Medical Device Link.

Originally Published IVDT September 2002

MOLECULAR DIAGNOSTICS

Recent advancements in proteome analysis will help in the development of diagnostic tools.

Louisa B. Tabatabai

Proteomics, or analysis of the proteome, offers a relatively new approach to protein expression profiling and cellular or tissue protein identification from samples that are obtained under various specified conditions.¹ Proteome analysis allows the investigator to obtain information on protein identity, protein-protein interaction, the level of protein expression and protein expression profiling, protein trafficking and turnover, protein variants, and protein post-translational modifications.

Traditionally, proteomics combines two-dimensional electrophoresis (2-DE), a high-resolution protein separation technique, with mass spectrometry (MS)—either matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF), electrospray ionization (ESI) mass spectrometry, or hybrid instrumentation.^2-7 Even though the 2-DE technique has inherent limitations, developments in 2-DE procedures and new techniques in sample preparation have overcome most of these obstacles that arose due to low protein abundance, the dynamic range of proteins to be separated, and solubilization of integral membrane proteins. The advantage of the 2-DE approach is that it is a high-resolution separation technique that can provide a snapshot of the cell's protein expression status and will likely remain a useful tool for quantitative comparisons of protein profiles.

Because of the need to automate fully the entire proteome analysis process, new approaches have been explored, including on-line multidimensional liquid chromatography and affinity chromatography methodologies for protein or peptide separation, followed by mass spectrometry.^8,9 Most of the gel- and chromatography-based approaches have been used for the discovery phase in research applications. For commercial applications, the direction appears to be toward developing disease-specific biomarker protein chips (see Table I).¹⁰

This article examines proteomics methodologies as well as alternative technologies, including biomolecular interaction analysis–mass spectrometry (BIA-MS) and surface-enhanced laser desorption-ionization (SELDI), which in conjunction with protein chips has commercial applications for disease biomarker detection (see Table I).^11,12

Proteomics Methodologies

Protein expression profiling using 2-DE is an old technique that was first published in 1975.¹ Since then, improvements to the technique have been reviewed recently.^3,13 With the introduction of commercially produced immobilized pH gradient (IPG) strips—together with improved protein solubilization techniques—comparisons could be made between laboratories, and the basic 2-DE technique became widely used for proteomics research.

The potential and application of 2-DE to proteomics research became a reality because of developments in other techniques, such as genomic sequencing, protein microsequencing and mass spectrometry, and associated database bioinformatics. For the first time, one could examine cells or tissues grown under different conditions (e.g., nutrient supplementation and deprivation), evaluate cellular protein changes as a result of drug administration, examine protein changes in normal versus diseased tissues, and evaluate the total proteome of an organism for establishing baseline parameters. Once the basic genomic information was available in various databases, proteins could be identified based on either accurate fragment molecular weight analysis by mass spectrometry and database searching, or a combination of fragment analysis and sequencing by tandem mass spectrometry followed by database searching (see Table II). Thus, the new field of proteomics emerged.

Sample Preparation

Sample preparation is one of the most critical steps in proteome analysis, whether the approach is protein separation by 2-DE or liquid chromatography (LC). Typically, membrane proteins are the most difficult proteins to solubilize and release into an aqueous environment. For 2-DE separations, the first-dimension isoelectric-focusing separation step depends on the intrinsic charge of the polypeptide. Reagents such as sodium dodecyl sulfate, which results in a charge shift and is normally used to solubilize membrane proteins for 1-DE separations, is not appropriate for the isoelectric-focusing step. New reagents have been introduced that are compatible with both the 2-DE and LC techniques, including chaotropes, surfactants, and reducing reagents.¹³

Protein solubilization solutions that contain both urea and thiourea have improved solubilization of integral membrane proteins, compared with standard solubilization solutions without thiourea.¹⁴ Surfactants are also used in solubilization solutions because they act synergistically with the chaotropes to solubilize membrane proteins. Surfactants are necessary because chaotropic agents unfold proteins, thereby exposing hydrophobic regions of the protein which can cause aggregation, precipitation, or adsorption to the IPG or column matrix. The surfactant therefore binds to the hydrophobic domains, keeping the protein solubilized.

