the USDA's Animal and Plant Health Inspection Service (APHIS). If either confirmatory test is positive, the sample will be considered positive for BSE.
The BSE case underscores the continuing importance of conventional tests, as well as the frequent need to use more than one tool to study proteins, which by nature are more complex than nucleic acids. Many advanced genomics tools already are available as a result of the race to sequence and study the human genome. But proteomics lacks an amplification technology similar to polymerase chain reaction (PCR) used for DNA and protein arrays are harder to develop because of the comparatively broad range of binding conditions for target proteins. Those are some of the reasons why ELISA, IHC, Western blot, one-dimensional (1D) and two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) persist in the market as protein detection and characterization techniques for diagnostics and drug discovery and development. These technologies are being improved by using capillaries to speed testing, and they are also being used with complementary technologies such as liquid chromatography (LC) and MS. "Proteins are so important that you're going to see everybody developing new techniques constantly," says Linda Cahill, president and CEO of Cell Biosciences Inc., Palo Alto, Calif.
Simplifying Western blots
The mere mention of a Western blot can elicit moans from graduate students who serve as free labor for a tedious process that has changed little since it was first published
Western blotting requires about 500,000 cells for analysis and highly specific antibodies for phosphoprotein differentiation. By contrast, the Blotless Nano Western requires fewer than 4,000 cells, and it separates and detects phospho and non-phospho species in one run with a single antibody. (Source: Cell Biosciences Inc.)
in 1979. The paper explained how to transfer proteins from a polyacrylamide gel to a sheet of nitrocellulose to obtain a faithful replica of the original gel pattern. No longer trapped inside the gel, the immobilized protein could be detected by antibodies and studied with a variety of analytical procedures. At the time, it was a breakthrough technology and is still a critical confirmatory assay. Cahill conducted an informal study of the test's prevalence. She looked at life science journals for four months and tracked each time a Western blot was used as the confirmatory assay: it was 85% of the time.
"With all the new protein techniques coming along, you have to ask yourself why Westerns are the dominant method," says Cahill. "The reason is they give you the physical separation of the protein so you can get more specificity out of your antibodies." But a Western blot protocol can seem like something from the 17th century, she says: one to two days of manual labor with as many as 100 to 200 different pipetting steps, each of which could lead to mistakes. "But it works."
Cell Biosciences, which develops instruments and reagents, will start beta testing a capillary-based system inspired by the Western blot later this summer. It will run smaller samples automatically and quickly, with reproducible results, Cahill says. "A Western blot might take you two days, but this takes 30 minutes."
The new system uses what the company calls Blotless Nano Western technology. The technique is based on a capillary immunoassay format that automates the basic steps of Western blotting using nanoliter volumes and physically separating the protein in the capillary. The initial system will require 500 to 1,000 cells in a sample, and it will be able to accommodate multiple capillaries running simultaneously. "We're going to look for whether the protein is there, if it is a good druggable target, and even if it isn't, how the protein is interacting in cells." It is scheduled for introduction in early 2006.
Faster, more sensitive
According to a Cell Biosystems comparison, Western blotting requires about 500,000 cells for analysis and highly specific antibodies for phosphoprotein differentiation. It can
Anatomy of BSE Detection
In June, the tissue of a cow in Texas tested positive for mad cow disease following a battery of time-honored protein tests The bovine spongiform encephalopathy (BSE)-positive cow was selected for testing at a Texas pet-food plant because it was non-ambulatory; its remains were incinerated, so it never entered the human or animal food chains.
According to the Animal and Plant Health Inspection Service, a part of the US Department of Agriculture (USDA), the first postmortem test on the cow's tissues in November 2004 was a Bio-Rad Laboratories Inc. ELISA rapid test that was inconclusive. The sample was then sent to the USDA's National Veterinary Services Laboratories in Iowa, where two internationally accepted confirmatory immunohistochemistry (IHC) tests were conducted in November, 2004. Both were negative for BSE.
The USDA's Office of the Inspector General recommended further testing in early June using another internationally recognized confirmatory test, the Western blot. It came back positive. The following week the USDA conducted another IHC test using different antibodies than the November, 2004 test. It was weakly positive for BSE. In addition, tissue samples were sent to a world reference laboratory for BSE, the Veterinary Laboratories Agency in Weybridge, UK, which conducted rapid enzyme-linked immunosorbent assays, IHC and Western blot tests, and concluded the sample was positive. Those IHC tests used different antibodies than those used by the USDA in November, 2004.
