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April 8, 2002
Volume 80, Number 14
CENEAR 80 14 pp. 29-33
ISSN 0009-2347
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Chemists are embracing the analytical challenges of proteomics research in a big way


NEW AND IMPROVED Speicher and coworkers' prefractionation technique improves 2-D gel analyses.
Proteomics clearly represents the next major development stage in understanding the mechanisms of life, following the completion of the Human Genome Project," said chemistry professor D. Jed Harrison of the University of Alberta, who arranged a Pittcon 2002 symposium on proteomics applications of chemical microinstrumentation. "Analytical chemists have recognized how important the contributions of their field were to the sequencing project, and many appear to have reached the conclusion that they could play a significant role in the burgeoning field of proteomics, too."

There's been an evolution toward the use of proteomics "to look at many proteins at once," said professor of chemistry and biochemistry Catherine C. Fenselau of the University of Maryland. "This will have enormous implications for science and will create a range of new business opportunities."

Fenselau said she recently saw an ad "stating that big pharma was now going to examine every protein in cells to discover new drugs. How will universities participate in this effort?" she asked. For one thing, "there will be an increase in proteomics centers with shared facilities" to distribute the high costs of proteomics instrumentation among many users. And Fenselau noted that the Human Proteome Organisation has been formed to coordinate proteomics efforts internationally.

"We can hardly keep the proteomics students we're training in the lab," she said, "because there's such aggressive recruitment for them. So we need to train more students."

And according to chemistry professor Milos V. Novotny of Indiana University, Bloomington, "Proteomics will be with us for a long time. Besides structural identification of proteins and their quantification, it is concerned with where the proteins are in cells and how they interact with other molecules and each other. It poses a lot of challenges for analytical chemists."

Novotny, who spoke at a Pittcon 2002 symposium titled "Analysis of Complex Systems," exemplifies the growing number of analytical chemists who are increasingly embracing the analytical challenges of proteomics. His group recently joined the Indiana Proteomics Consortium, a newly established collaboration among researchers at Eli Lilly, Indiana University, and Purdue University that aims to develop novel analytical techniques and instruments for proteomics. The complex systems symposium was arranged by associate professor of chemistry Adrian C. Michael of the University of Pittsburgh.

WELL EQUIPPED Clemmer with LC-IMS-TOF-MS/MS instrument.
There are 50,000 to 200,000 proteins in the human proteome, noted chemistry professor David E. Clemmer of Indiana University, and analytical chemists are trying to develop better ways to separate and identify them. At the session, Clemmer received the first Pittcon Achievement Award, honoring outstanding achievements during the early stages of one's independent scientific career.

Clemmer and coworkers are developing gas-phase ion mobility strategies for proteomics use. In ion mobility spectrometry (IMS), mixtures of ions are separated in a gas as they traverse an electric field in a "drift tube." The separation is based on differences in the ions' charge, mass, and shape. Clemmer's group uses IMS as a highly parallel way to prepare samples for analysis by time-of-flight tandem mass spectrometry (TOF-MS/MS). "Our strategy is to take whole cells, digest the cell proteins to form peptides, separate the peptides by standard liquid chromatography (LC)--yielding typically 100 or more fractions--separate them in a drift tube by IMS, and then analyze them by reflectron time-of-flight MS/MS," Clemmer said.

The multidimensional technique is thus called LC-IMS-TOF-MS/MS. It makes it possible to carry out MS/MS experiments in parallel--that is, more quickly--whereas "up to now it's been a serial process," he said.

THIS TYPE of multidimensional separation is analogous to lining up the residents of New York City by height, separating them further by weight, and then distinguishing among them once again by hair color, Clemmer explained. "You can separate a lot of people pretty effectively by using just these simple characteristics."

LC-IMS-TOF-MS/MS actually provides four degrees of separation, because MS/MS is two-dimensional. "We think we have the capacity to separate 10 million peptides," he predicted.

Clemmer and coworkers recently used the technique to compare peptides from stressed and nonstressed cells after derivatizing them with two different isotopic labels [Anal. Chem., 74, 950 (2002)]. Beyond Genomics, a Boston-based company Clemmer cofounded, is also planning to use it in proteomics studies.

"One of the major complications of analyzing proteomes is that a lot of proteins are glycosylated," said Novotny in his presentation at the session. Glycosylated proteins participate in a multitude of biochemical interactions and play an important role in immunity and disease, but researchers "are only at the beginning of understanding the physiological functions of glycoproteins," he said.