Surfactants may be nonionic and are normally used at concentrations between 0.4 and 4%. However, zwitterionic surfactants and other sulfobetaines are preferred because of their excellent solubilization properties, compared with nonionic surfactants, and are used at concentrations ranging from 0.5 to 4%.³ While one drawback of most sulfobetaines is their low compatibility with urea, a new sulfobetaine that contains a carboxy-amido group and a quaternary ammonium group separated by a three-carbon spacer, has been introduced and has proven to be superior for use in high-urea-containing solubilization solutions for membrane proteins.¹⁵

Another reagent that can be used to solubilize proteins is a reducing agent which cleaves intra- and intermolecular disulfide bonds. Reducing disulfide bonds is critical to the solubilization of proteins, and the most commonly used reducing agents are dithiothreitol (DTT) or dithioerythritol. Once the reduction of the protein disulfides has been completed, these dithiols form stable cyclic disulfides, therefore low concentrations of 2 mmol per liter usually suffice. The use of a newer reducing agent, tributyl phosphine (TBP), in 2-DE has been documented. According to studies, TBP provided even better protein solubility properties and reduced the streaking that is sometimes observed with DTT.¹⁶

Differential solubilization of proteins using a sodium carbonate wash also resulted in improved representation of outer membrane proteins.¹⁵ Other prefractionation methods have been described as well, including narrow-range isoelectric focusing strips and a trichloroacetic acid precipitation step to enhance the representation of low-abundance proteins that are present in mouse liver.³

Applications of Proteomics Technologies

Immunoblotting procedures can be used to identify subsets of microbial proteins. In this manner, proteins that are important in the immune response of the host to the pathogen have been identified.¹⁷ Another important factor for developing diagnostic proteins and designing vaccines is understanding the differences between virulent and attenuated strains of pathogens. A comparison of the proteomes of the virulent M. tuberculosis with the attenuated M. bovis organisms identified 56 protein spots in the virulent strain and 40 spots in the attenuated strain.¹⁸ Several vaccine candidates were also identified by using the comparative or subtractive proteome analysis approach.

Figure 1. A comparative analysis in the minigel format of H. parasuis serovar 4 (a) and serovar 5 (b). The numbers indicate the proteins that are present in both serovars: antigenic heat-stable protein (1), xanthine dehydrogenase (2), and neuraminidase (3). The lower-case letters (a, b, c, and d) indicate the proteins that react with pig sera from infected animals. Only one immunoreactive protein (d) is similar between the two serovars. (click to enlarge)

Similarly, exposing the microorganisms to temperature changes as well as to organic and inorganic chemicals will induce a cellular adaptation response, which can be measured with proteomics analysis. One example is a comparative 2-DE study in the minigel format of Haemophilus parasuis spheroplasts (see Figure 1). A MALDI-TOF mass spectrum of a tryptic digest of one of the proteins, neuraminidase, has been done (see Figure 2).

An interesting study in comparative proteomics showed the time-dependent expression of proteins in response to infection by a viral agent.¹⁹ This study showed that infection of alveolar macrophages with African swine fever virus produces a general shutdown of cellular protein synthesis which affects approximately 65% of the total proteins (152 proteins) over a 6-hour period. The expression of new viral proteins (100 proteins) was observed over the same time period.

The 2-DE method has numerous other applications, particularly in disease biomarker identification (see Table III).^20–23 For example, comparisons of normal tissues with tumor tissues identify both up- and down-regulated proteins. The up regulated proteins are studied further for their diagnostic potential (see Table I).

Alternative Methods to 2-DE

In the mid-1990s, as other approaches to improve 2-DE technologies were being developed, alternative methods to 2-DE were investigated. These alternatives were designed to overcome various problems, such as protein solubility and low-abundance proteins.

