New Directions in Proteomics

15.1. Evolving Techniques, Emerging Technologies

When we ask ourselves what we care about most in analytical proteomics work, two things spring to mind: sensitivity and throughput. Sensitivity is important because proteomics demands analysis of proteins at their natural abundance. In many cases, this forces us to analyze proteins that are present at very low levels. Throughput is important because, to really analyze proteomes (as opposed to proteins), we must be able to perform many analyses as rapidly as possible. Not surprisingly, proteomics technology is evolving toward both improved sensitivity and improved throughput.

These improvements are owing to evolution in both technologies and techniques. In this context, "technologies" refers to those instruments or instrumental approaches that provide fundamental capabilities, such as MS instruments, sources, chromatographic instrumentation, and so on. "Techniques," on the other hand, are the procedures we use to get the most out of the available instrumentation. It is important to distinguish these two areas, because improvements in both will drive future progress in proteomics.

Most of the instrumentation described earlier in this book has been available for at least five years. ESI-LC-MS-MS instruments have been in laboratories since the early 1990s and have enjoyed widespread use since 1996. MALDI-TOF instruments have been in use throughout

From: Introduction to Proteomics: Tools for the New Biology By: D. C. Liebler © Humana Press, Inc., Totowa, NJ

the same period and the new high-resolution TOF analyzers have become widely used over the past five years. The capabilities of MS instruments have improved dramatically over this time period. Indeed, a typical MALDI-TOF or LC-MS system is over an order of magnitude more sensitive than the same instrument sold five years ago. This reflects improvements in mass analyzer technology, ESI and MALDI source design, detector sensitivity, and system electronics.

Much of the improvement in sensitivity for proteomics analyses has come from improved techniques for sample preparation and introduction for both MALDI-TOF and ESI-LC-MS. More efficient protein extraction and digestions produce better yields of proteins and peptides from complex samples. Improved sample cleanup procedures remove contaminating detergents and salts that interfere with ionization and MS analysis. Newer low-flow and low dead-volume LC systems ensure more efficient delivery of small sample amounts to MS instruments, which often perform best in conditions of low flow (see below). Thus, the community of researchers doing proteomics work and analytical protein biochemistry continues to develop better techniques that provide better sensitivity and, in some cases, better throughput.

These changes will continue to make proteome analyses more sensitive and will fuel continued rapid growth of proteomics. Nevertheless, other emerging technologies will produce even more impressive improvements in our ability to analyze proteomes. Four areas of emerging technologies are highlighted in the following sections.

15.2. New MS Instruments

Instrumentation for MS analysis of peptides and proteins is evolving at an impressive rate. This evolution involves both techniques and technologies. Although the sensitivity of mass analyzers and detectors has improved significantly, additional improvements in sensitivity have come from new approaches to sample preparation and introduction. Most notable among these "front-end" innovations is the introduction of ultra-low flow sample inlet systems for ESI-LC-MS instruments. The term "nanospray" is most commonly used to describe these techniques, in which sample flows into the ESI source are in the range of 50-500 nanoliters per minute (as opposed to 50-500 microliters per minute with more commonly used narrow-bore columns). A reduced flow rate for sample introduction results in more efficient transfer of peptide ions from the solution to the mass analyzer. This allows MS-MS analyses to be done on samples in the high attomole to low femtomole range, which is about two orders of magnitude lower than can be done with higher flow systems. Nanospray may be done with fused silica capillaries that are packed with HPLC separation media and thus function both as LC column and electrospray needle. Alternatively, samples may be simply loaded into unpacked nanospray needles and the peptides in the needle then are directed into the MS without in-line separation. In the application of multidimensional chromatography to tandem LC-MS-MS analyses, Yates and colleagues use fused silica capillaries packed with both ion exchange and RP LC separation media (see Chapter 4). Nanospray sources have been in use by many proteomics laboratories for the past 2-3 years. However, more widespread use of this approach has been limited in the past by the relatively delicate nature of available nanospray sources, the difficulty in interfacing these sources with automation tools (e.g., autosamplers), and a general lack of robustness of the available instrumentation. The outstanding sensitivity advantage offered by nanospray has led to the recent introduction of more reliable, user friendly commercial nanospray sources and accessories. The increasingly widespread use of nanospray techniques suggests that this will become the default LC-MS-MS mode for proteomics analyses.