However, Weybridge also confirmed the accuracy of the results of the USDA's November confirmatory IHC tests, and concurred that the case could not have been confirmed on the basis of the sample. USDA and Weybridge scientists are trying to determine the cause of the conflicting IHC results. Several factors could have contributed, one being that the cow had a very low level of infectivity and the USDA's routine IHC test might not have been sensitive enough to detect the disease. In addition, Weybridge experts indicated that the deposits of the abnormal prion were not uniformly distributed in the brain tissue and were present at a low concentration. That means adjacent samples of brain tissue might have yielded different results.
take two days to complete, and it has limited quantitative capability. It is a manual procedure that has a difficult blotting step that works differently for every protein. By contrast, the Blotless Nano Western requires fewer than 4,000 cells, and single-cell sensitivity is anticipated in the future. It separates and detects phospho and non-phospho species in one run with a single antibody.
"We get better separation because we're doing it on a capillary electrophoresis device, which gives us much better control," Cahill says. Presorting is done by isoelectric focusing, so it is done by the charge of the protein and not just its size. "The ability to do this repeatedly with standard measurements against it, so that what you do on Monday morning is the same as what you do on Thursday afternoon, is critically important to biology." And with Western blot technology being used more often to confirm BSE cases, the market potential is large.
Roche Applied Science, Indianapolis, has taken an incremental approach to improving technology. Their LumiLight and LumiLight Plus products have improved the sensitivity and length of the chemiluminescence signal possible when developing a Western blot. "But we're putting a lot of our attention on the production of the specific proteins that are detected using Western blot technology," says Jeffrey Emch, a product manager at Roche. "Customers are struggling to produce the proteins to detect, to isolate. That's one of the significant bottlenecks we hear from academia, pharma, and biotech."
Roche developed a technology for in vitro protein expression called Rapid Translation System. It uses Escherichia coli or a wheat germ lysate for protein production. "When you're producing a protein in vitro it can be the dominant protein, but when you're isolating it from a cellular system you have additional proteins that need to be stripped away from your protein of interest," Emch says. "It's essentially reducing the size of the haystack to find your needle."
Improving gels
Like Western blots, gels have also made grad students cringe: pouring gels, staining, and doing quantitation to get protein characterization is time-consuming. Bio-Rad Laboratories Inc., Hercules, Calif., is working on improving a variety of products including Western blots and 1D and 2D gels. The 1D gels detect proteins by size and are often used with Western blots, and the 2D gels detect proteins by size and charge. The 2D gels can look at thousands of proteins in one experiment.
"The 2D gels today still represent the highest resolution technique available for the greatest number of proteins in one assay that you can do," says Gustavo Salem, division manager of Bio-Rad's protein separations division. "We don't see any specific technology that will displace 2D gels, but there are opportunities to continue to improve how they are used, especially as it relates to sample preparation to try to provide greater resolution of specific proteins." When looking for low-abundance proteins, for example, the sample preparation step is vital to how clean a gel is and how reproducible it will be, especially when trying to make comparisons.
While many in the industry believe Western blots are here to stay, some question the longevity of 2D gels. "At thousands of different proteins in a sample, it hits a ceiling," says Gavin MacBeath, PhD, assistant professor of chemistry and chemical biology at Harvard University, Cambridge, Mass. He believes LC and capillary multidimensional column chromatography (MDCC) coupled with MS will give researchers as many dimensions as they want. "You have smaller sample sizes with MDCC and MS, so you can drill deeper into the proteome," he says.
"I see a lot of interest to move away from 2D gels and replace it with chromatographic techniques," says Jeffrey Jensen, president of Eksigent Technologies LLC, Livermore, Calif. His company has multidimensional LC separation products, but he doesn't see them as direct replacements for 2D gels. The products are focused at detecting low-abundance proteins when used with mass spectrometry. To get at the low concentration proteins requires an extremely low flow rate, which Eksigent says it gets with its Microfluidic Flow Control (MFC) technology. MFC uses meters to continuously monitor the flow rate of each mobile phase with microprocessor feedback to a pressure source, so nanoscale flow rates as low as 20 nL/min can be obtained without flow splitting. Flow splitting involves trying to control the flow rate by leaking off 99% or more of the sample in an effort to get the tiny amount they want into the mass spectrometer.