Having the ability to analyze the structures of oligosaccharide groups on glycosylated proteins would help, but it remains a tremendous challenge. Glycosylated proteins are enormously complex, each type is typically present in cells in vanishingly small amounts, and no technique like polymerase chain reaction (used to amplify DNA) is available to amplify them. "Analytical chemistry advances will be needed to address this problem," Novotny said. "There's a need for new instrumentation that is faster and more informative."

He and his coworkers are developing microscale enzymatic cleavage procedures to remove oligosaccharides from glycosylated proteins and novel capillary electrochromatography (CEC) media to separate the cleaved sugar groups [Chem. Rev., 102, 321 (2002)].

They recently isolated glycosylated proteins from the tiny vomeronasal organ of rodents, which senses pheromones. They washed the proteins, separated them on gels, isolated the spots, removed the O- and N-linked oligosaccharides, separated them by capillary electrochromatography, and then analyzed them by MS/MS. "We found major differences between male and female mice in expression levels and glycosylation patterns of vomeronasal proteins," Novotny said. "These distinctions can potentially be related to differences in male and female responses to pheromones."

Chemistry professor Robert T. Kennedy and coworkers at the University of Florida, Gainesville, are also developing novel analytical methods for studying neurological peptides. They use in vivo sampling techniques, capillary LC, and electrospray ionization (ESI) MS/ MS to study tiny quantities of peptide neurotransmitters in the brain--to identify neurotransmitters released during stimulation, for example.

PEPTIDE neurotransmitters, such as Met-enkephalin, tend to be present at extracellular concentrations only a hundredth or thousandth of those of small-organic neurotransmitters, such as glutamate, dopamine, and g-aminobutyric acid. Neuropeptides can be detected in vivo by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), but the approximately 100-attomole detection limits of these techniques are too high to detect peptide neurotransmitters without necessitating inordinately long sample collection times. RIA and ELISA also require that you know in advance what peptide you're looking for, making them inapplicable to the analysis of unknown peptides, and they're limited to the determination of one peptide at a time, making them impractical for use in a proteomics environment.

"Analytical chemists have recognized that they are well situated to play a key role in developing the tools required to succeed in this complex enterprise."
Kennedy, graduate student William E. Haskins, and coworkers have been developing a high-sensitivity capillary LC-ESI-MS/MS technique that helps address these problems. The technology is based in part on earlier work by biochemistry professor Richard M. Caprioli and coworkers at Vanderbilt University School of Medicine, who developed miniaturized ESI systems and high-sensitivity ESI-MS/MS methods for analyzing LC-separated compounds. Kennedy's group has now miniaturized LC columns and ESI devices still further for use in trace neuropeptide analysis.

Kennedy and coworkers have improved LC-ESI-MS/MS detection limits from about 100 attomoles to the 2- to 5-attomole range and have demonstrated that their system can identify endogenous neuropeptides at those levels by their MS/MS fragmentation patterns [Anal. Chem., 73, 5005 (2001)]. At Pittcon, Kennedy reported preliminary results of a collaborative study with assistant professor of psychiatry Robert E. Strecker's group at Har- vard Medical School in which differences in the chemical composition of extracellular regions of rat hypothalamus tissue were detected in sleeping versus alert states.

Chemistry professor Jack Beauchamp and coworkers at California Institute of Technology are developing biomimetic reagents to cleave proteins selectively in the gas phase prior to sequencing them by MS. The goal is to be able to characterize proteins in the gas phase using MS without having to first use a solution-phase process like tryptic digestion to cut them into manageable fragments.

The researchers have designed a number of reagents that bind to specific sites on proteins and then induce cleavage at or adjacent to those sites. For example, the ligand 18-crown-6 ether forms noncovalent complexes with proteins by binding to lysine residues with high specificity [Int. J. Mass Spectrom., 210, 613 (2001)], while the larger 30-crown-10 ether preferentially binds to arginine, even in the presence of lysine.

MULTIDIMENSIONAL Clemmer and coworkers generated these LC-IMS-MS plots of a mixture of peptides from digestion of 18 proteins. IMS spectrum of peptide ion from aldolase is highlighted in the ion mobility plot. LC-IMS-MS generates some 500 mass spectra for each chromatographic slice (red bar)--demonstrating the extremely high resolving power that makes it very promising for proteomics analysis.
Such ligands can be converted into "lariat ethers"--crown ethers with attached acidic groups capable of attacking the protein at or near the binding site. But the energy required to cleave the protein is generally greater than the binding energy of the ligand-protein complex. So when the ligand tries to induce cleavage, it ends up falling off the protein instead of cutting it.