Figure 2. A MALDI-TOF mass spectrum of a tryptic digest of neuraminidase. (click to enlarge)

Affinity Capture of Proteins. One of these alternatives is based on the affinity capture of labeled protein digests prior to an analysis by mass spectrometry. This technology is also referred to as the isotope-coded affinity tag (ICAT) procedure and is based on the use of a new sulfhydryl reagent, N-(13-amino-4,7,10-trioxatridecanyl) biotinamide, which modifies cysteinyl residues.⁹ The linker reagent between the thiol-specific moiety and the biotin moiety is either in the light form (eight hydrogen atoms) or heavy form (eight deuterium atoms). For a singly charged adduct, the heavy peptide would be eight mass units heavier than its light form.

Protein extracts in two different states are covalently modified with either the light form or the heavy form of the reagent. The protein samples are mixed and digested with trypsin, and the labeled peptides are separated by avidin-affinity chromatography. A full mass spectrum then identifies all doublets of peptides that differ by four or eight mass units (doubly or singly charged peptides, respectively). The relative abundance of each peptide in a doublet can be accurately quantified, providing an indication of either up- or down-regulation of a particular protein. Selected peptide ions are then subjected to µLC-MS/MS for sequence determination. This approach eliminates the problems that are posed by low-abundance proteins because the affinity selection process is neutral to concentration effects.

An application of the ICAT technique for cancer biomarker discovery seems promising.²⁴ However, this approach to protein analysis unfortunately has its drawbacks, as it has been estimated that one in seven proteins does not contain cysteinyl residues.

Multidimensional Protein Identification Technology. A different approach to 2-DE was used for a large-scale analysis of the yeast proteome.²⁵ While cells were fractionally solubilized and digested sequentially with endoproteinase Lys-C and immobilized trypsin, the insoluble fraction was digested with cyanogen bromide. All samples were then subjected to multidimensional protein identification technology (MUDPIT), which involves a sequential separation of the peptide fragments by on-line biphasic microcapillary chromatography (strong ion exchange, then C-18 separation), followed by tandem mass spectrometry (MS-MS).²⁵ A total of 1484 proteins were detected and analyzed, including 131 proteins with three or more transmembrane domains. These domains were typically difficult to identify before the new 2-DE solubilization techniques were implemented.¹³

Figure 3. A partial map of UV versus pI protein profiles of a colon cancer cell line. The protein profiles were obtained from untreated and treated colon cancer cell preparations. The far left panel shows UV absorbance of pI 6.2–6.0 chromatofocusing fraction. Column 1 of the untreated panel shows UV absorbance of pI 6.2–6.0 chromatofocusing fraction from untreated cells. Column 1 of the difference panel is the difference profile between the untreated and treated panels and shows UV absorbance of pI 6.2–6.0 chromatofocusing fraction. Column 1 of the treated panel shows UV absorbance of pI 6.2–6.0 chromatofocusing fraction from treated cells. The far right panel shows UV absorbance of pI 6.2–6.0 chromatofocusing fraction from treated cells. The stars indicate the bands of potential diagnostic interest. (click to enlarge)

Another recently developed alternative to 2-DE is ProteoSep, a commercialized application by Eprogen Inc. (Darien, IL). This technology separates groups of proteins based on their iso-electric pH (pI) using chromatofocusing, followed by reverse-phase high-performance LC of each fractionated pH range. The advantage of this up-front protein separation technology is that the proteins remain solubilized at all times, allowing direct mass spectrometry. A UV/pI map of protein profiles has been obtained from a treated and untreated colon cancer cell line (see Figure 3).

Other Alternative Technologies

Alternative technologies to 2-DE, MUDPIT, and ICAT for quantifying protein expression, detection, and analysis have been developed in order to identify specific proteins for commercial applications. These technologies address issues involving multiple protein interactions in signaling pathways as well as applications in automated disease biomarker detection and analysis, including developments of protein microarrays.