Complementary technologies
Another trend is the combination of different separation technologies to try to get fractionation of a particular class of proteins. For example, an LC system and a gel
When studying proteins, researchers begin by expressing their protein of interest or isolating the protein from biological tissue. Then they prepare and simplify their sample, then characterize it using multiple techniques. (Source: Invitrogen Corp.)
could be used to get the purification capability of an ion-exchange resin on a column and then a size-based separation on a gel, and then it can be taken to a mass spectrometer, Salem says. There is more and more demand lately for isoelectric focusing separations, and Bio-Rad has a system called the Rotofor that can be used with gels and LC.
Bio-Rad is not alone in bringing together complementary technologies. Invitrogen Corp., Carlsbad, Calif., recently launched a SILAC (stable isotope labeling using amino acids in cell culture) kit for protein identification and quantitation. Previously, most SILAC technology was home brewed. SILAC can be used, for example, to purify proteins going through a 1D gel and then to a mass spectrometer. SILAC is a metabolic labeling method that can measure different protein levels between a normal and diseased state. "One isotope is heavy and one is light, so you can run it through a mass spectrometer and see small-fold changes that you couldn't see with a 2D gel," says Cheri Walker, PhD, vice president of proteomics at Invitrogen.
"With consumables our plan is to put together matched reagents that work together. We have some protein expression systems that help with hard-to-express proteins like membrane proteins." Invitrogen also has a system called Zoom Benchtop Proteomics that includes a mini-gel format to profile proteins and 2D gel electrophoresis. It performs sample fractionation, first and second dimension gel electrophoresis, and staining with MS-compatible stains. It uses wide and narrow Zoom Strips, with the narrow range strips running from pH 4 to pH 5.5. Walker says companies with big genomics groups 10 years ago are rapidly changing to more proteomics-focused groups. "They're looking for these types of tools because the workflows aren't standardized today."
High-performance chromatography is being adopted along with MS and moving from experts' laboratories into biologists' labs. "There is a revolution going on, being driven by what you can do with MS today," says Patrick Carberry, LC-MS marketing manager, proteomics, Agilent Technologies Inc., Santa Clara, Calif. Carberry predicts that within the next year, there will be systems on the market priced around $125,000 that incorporate a chromatographic front and a high-performance MS back end with easy-to-use software.
Agilent has a variety of technologies, including a high-performance LC (HPLC) system line whose most recent member is an HPLC chip that can produce narrower peaks that it says are better resolved than a standard nano-column. But the company is taking a systems approach to proteins. "We'll evolve separation equipment and technologies around proteins and peptides along with MS and MS analyzers, informatics and data handling tools that will be integrated with common control, application, and informatics software. And you'll find all these things will become miniaturized. The current HPLC may be able to fit in the palm of your hand and be made of microfluidics components."
Deeper into the proteome
Beckman Coulter Inc., Fullerton, Calif., is working on improving its technology offerings. Its hallmark system is the ProteomeLab PF 2D, a protein fractionation system that allows for 2D chromatography to fractionate the target sample and obtain fractions in a liquid format. It can detect low-abundance proteins and characterize them. The company's ProteomeLab PA 800 protein characterization system resolves and quantifies proteins by their isoelectric point and molecular weight. It can serve as a front-end separation to MS. The ProteomeLab IgY, partitions away the 12 most abundant proteins that make up to 96% of the protein mass in plasma or serum.
Beckman Coulter's aim, like that of many companies in proteomics, is to make it easier for scientists to conduct extensive protein research. And when instruments give scientists an extra inch, they want an extra mile. "To me, proteins are like real estate. It's location, location, location," says Kenneth Bloom, MD, chief medical officer of Clarient Inc., a San Juan Capistrano, Calif., company that provides products and services to characterize, assess and treat cancer. "It's really not just a question of whether a protein is or isn't there. It's a question of how much protein is there and more importantly, where it is in the cell."
Old tools will likely remain a mainstay for the foreseeable future, but new approaches aim to get data faster, reproducibly, and with smaller samples
Lori Valigra
Valigra is a freelance writer
based in Cambridge, Mass.
A Texas cow whose tissue tested positive for mad cow disease in June turned some time-honored protein tests into household words. Consumers and the cattle industry alike debated whether the US Department of Agriculture (USDA) had performed rigorous enough tests on the animal, which initially was deemed negative for bovine spongiform encephalopathy (BSE).