Among the newer, more powerful MS instruments mentioned at the end of Chapter 6 are the Q-TOF (quadrupole-time of flight) and FT (Fourier transform) mass analyzers. FT instruments offer the ultimate in mass resolution and have been used increasingly to analyze complex mixtures of peptides. Accurate, high-resolution measurements of the masses of peptides in a complex mixture can permit protein identification by a variant of the peptide mass fingerprinting approach discussed in Chapter 7. Known peptide masses, termed "accurate mass tags" in FT analyses, can be used as unique identifiers in some cases. In principle, the accurate mass tag approach to proteome analysis can be very powerful and comprehensive. Major limitations to wider use of FT instruments are their great expense and rather delicate nature.

Q-TOF MS instruments (most commonly equipped with ESI sources) are becoming very widely used in proteomics work. A key advantage of the Q-TOF over ion traps and triple quad instruments is high mass resolution provided by the TOF mass analyzer. This provides higher resolution and (with an appropriate calibration) high mass accuracy, which enables easier de novo sequence interpretation from peptide MS-MS data. High accuracy for precursor ion selection and product ion analysis greatly facilitates accurate identification of protein sequences from MS-MS spectra with database correlation algorithms (see Chapter 9). Q-TOF instruments also offer high sensitivity that equals or exceeds that of the best ion trap and MALDI-TOF instruments. A recent extension of Q-TOF technology is the recent coupling of this mass analyzer to a MALDI rather that ESI source. This hybrid couples the advantages of MALDI ionization with the ability to perform true MS-MS analyses. This is in contrast to most MALDI-TOF instruments, which cannot do true MS-MS (see Chapter 6). Application of new quadrupole designs has recently given rise to a new generation of triple quadrupole instruments that will rival Q-TOF mass analyzers for resolution, mass accuracy, and sensitivity.

In addition to these improvements or further developments of existing MS technologies, new tandem mass analyzers are emerging. Most interesting among these is the TOF-TOF mass analyzer, which is a tandem mass analyzer in which two different time of flight analyzers are used for high-resolution precursor selection and product ion detection. In contrast to MALDI-TOF instruments, which cannot perform true MS-MS experiments, the TOF-TOF analyzer offers the prospect of high-throughput, MALDI-based MS-MS with exceptional resolution for precursor and product ions in MS-MS.

Another noteworthy development is the application of MS as a "virtual imaging" approach to the analysis of protein distributions in cell and tissue samples. Recent work indicates that it is possible to blot tissue slices onto a polyethylene membrane, coat with a MALDI matrix, and then perform a series of MALDI analyses distributed over the surface of the blot. Alignment of an ordered series of MALDI laser "shots" and recording of the spectra allows spectral patterns of peptide and protein masses to be recorded over the entire blot surface. Representation of the data for any particular mass will then indicate the spatial distribution of the corresponding protein or peptide in the tissue slice. Further development of this technology and eventual integration with tandem mass analyzers offers a powerful new tool to integrate proteomics and imaging in biological samples.

15.3. Automation and Robotics

In describing the analytical proteomics techniques in this book, we have focused on fundamental aspects. For example, in the analysis of 2D gels, we may select a number of protein spots for MS analysis. This implies that we will cut out each spot, subject each of these samples to in-gel digestion and MS analysis of the resulting peptides, then analyze the data with appropriate software. Although this approach is workable, it is limited in throughput. Moreover, individual differences in sample quality arise through inevitable variability in manual sample processing. Of course, the answer to this problem is the automation of as many steps in the analytical process as possible. Indeed, this is being done to an increasing extent to enhance the speed and reliability of proteome analyses.

In the case of 2D gels, several companies sell software to facilitate automated imaging of the protein spots in gels. Spot selection for subsequent analysis can be automated to a significant degree with the aid of pattern recognition and comparison algorithms. This software then drives automated "spot cutters," which harvest gel pieces and transfer them to robotic apparatus for automated digestion and preparation for MS analysis. In many cases, these robots actually can transfer the prepared samples to MALDI targets or to autosampler vials for LC-MS analyses. The automation of the entire process greatly improves the overall speed of proteome analyses. The high reproduc-ibility of digestion and other sample preparation steps by robots diminishes sample-to-sample variability that inevitably accompanies manual sample preparation. In addition, the software controlling these automated systems provides automated sample tracking and related aspects of quality control, which is crucial to high-throughput analyses.