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a suitable technique for detecting interacting protein complexes.²⁶ This technology detects bound molecules with a ligand that is covalently attached to a surface. As the density of biomaterial on the surface increases, changes occur in the refractive index at the solution or surface interface. This change in the refractive index is detected by varying the angle or wavelength at which the incident light is absorbed at the surface. The difference in the angle or wavelength is proportional to the amount of material bound on the surface, giving rise to a signal that is termed surface plasmon resonance (SPR).

The SPR biosensing technology has been combined with MALDI-TOF mass spectrometry for the desorption and identification of biomolecules. This technology has been used to demonstrate the analysis of multiprotein complexes bound by immobilized antibodies, ligands for receptor complexes, and similar biomolecular capture and interactions of proteins in signaling pathways. Although this proteomics technology has potential commercial applications, it is currently used only in research laboratories.

In a chip-based approach to BIA-MS, a ligand or receptor is covalently immobilized on the surface of a chip.²⁶ A tryptic digest of solubilized proteins is routed over the chip, and the relevant peptides are bound by the ligand. After a washing step, the eluted peptides are analyzed by MALDI-TOF mass spectrometry. The advantage of this system is that it lends itself to a fully automated process and is applicable to detecting and characterizing proteins present in complex biological fluids and cell extracts at low- to subfemtomol levels. This technology is currently being developed for detecting diagnostic proteins.

A similar approach is surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS).¹² Using this technique, both chemically derived surfaces (i.e., hydrophobic, ionic, metal affinity, or mixed modes) and biochemically derived surfaces (i.e., antibody, DNA, enzyme, receptor) have been prepared. In addition to providing the basis for protein chip technology, SELDI-TOF MS applications include antibody-mediated capture of femtomol amounts of tumor necrosis factor-a and detection of prostate cancer–specific proteins using either an Ni(II)-modified surface or a hydrophobic surface to capture the proteins.¹² Applications of this technology are also undergoing clinical trials for detecting ovarian cancer (see Table I).

Laser capture microdissection (LCM) is another technology that has been employed to address the difficulties with heterogeneous cell types in tissue samples. Often the relevant cell types of the tissue to be examined are available only in small amounts. Tissue extracts could mask the particular changes in the protein expression or disease markers of the relevant cell type. However, by using LCM, dissected material can be solubilized in an appropriate protein solubilization cocktail for subsequent analysis by either 2-DE or SELDI. The applications and limitations of LCM in conjunction with 2-DE or SELDI have been reviewed in the literature.²⁷

Protein Expression Profiling

While protein microarrays have been developed for miniaturized protein expression assays to determine protein function, the application and potential of this technology for protein identification is enormous. Methodologies have been developed for protein spotting on glass slides treated with an aldehyde-containing silane reagent or bovine serum albumin-N-hydroxysuccinimide.²⁸ Applications have also been developed for detecting cancer biomarkers using a spotting technology which deposits 1600 spots per square cm.²⁹

Mass Spectrometry Instrumentation

Mass spectrometry has become a major analytical tool for proteomics research, mainly because of recent advancements in the instrumentation used for biomolecular ionization, electrospray ionization (ESI), and matrix-assisted laser desorption-ionization (MALDI).³⁰ MALDI is usually combined with a time-of-flight (TOF) mass analyzer, which has an unlimited mass-to-charge ratio range and is highly efficient.⁴ Typically, 0.5 µ1 of sample that contains 1–10 pmol of protein or peptide is mixed with an equal volume of a saturated matrix solution and allowed to dry. This process results in the co-crystallization of the analyte with the matrix. Typical matrix compounds that are used include sinapic acid with proteins and a-hydroxycinnamic acid with peptides. The cocrystallized material on the target plate is irradiated with a nitrogen laser pulse at a wavelength of 337 nm, resulting in volatilization and ionization of the protein or peptide molecules. A strong acceleration field is switched on, and the kinetic energy of the ions causes the ionized molecules to travel down the flight tube to a detector. The amount of time required to reach the detector is related to the mass-to-charge ratio.