But it wasn't biosensors or other cutting-edge technologies that confirmed the deposits of abnormal prions in a postmortem tissue sample. Instead, it was a positive result
About 12 proteins comprise up to 96% of the protein mass in plasma. (Source: Plasma Proteome Institute and Beckman Coulter)
from an old standby, the Western blot, that prompted the USDA to dig beyond earlier inconclusive and negative results from enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry (IHC) tests (see sidebar on "Anatomy of BSE Detection"). The USDA and The Veterinary Laboratories Agency, a world reference lab for BSE in Weybridge, UK, conducted additional tests and concurred the sample was positive. The Texas case led the USDA to change its BSE protocol. All inconclusive ELISA tests now will be followed by both an IHC and Western blot rather than just the IHC alone, says Jim Rogers, a spokesman for the USDA's Animal and Plant Health Inspection Service (APHIS). If either confirmatory test is positive, the sample will be considered positive for BSE.
The BSE case underscores the continuing importance of conventional tests, as well as the frequent need to use more than one tool to study proteins, which by nature are more complex than nucleic acids. Many advanced genomics tools already are available as a result of the race to sequence and study the human genome. But proteomics lacks an amplification technology similar to polymerase chain reaction (PCR) used for DNA and protein arrays are harder to develop because of the comparatively broad range of binding conditions for target proteins. Those are some of the reasons why ELISA, IHC, Western blot, one-dimensional (1D) and two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) persist in the market as protein detection and characterization techniques for diagnostics and drug discovery and development. These technologies are being improved by using capillaries to speed testing, and they are also being used with complementary technologies such as liquid chromatography (LC) and MS. "Proteins are so important that you're going to see everybody developing new techniques constantly," says Linda Cahill, president and CEO of Cell Biosciences Inc., Palo Alto, Calif.
Simplifying Western blots
The mere mention of a Western blot can elicit moans from graduate students who serve as free labor for a tedious process that has changed little since it was first published
Western blotting requires about 500,000 cells for analysis and highly specific antibodies for phosphoprotein differentiation. By contrast, the Blotless Nano Western requires fewer than 4,000 cells, and it separates and detects phospho and non-phospho species in one run with a single antibody. (Source: Cell Biosciences Inc.)
in 1979. The paper explained how to transfer proteins from a polyacrylamide gel to a sheet of nitrocellulose to obtain a faithful replica of the original gel pattern. No longer trapped inside the gel, the immobilized protein could be detected by antibodies and studied with a variety of analytical procedures. At the time, it was a breakthrough technology and is still a critical confirmatory assay. Cahill conducted an informal study of the test's prevalence. She looked at life science journals for four months and tracked each time a Western blot was used as the confirmatory assay: it was 85% of the time.
"With all the new protein techniques coming along, you have to ask yourself why Westerns are the dominant method," says Cahill. "The reason is they give you the physical separation of the protein so you can get more specificity out of your antibodies." But a Western blot protocol can seem like something from the 17th century, she says: one to two days of manual labor with as many as 100 to 200 different pipetting steps, each of which could lead to mistakes. "But it works."
Cell Biosciences, which develops instruments and reagents, will start beta testing a capillary-based system inspired by the Western blot later this summer. It will run smaller samples automatically and quickly, with reproducible results, Cahill says. "A Western blot might take you two days, but this takes 30 minutes."
The new system uses what the company calls Blotless Nano Western technology. The technique is based on a capillary immunoassay format that automates the basic steps of Western blotting using nanoliter volumes and physically separating the protein in the capillary. The initial system will require 500 to 1,000 cells in a sample, and it will be able to accommodate multiple capillaries running simultaneously. "We're going to look for whether the protein is there, if it is a good druggable target, and even if it isn't, how the protein is interacting in cells." It is scheduled for introduction in early 2006.
Faster, more sensitive
According to a Cell Biosystems comparison, Western blotting requires about 500,000 cells for analysis and highly specific antibodies for phosphoprotein differentiation. It can
Anatomy of BSE Detection
In June, the tissue of a cow in Texas tested positive for mad cow disease following a battery of time-honored protein tests The bovine spongiform encephalopathy (BSE)-positive cow was selected for testing at a Texas pet-food plant because it was non-ambulatory; its remains were incinerated, so it never entered the human or animal food chains.
According to the Animal and Plant Health Inspection Service, a part of the US Department of Agriculture (USDA), the first postmortem test on the cow's tissues in November 2004 was a Bio-Rad Laboratories Inc. ELISA rapid test that was inconclusive. The sample was then sent to the USDA's National Veterinary Services Laboratories in Iowa, where two internationally accepted confirmatory immunohistochemistry (IHC) tests were conducted in November, 2004. Both were negative for BSE.
