July 1, 2008 (Vol. 28, No. 13)

Collection and Processing Activities Are Under Increasing Scrutiny

Proteomics continues to have an increasing role in drug discovery and diagnostics development and also provides a better understanding of disease. Advances in technology are providing innovative methods to enhance protein analysis with an increased focus on sample preparation. Cambridge Healthtech’s recent “Proteomic Sample Prep” conference shed some light on what researchers are focusing on to help bring protein analysis to the next level.

One of the challenges in protein sample prep is avoiding degradation. The most common method currently used for inactivation of denaturation is chemical inhibitors. These only provide a reduction in degradation, however. “The diversity of proteins is large, and it’s not fully known which chemical inhibitors work well for some subgroups, proteases, or other fully active groups,” explained Mats Boren, Ph.D., head of research, Denator Biotechnology (www.denator.com).

“Degradation potentially occurs during the entire work flow unless you stabilize the sample.”

Denator’s Stabilizor T1™ was designed to provide rapid, controlled processing of tissue samples. The beta-stage product was developed by company founders via a research project at Uppsala University and the Karolinska Institute. According to Dr. Boren, it works quickly to stop all biological activity and maintain the primary structure of the protein. It can be used for solid tissue (fresh or frozen) samples less than 5 mm thick.

“The Stabilizor enables efficient detection of endogenous peptides, analyzes phosphorylations in a state as in vivo as possible, lowers intragroup variability, and provides fast detection of novel peptide targets or biomarkers,” said Dr. Boren. The current application is for basic research, but it has the potential for use in diagnostics with nonsolid tissues (e.g., blood and urine). The company anticipates launching the Stabilizor T1 in late August.

Researchers at Millipore (www.millipore.com) have developed a new strategy for Amicon® Ultra Centrifugal Filter Units to deplete some of the high-abundant proteins and enrich for low-abundant proteins, potential targets for biomarkers.

Janice Simler, Ph.D., research scientist, presented information at the meeting on using these filter units with serum. “Separating out the low-abundant proteins, especially in matrices like serum, is one of the challenges for biomarker research. We’re trying to empower people who are doing this type of research with a different method that is something they could easily adapt with the equipment they already have in the lab—a centrifuge,” noted Dr. Simler.

Her group presented a model system of human serum to demonstrate the efficiency of Amicon Ultra Centrifugal Filter Units in separation of high molecular weight proteins from low molecular weight proteins. The units are scalable, allowing researchers to start with 50 or 100 microliters and scale up as needed (4 mL and 15 mL versions are available). “These devices save time because you can concentrate and separate in one step, and do a buffer-exchange within the devices, so you don’t have to do an acetone-precipitation, you can just buffer exchange into the buffer you would need for your 2-D analysis.”

The filter units can be used in several steps of protein research. “We feel you can combine this approach with others in order to get optimal depletion, because right now it doesn’t seem like there’s one unique solution,” added Dr. Simler.

In addition, Amicon Ultra-4 filters allow the researcher access to different protein fractions, covering the whole proteome. “I think it’s key to have the whole proteome available. You may initially think your target is in a certain MW range, and then you realize you have to look at higher MW proteins.”

This method is adaptable to any lab and does not require a new skill set, according to Dr. Simler. “Anyone can easily use this method and optimize it to answer the questions they are looking at—either low-abundance proteins or molecular proteins in the high molecular weight range.”

New Approaches for Protein Capture

Hongshan Li, Ph.D., senior principle scientist, proteomics, Pall Life Sciences (www.pall.com), presented a new application of protein capturing under denatured conditions, which avoids precipitation to increase protein solubility and separation. He also discussed the benefits of new solvents (HEA, PPA, and MEP) in combination with ion-exchange chromatography. “Most scientists that work on protein capture are hesitant to add the denaturants. They are afraid the protein will break apart, resulting in less interaction, less affinity, and reduced efficiency of capturing.”

Specific protein capturing under denaturing conditions is achieved using the company’s Enchant™ Multi-Protein Affinity Separation Kit to help avoid protein precipitation and aggregation and to increase protein solubility and separation. This benefits protein-protein interaction studies, drug discovery, and proteomics, according to Dr. Li, who added that the combination of depletion under denaturing conditions with ion-exchange chromatography enhances sample concentration and increases detection of more peaks.

All three solvents operate on a mixed-mode mechanism, meaning their chromatographic behavior is based on a combination of electrostatic, hydrophobic, and affinity properties of the protein and ligands. This mechanism can be exploited to achieve discrimination of proteins having similar or very close isoelectric points—a separation that cannot be performed by other methods like ion exchange.