ESI has most often been used with quadrupole mass filters or ion traps, a common configuration being the ESI triple-quadrupole combination.⁶ The recent introduction of orthogonal injection has also made it possible to couple an ESI source directly to a TOF instrument.⁷ In ESI, a liquid is pumped through a hypodermic needle at low microliter- or nanoliter-per-minute flow rates that are maintained at high voltage and atmospheric pressure. This flow rate causes the liquid to disperse electrostatically into small micrometer-sized droplets that evaporate rapidly, causing the analyte to become charged. Since this process does not cause fragmentation of the analyte, the ionized molecules enter the vacuum system and are focused into the first quadrupole section. They can then be mass-separated in the second quadrupole section, and selected ions can be dissociated in the third quadrupole section before being transferred to the TOF mass analyzer.

Some improvements that have been introduced to this technology include a collision-induced cell to produce peptide fragments, which are subsequently analyzed by MS-MS for determining peptide sequence.³¹

The latest technological development for high-throughput proteomics and mass spectrometry is the Voyager TOF-TOF, a tandem time-of-flight (TOF-TOF) mass spectrometer developed by Applied Biosystems (Foster City, CA). Other tandem TOF systems that are being developed include the UltraFlex by Bruker Daltronics (Billerica, MA) and the Ettan by Amersham Pharmacia (Sunnyvale, CA).

Conclusion

In the present postgenomics era, technological developments in sample solubilization and separation procedures, advancements in instrumentation for biomolecular interaction measurements, protein microarray technologies, cell microdissection, and mass spectrometry instrumentation, have given researchers unique tools to begin understanding the complexities of the cell's proteome. As the cell's proteome becomes defined in terms of its normal and altered states, developing diagnostic tools becomes a reality. An example is the development of a protein-chip-based technology for detecting ovarian cancer, which is currently in clinical trials. Protein-chip-based technologies for disease biomarker detection should be an achievable goal within the next decade.

References

1. MR Wilkins et al., "Progress with Proteome Projects: Why All Proteins Expressed by a Genome Should Be Identified and How to Do It," Biotechnology and Genetic Engineering Review 13 (1995): 19–50.

2. PH O'Farrell, "High Resolution Two-Dimensional Electrophoresis of Proteins," Journal of Biological Chemistry 250 (1975): 4007–4021.

3. A Görg et al., "The Current State of Two-Dimensional Electrophoresis with Immobilized pH Gradients," Electrophoresis 21 (2000): 1037–1053.

4. M Karas and F Hillenkamp, "Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons," Analytical Chemistry 60 (1988): 2299–2301.

5. JB Fenn et al., "Electrospray Ionization for Mass Spectrometry of Large Biomolecules," Science 246 (1989): 65–71.

6. AV Loboda et al., "A Tandem Quadrupole/ Time-of-Flight Mass Spectrometer with a Matrix-Assisted Laser Desorption/Ionization Source: Design and Performance," Rapid Communications in Mass Spectrometry 14 (2000): 1047–1057.

7. MA Baldwin et al., "Matrix-Assisted Laser Desorption/Ionization Coupled with Quadrupole/ Orthogonal Acceleration Time-of-Flight Mass Spectrometry for Protein Discovery, Identification, and Structural Analysis," Analytical Chemistry 73 (2001): 1707–1720.

8. H Liu, D Lin, and JR Yates III, "Multidimensional Separations for Protein/Peptide Analysis in the Post-Genomic Era," Biotechniques 32, no. 4 (2002): 898–911.

9. F Turecek, "Mass Spectrometry in Coupling with Affinity Capture-Release and Isotope-Coded Affinity Tags for Quantitative Protein Analysis," Journal of Mass Spectrometry 37 (2002): 1–14.

10. CD Rathin, "Progress and Partnerships in Proteomics," American Biotechnology Laboratory February (2002): 42–48.

11. RW Nelson et al., "BIA/MS of Epitope-Tagged Peptides Directly from E. coli Lysate: Multiplex Detection and Protein Identification at Low-Femtomole to Subfemtomole Levels," Analytical Chemistry 71 (1999): 2858–2865.