The USDA's Office of the Inspector General recommended further testing in early June using another internationally recognized confirmatory test, the Western blot. It came back positive. The following week the USDA conducted another IHC test using different antibodies than the November, 2004 test. It was weakly positive for BSE. In addition, tissue samples were sent to a world reference laboratory for BSE, the Veterinary Laboratories Agency in Weybridge, UK, which conducted rapid enzyme-linked immunosorbent assays, IHC and Western blot tests, and concluded the sample was positive. Those IHC tests used different antibodies than those used by the USDA in November, 2004.
However, Weybridge also confirmed the accuracy of the results of the USDA's November confirmatory IHC tests, and concurred that the case could not have been confirmed on the basis of the sample. USDA and Weybridge scientists are trying to determine the cause of the conflicting IHC results. Several factors could have contributed, one being that the cow had a very low level of infectivity and the USDA's routine IHC test might not have been sensitive enough to detect the disease. In addition, Weybridge experts indicated that the deposits of the abnormal prion were not uniformly distributed in the brain tissue and were present at a low concentration. That means adjacent samples of brain tissue might have yielded different results.
take two days to complete, and it has limited quantitative capability. It is a manual procedure that has a difficult blotting step that works differently for every protein. By contrast, the Blotless Nano Western requires fewer than 4,000 cells, and single-cell sensitivity is anticipated in the future. It separates and detects phospho and non-phospho species in one run with a single antibody.
"We get better separation because we're doing it on a capillary electrophoresis device, which gives us much better control," Cahill says. Presorting is done by isoelectric focusing, so it is done by the charge of the protein and not just its size. "The ability to do this repeatedly with standard measurements against it, so that what you do on Monday morning is the same as what you do on Thursday afternoon, is critically important to biology." And with Western blot technology being used more often to confirm BSE cases, the market potential is large.
Roche Applied Science, Indianapolis, has taken an incremental approach to improving technology. Their LumiLight and LumiLight Plus products have improved the sensitivity and length of the chemiluminescence signal possible when developing a Western blot. "But we're putting a lot of our attention on the production of the specific proteins that are detected using Western blot technology," says Jeffrey Emch, a product manager at Roche. "Customers are struggling to produce the proteins to detect, to isolate. That's one of the significant bottlenecks we hear from academia, pharma, and biotech."
Roche developed a technology for in vitro protein expression called Rapid Translation System. It uses Escherichia coli or a wheat germ lysate for protein production. "When you're producing a protein in vitro it can be the dominant protein, but when you're isolating it from a cellular system you have additional proteins that need to be stripped away from your protein of interest," Emch says. "It's essentially reducing the size of the haystack to find your needle."
Improving gels
Like Western blots, gels have also made grad students cringe: pouring gels, staining, and doing quantitation to get protein characterization is time-consuming. Bio-Rad Laboratories Inc., Hercules, Calif., is working on improving a variety of products including Western blots and 1D and 2D gels. The 1D gels detect proteins by size and are often used with Western blots, and the 2D gels detect proteins by size and charge. The 2D gels can look at thousands of proteins in one experiment.
"The 2D gels today still represent the highest resolution technique available for the greatest number of proteins in one assay that you can do," says Gustavo Salem, division manager of Bio-Rad's protein separations division. "We don't see any specific technology that will displace 2D gels, but there are opportunities to continue to improve how they are used, especially as it relates to sample preparation to try to provide greater resolution of specific proteins." When looking for low-abundance proteins, for example, the sample preparation step is vital to how clean a gel is and how reproducible it will be, especially when trying to make comparisons.
While many in the industry believe Western blots are here to stay, some question the longevity of 2D gels. "At thousands of different proteins in a sample, it hits a ceiling," says Gavin MacBeath, PhD, assistant professor of chemistry and chemical biology at Harvard University, Cambridge, Mass. He believes LC and capillary multidimensional column chromatography (MDCC) coupled with MS will give researchers as many dimensions as they want. "You have smaller sample sizes with MDCC and MS, so you can drill deeper into the proteome," he says.