The solvents also can work under physiological conditions, meaning there won’t be too much salt or other reagents in sample processing, making it cost effective and environmentally beneficial, reported Dr. Li. The company says it will soon offer all three solvents in a 1 mL prepacked column (AcroSep™). Dr. Li said this new format works well for high-throughput screening and offers flexibility for syringe-method users.

Analyzing Intact Proteins

Mass spec immunoassay was developed by scientists at The Biodesign Institute at Arizona State University (www.biodesign.asu.edu) over a decade ago, and applications continue to multiply.

When researchers realized that a protein is present in various qualitative forms, “we began to look at different disease states to determine whether these differences, or microheterogeneity, within proteins of a population would serve as biomarkers of disease,” explained Chad Borges, Ph.D., assistant research scientist.

Analysis of intact proteins in a targeted manner guarantees a comprehensive analysis of the microheterogeneity, added Dr. Borges. The current way to analyze intact proteins clinically is via immunoassays, which only look at quantitative differences, not qualitative modulations in the protein.

Dr. Borges presented a study where his group discovered two different biomarkers from the vitamin D binding protein, which serves as biomarker platform for type 2 diabetes. The first biomarker is a genotype of the protein. The team was able to obtain the genotype information from the protein level. “This works because an exact genotype will define an exact amino acid sequence,” Dr. Borges explained. “So when you calculate the exact molecular weight of that sequence, it gives you a target or calculated value.”

The vitamin D binding protein had SNPs in the three common alleles, resulting in mass changes between all three alleles. The mass differences were resolved using mass spectrometry. Dr. Borges continued, “In addition to determining genotype, we’re able to look at additional qualitative features like post-translational modifications that may be present on the protein.” Also, MS provided information on additional peaks, which describe a separate subpopulation of the proteins that are modified with glycosylation.

“By having the MS info, we get the genotype, and the shifted mass peaks give us info on the phenotype.” A pilot study of ten verified biomarkers using this analytical platform will be conducted with a collaborator at the University of Arizona.

Dr. Borges said that ultimately it is the goal to see MS-based diagnostics in the clinic, but that it is going to take time for the medical community to adapt. “I think it has value in terms of being able to monitor these qualitative variants, which may serve as biomarkers, and which are almost impossible to address by other techniques.”

Some of the obstacles encountered in deciphering results from genomic and proteomic investigations include pre-analytical considerations such as sample collection, handling, processing, preservation and use, as well as history of the tissue collection.

Critical Role

James Wittliff, Ph.D., M.D., professor of biochemistry and molecular biology, at the James Graham Brown Cancer Center at the University of Louisville (www.browncancercenter.org), presented guidelines to enhance sample prep.

Dr. Wittliff explained that pre-analytical considerations of tissue specimens don’t get enough attention, and that few investigators gather the history of the specimens. “Use of inadequately prepared and treated specimens in research is more prevalent than many believe, in my opinion.”

Standardization of sample freezing and storage conditions is important to maintain sample integrity. Dr. Wittliff is currently working with the NCI on a new program, the Biospecimen Research Network, that is addressing standardization of tissue collection and handling, as well as extraction and storage protocols.

Lab Proficiency Surveys

Laboratory proficiency surveys are important and should be conducted with reference specimens for genomic and proteomic analyses. Dr. Wittliff set up a QA Survey with the College of American Pathologists for estrogen and progestin receptor assays using standardized tissue-collection protocols and assay procedures, which proved useful in establishing uniformity. “The lessons learned there can be transferred to new applications for genomics and proteomics.”

Two of the main obstacles in deciphering the results from genomic and proteomic investigations include biological (unique genetic composition of each specimen and its cellular heterogeneity) and technical variations. “Laser capture microdissection can significantly help in discovery,” added Dr. Wittliff, “and may contribute to new diagnostics since specific cell types can be collected nondestructively.” Technical variation includes operator-dependent variables, protocol conditions for extracting macromolecules, integrity of RNA/DNA, and methods for handling bioinformatics and statistical analyses.

Sample collection and processing must be compatible with the clinical setting so it does not compromise diagnosis or surgery. Clinical tests have to be easily performed with quality control measures by lab personnel and results presented in an easily interpreted format.

“Clearly, we need to initiate efforts to develop practice guidelines,” stated Dr. Wittliff. He is confident that standardization for this new generation of genomic- and proteomic-based tests is obtainable thanks to efforts by industry and a growing number of investigators in groups like the National Academy of Clinical Biochemistry and the NCI Biospecimen Research Network.

“I feel very strongly that we can make this happen, and we need to get all the parties in the same room. The benefits to successful research outcomes and cost savings are small compared to those that could be realized for improved patient care.”

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