12. M Merchant and SR Weinberger, "Recent Advancements in Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry," Electrophoresis 21 (2000): 1164–1177.

13. MP Molloy et al., "Proteomic Analysis of the Escherichia coli Outer Membrane," European Journal of Biochemistry 267 (2000): 2871–2881.

14. T Rabilloud, "Use of Thiourea to Increase the Solubility of Membrane Proteins in Two-Dimensional Electrophoresis," Electrophoresis 19 (1998): 758–760.

15. MP Molloy et al., "Two-Dimensional Electrophoresis and Peptide Mass Fingerprinting of Bacterial Outer Membrane Proteins," Electrophoresis 22 (2001): 1686–1696.

16. BR Herbert et al., "Improved Protein Solubility in Two-Dimensional Electrophoresis Using Tributyl Phosphone as Reducing Agent," Electrophoresis 19 (1998): 845–851.

17. PR Jungblut, "Proteome Analysis of Bacterial Pathogens," Microbes & Infection 3 (2001): 831–840.

18. J Mattow et al., "Identification of Proteins from Mycobacterium Tuberculosis Missing in Attenuated Mycobacterium bovis BCG Strains," Electrophoresis 21 (2001): 2936–2946.

19. JM Rodriguez et al., "African Swine Fever Virus-Induced Polypeptides in Porcine Alveolar Macrophages and in Vero Cells: Two-Dimensional Analysis," Proteomics 1 (2001): 1441–1446.

20. SJ Schonberger et al., "Proteomic Analysis of the Brain in Alzheimer's Disease: Molecular Phenotype of a Complex Disease Process," Proteomics 1 (2000): 1519–1528.

21. M Ostergaard et al., "Psoriasin (S100A7): A Putative Urinary Marker for the Follow-Up Patients with Bladder Squamous Cell Carcinomas," Electrophoresis 20 (1999): 2100–2110.

22. SC Prasad et al., "Protein Expression Changes Associated with Radiation-Induced Neoplastic Progression of Human Prostate Epithelial Cells," Electrophoresis 18 (1997): 629–637.

23. SM Hanash et al., "Integrating Cancer Genomics and Proteomics in the Post-Genome Era," Proteomics 2 (2002): 69–75.

24.DB Martin, and PS Nelson, "From Genomics to Proteomics: Techniques and Applications in Cancer Research," Trends in Cell Biology 11 (2001):S60–S65.

25. MP Washburn, D Wolter, and JR Yates, "Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology," Nature Biotechnology 19 (2001): 242–247.

26. D Nedelkov and RW Nelson, "Analysis of Native Proteins from Biological Fluids by Biomolecular Interaction Analysis Mass Spectrometry (BIA/MS): Exploring the Limit of Detection, Identification of Non-Specific Binding and Detection of Multiprotein Complexes," Biosensors and Bioelectronics 16 (2001): 1071–1078.

27. RA Craven and RE Banks, "Laser Capture Microdissection and Proteomics: Possibilities and Limitation," Proteomics, 1 (2001): 1200–1204.

28. G MacBeath and SL Schreiber, "Printing Proteins as Microarrays for High-Throughput Function Determination," Science 289 (2000): 1760–1763.

29. J Madoz-Gdrpide, H Wang, and DE Misek, "Protein-Based Microarrays: A Tool for Probing the Proteome of Cancer Cells and Tissues," Proteomics 1 (2001): 1279–1287.

30. S Patterson, "Mass Spectrometry and Proteomics," Physiological Genomics 2 (2000): 59–65.

31. A Schevchenko et al., "Maldi Quadrupole Time-of-Flight Mass Spectrometry: A Powerful Tool for Proteomic Research," Analytical Chemistry 72 (2000):2132–2141.

Louisa B. Tabatabai, PhD, is a research chemist at the National Animal Disease Center (Ames, IA) and a professor of biochemistry at Iowa State University (Ames, IA). She can be reached via lbt@iastate.edu.

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