"I see a lot of interest to move away from 2D gels and replace it with chromatographic techniques," says Jeffrey Jensen, president of Eksigent Technologies LLC, Livermore, Calif. His company has multidimensional LC separation products, but he doesn't see them as direct replacements for 2D gels. The products are focused at detecting low-abundance proteins when used with mass spectrometry. To get at the low concentration proteins requires an extremely low flow rate, which Eksigent says it gets with its Microfluidic Flow Control (MFC) technology. MFC uses meters to continuously monitor the flow rate of each mobile phase with microprocessor feedback to a pressure source, so nanoscale flow rates as low as 20 nL/min can be obtained without flow splitting. Flow splitting involves trying to control the flow rate by leaking off 99% or more of the sample in an effort to get the tiny amount they want into the mass spectrometer.
Complementary technologies
Another trend is the combination of different separation technologies to try to get fractionation of a particular class of proteins. For example, an LC system and a gel
When studying proteins, researchers begin by expressing their protein of interest or isolating the protein from biological tissue. Then they prepare and simplify their sample, then characterize it using multiple techniques. (Source: Invitrogen Corp.)
could be used to get the purification capability of an ion-exchange resin on a column and then a size-based separation on a gel, and then it can be taken to a mass spectrometer, Salem says. There is more and more demand lately for isoelectric focusing separations, and Bio-Rad has a system called the Rotofor that can be used with gels and LC.
Bio-Rad is not alone in bringing together complementary technologies. Invitrogen Corp., Carlsbad, Calif., recently launched a SILAC (stable isotope labeling using amino acids in cell culture) kit for protein identification and quantitation. Previously, most SILAC technology was home brewed. SILAC can be used, for example, to purify proteins going through a 1D gel and then to a mass spectrometer. SILAC is a metabolic labeling method that can measure different protein levels between a normal and diseased state. "One isotope is heavy and one is light, so you can run it through a mass spectrometer and see small-fold changes that you couldn't see with a 2D gel," says Cheri Walker, PhD, vice president of proteomics at Invitrogen.
"With consumables our plan is to put together matched reagents that work together. We have some protein expression systems that help with hard-to-express proteins like membrane proteins." Invitrogen also has a system called Zoom Benchtop Proteomics that includes a mini-gel format to profile proteins and 2D gel electrophoresis. It performs sample fractionation, first and second dimension gel electrophoresis, and staining with MS-compatible stains. It uses wide and narrow Zoom Strips, with the narrow range strips running from pH 4 to pH 5.5. Walker says companies with big genomics groups 10 years ago are rapidly changing to more proteomics-focused groups. "They're looking for these types of tools because the workflows aren't standardized today."
High-performance chromatography is being adopted along with MS and moving from experts' laboratories into biologists' labs. "There is a revolution going on, being driven by what you can do with MS today," says Patrick Carberry, LC-MS marketing manager, proteomics, Agilent Technologies Inc., Santa Clara, Calif. Carberry predicts that within the next year, there will be systems on the market priced around $125,000 that incorporate a chromatographic front and a high-performance MS back end with easy-to-use software.
Agilent has a variety of technologies, including a high-performance LC (HPLC) system line whose most recent member is an HPLC chip that can produce narrower peaks that it says are better resolved than a standard nano-column. But the company is taking a systems approach to proteins. "We'll evolve separation equipment and technologies around proteins and peptides along with MS and MS analyzers, informatics and data handling tools that will be integrated with common control, application, and informatics software. And you'll find all these things will become miniaturized. The current HPLC may be able to fit in the palm of your hand and be made of microfluidics components."
Deeper into the proteome
Beckman Coulter Inc., Fullerton, Calif., is working on improving its technology offerings. Its hallmark system is the ProteomeLab PF 2D, a protein fractionation system that allows for 2D chromatography to fractionate the target sample and obtain fractions in a liquid format. It can detect low-abundance proteins and characterize them. The company's ProteomeLab PA 800 protein characterization system resolves and quantifies proteins by their isoelectric point and molecular weight. It can serve as a front-end separation to MS. The ProteomeLab IgY, partitions away the 12 most abundant proteins that make up to 96% of the protein mass in plasma or serum.
Beckman Coulter's aim, like that of many companies in proteomics, is to make it easier for scientists to conduct extensive protein research. And when instruments give scientists an extra inch, they want an extra mile. "To me, proteins are like real estate. It's location, location, location," says Kenneth Bloom, MD, chief medical officer of Clarient Inc., a San Juan Capistrano, Calif., company that provides products and services to characterize, assess and treat cancer. "It's really not just a question of whether a protein is or isn't there. It's a question of how much protein is there and more importantly, where it is in the cell."