BUT BEAUCHAMP and coworkers, in collaboration with Caltech assistant professor of chemistry Brian M. Stoltz's group, recently prepared lariat ethers containing two crown ethers with a cleaving agent in between, and some of these cut proteins successfully. Once the technology matures, Beauchamp hopes it can be used to facilitate protein sequencing and to achieve selective recognition of functional protein motifs.

Another proteomics symposium, "Comparative Proteomics for Studies of Cell Function," was arranged by Fenselau. It focused on the determination of differences in protein expression in different cell types, such as healthy versus sick cells.

Postdoc Xudong Yao explained at the session that Fenselau's group has developed a technique in which proteins are simultaneously cleaved by proteolytic enzymes and isotopically labeled. Proteins from different cells can be labeled with different isotopes, making it possible to analyze them together in a single run and determine the concentrations of proteins from different sources. Fenselau's group recently used high-performance LC (HPLC) and MS/MS to study changes in protein expression in different groups of human cancer cells.

Using MS as a readout, there are two general classes of approach to differential quantitation of protein levels and protein modifications for proteomics studies, explained chemistry professor Brian T. Chait of Rockefeller University, New York City. "One uses metabolic labeling with heavy stable isotopes, while the other uses some kind of chemical labeling," such as isotope-coded affinity tagging (ICAT). "They each have pros and cons." Chait reported on a metabolic route to protein labeling in which cells are grown in an isotopically enriched culture medium.

Assistant professor of cell biology Steven P. Gygi of Harvard Medical School helped develop the ICAT strategy when he was a postdoc with proteomics researcher Ruedi Aebersold, who is now at the Institute for Systems Biology, in Seattle. ICAT is a chemical isotope labeling technique that is used "to compare on a very large scale the difference between protein levels for two different cell states," Gygi said.

He and his coworkers recently developed another labeling strategy called AQUA (an acronym for "absolute quantitation") that measures a protein's absolute, as opposed to relative, abundance. In this technique, a chemically synthesized version of a targeted protein is used as a standard to determine the absolute amount of that protein in a biological sample. Gygi and coworkers recently used it to obtain the absolute quantification of phosphorylated versus nonphosphorylated protein directly from total cell lysates.

Chemistry professor Fred E. Regnier and coworkers at Purdue University use a combination of deuterium labeling and affinity selection to identify proteins in cancer cells that change concentration in response to chemotherapy. "The big difference between ICAT and our approach is that we can target posttranslational modifications in addition to protein expression," Regnier said. "This is critical in many diseases." His group was able to detect "15 to 20 specific proteins associated with non-Hodgkin's lymphoma in the serum of dogs and to show that their concentration either decreased or increased with chemotherapy," he said. "This was possible because stable isotope technology is a way to find needle-in-a-haystack-type proteins."

MICROSPRAYER This combined capillary LC column and electrospray emitter tip is a key part of the LC-ESI-MS/MS system designed by Kennedy and coworkers for trace neuropeptide analysis.
A third proteomics symposium at Pittcon--"Cancer Diagnostics Based on MS, Protein Chips, and Microarrays for Detection of Biomarkers"--also focused on the proteomics analysis of different types of cells, in this case using protein chip technology. The symposium was arranged by chemistry professor David M. Lubman of the University of Michigan, Ann Arbor.

Gavin MacBeath, who will join Harvard University's department of chemistry and chemical biology as an assistant professor this July, reported at the session on techniques to discover small organic molecules capable of modulating protein function. "We are interfacing protein microarray technology with high-throughput screening to identify cell-permeable small molecules that disrupt or otherwise modulate protein-protein or protein-peptide interactions in a highly specific manner," MacBeath said. "Multiplexed screening greatly increases the number of interactions that can be studied, dramatically decreases sample consumption, and enables us to assess the specificity of compounds during the screening process."

The group is also using microarrays of antibodies "to study how cell-permeable compounds modulate both the abundance and posttranslational modification state of multiple proteins simultaneously," MacBeath said. "These and other protein profiling technologies provide a more holistic understanding of how bioactive compounds modulate complex protein networks within the context of a living cell."

Lubman and coworkers combine 2-D liquid separations with MS to profile proteins in different types of tumor cells. They separate proteins using isoelectric focusing or chromatofocusing and separate them further by reversed-phase HPLC. They then use TOF-MS to obtain the molecular weights of the separated proteins. The result is a 2-D "mass map" that's analogous to a 2-D gel but has much higher mass accuracy--typically in the 100-parts-per-million range. Lubman's team recently used the technique to compare protein expression in chemotherapeutically treated versus untreated colon cancer cell lines and found significant differences between the two types. He believes the strategy could aid the discovery of medicines for different types of cancers.

David W. Speicher, professor and chair of the structural biology program at Wistar Institute, Philadelphia, reported on a prefractionation strategy that improves the analysis of 2-D gels. The procedure is capable of quantitatively comparing more than 10,000 protein components from two or more proteomes.

IT IS BASED on microscale solution isoelectric focusing (msol-IEF), a technique in which protein separation is achieved in tiny chambers separated by membranes that are maintained at different pH values. Protein samples can be prefractionated in this way prior to separation on 2-D gels to produce better quality gel separations with less protein overloading, fewer interferences, and less streaking.

"Compared with alternative methods, our strategy expands the number of proteins that can be reliably quantitatively compared and the dynamic range of detection, and it reduces the variability of subsequent 2-D gel separations," Speicher said. "It also solves several problems that have limited 2-D gel-based proteome comparisons" by making it possible to analyze large proteins, insoluble proteins, and very acidic and very basic proteins that are normally not accessible. "We are continuing to improve the method and expect to achieve quantitative comparisons of 15,000 to 20,000 proteins in the future," he noted.

CHOOSY Selective lariat ether binding agent synthesized by Beauchamp and coworkers is shown bound to a peptide. The lariat ether consists of a pair of 18-crown-6 ethers linked by a benzoic acid derivative capable of cleaving a peptide
or protein.
"Microfluidic Chips and Mass Spectrometry Meet the Proteomics Challenge" was yet another of this year's plethora of Pittcon proteomics programs. The symposium--arranged by the University of Alberta's Harrison--showed how microfluidic technologies can be used to miniaturize and automate protein preparation for MS analysis.

At the symposium, Richard Smith, leader of Pacific Northwest National Laboratory's proteomics group, discussed how one can apply "the tremendous resolving power of Fourier transform ion cyclotron resonance MS (FT-ICR-MS) to protein digests"--as Harrison described it for C&EN. Smith's group uses the FT-ICR-MS technique, in conjunction with capillary LC, to detect and resolve hundreds of thousands of peptide fragments from protein digests. "The sensitivity and quality of resolution achieved represents a significant advance compared to conventional
2-D gel separation and MS analysis methods," Harrison said.

Chemistry professor Liang Li of the University of Alberta described the use of capillaries as nanoliter reactors to handle low-abundance proteins expressed in cells. He and his coworkers use the capillaries for sample manipulation and protein digestion of 2-D gel extracts or LC eluents prior to MS or MS/MS analysis.

Microinstrumentation specialist J. Michael Ramsey of Oak Ridge National Laboratory was one of several speakers who discussed protein processing on microfluidic devices and ways to interface such devices with electrospray ion sources for MS analysis. He reported that electrophoresis and micellar electrokinetic chromatography can give nearly orthogonal separation of peptide digests, providing much more highly resolved 2-D peptide maps than with previous chip-based systems. "We have demonstrated peak capacities of 3,000 to 5,000 for peptides," Ramsey said.

Harrison reported on the results of a collaboration between his group and that of researcher Pierre Thibault of the National Research Council of Canada. In an on-chip protein processing study, the scientists identified different chip designs in which protein samples could be digested 10 to 100 times faster than in conventional laboratory systems. "The integrated devices reduce analysis time, increase the level of automation, and improve the sensitivity of protein analysis by MS," Harrison said.

And Frantisek Foret of the Academy of Sciences of the Czech Republic, in Brno, a visiting scientist at Northeastern University, described efforts to produce better chip-MS interfaces that are more easily manufactured. "Overall, the use of capillary and microchip devices to perform protein sample preparation is a rapidly developing field, with important ramifications for automating proteomics methods," Harrison said.

"Certainly, the challenge of measuring all the proteins in an organism on a quantitative level and comparing the effects of disease, drugs, age, or some other stimulus on the protein complement in a quantitative fashion is an enormous one," Harrison added. "Analytical chemists have recognized that they are well situated to play a key role in developing the tools required to succeed in this complex enterprise."

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