Postanalysis automation facilitates the analysis of data. For example, automated processing of MS datafiles permits completely automated or semi-automated protein identification from the data as it is being collected. Of course, the task of reviewing and interpreting the results of these analyses will always be with us. However, the power of automation tools for sample preparation, analysis, and data mining and organization is essential to the large-scale analysis of proteomes.

15.4. Micro- and Nanoscale Instrumentation

An important emerging theme in virtually all areas of technology is miniaturization. Miniature-scale technology is particularly applicable to high-sensitivity analytical work, because it brings the scale of the analytical tools closer to that of the targets of our analyses: proteins and peptides in cells. A major inefficiency in most of the techniques described in this book is that we are attempting to analyze picomoles or femtomoles of peptides in columns, gels, and MS sources with micrometer to millimeter internal dimensions. This difference in scale is like rolling a handful of marbles down the street and trying to recover them all at the other end. Losses are inevitable due to the many surfaces and components that peptides may interact with. Indeed, multiple material transfer steps (pipetting, chromatography, elution from gels, etc.) all present opportunities for peptides to be lost.

The idea of applying micro- or nanoscale separations and instrumentation is an effort to minimize the scale difference between analytes and apparatus and to minimize inefficiencies in analyses. Thus, much attention has recently been given to the development of microfluidic devices for extracting, digesting, and otherwise preparing proteins and peptides for MS analysis. Volumes for sample application to such devices are in the picoliter to microliter range. Numerous prototype devices have been reported in the public literature and others are in proprietary commercial development. A common characteristic of many of these devices is their construction on silicon chips similar to those used for microcircuits. This facilitates incorporation of electronic controls and of detectors into the devices.

New microscale analytical sample preparation or separation devices often employ parallel designs to facilitate the simultaneous processing and analysis of a number of samples at the same time. This addresses another imperative of proteome analysis: high throughput. As noted in earlier chapters, proteomics as described in much of this book fails to meet the standards for highly parallel analyses established by microarrays. Highly parallel devices that can facilitate digestion, separations, and MS analyses of multiple samples can greatly increase the speed of proteome analyses. Finally, microscale MS sources are being developed to efficiently couple microscale peptide separation devices to mass analyzers. Ionization methods used in prototypes for such sources include both MALDI and ESI. The sensitivity advantage offered by microscale sources is similar to that offered by nanospray. These microscale ionization sources offer highly efficient translation of sample ions to the mass analyzer.

15.5. Protein Arrays

Certainly the ultimate proteomics match to DNA microarrays would be protein arrays. Unfortunately, there is a major intrinsic problem with this approach. The basis for oligonucleotide array technology is the hybridization of complementary sequences via Watson-Crick base pairing. Unfortunately, proteins do not hybridize to complementary sequences. Thus, the one-to-one correspondence between targets and probes that makes oligonucleotide microarrays work is unavailable to proteomics researchers. Nevertheless, protein analysis by selective interaction of proteins or peptides with an array of different recognition elements is in development in a number of laboratories.

There are a number of different possible recognition elements for proteins. These range from relatively nonselective to highly selective recognition molecules. The former include ion exchange media, which bind proteins or peptides on the basis of charge under specific solution conditions, and immobilized metal affinity ligands, which recognize some protein functional groups, such as phosphoserine, phosphothreonine, and phosphotyrosine residues. The latter include antibodies, which are directed against specific proteins. MAbs directed against specific protein-sequence epitopes display the greatest selectivity for their protein targets. Nucleic acid aptamers represent another highly selective recognition element for proteins or peptides. Aptamers arise from the fact that different oligonucleotide sequences for unique arrangements of hydrogen-bonding donors and acceptors in three dimensional space. Thus, different oligonucleotide sequences may specifically bind to specific protein or peptide structural motifs.

These and other various recognition elements have been employed in proteome analyses as a step to extract specific protein or peptides from complex mixtures. Indeed, this approach has been extensively developed by Ciphergen Biosystems (www.ciphergen.com), which offers a large variety of customized surface "chips" for protein capture prior to MALDI-TOF MS analysis. Thus, the use of a relatively nonspecific capture surface harvests diverse proteins, whereas a more specific surface chemistry (e.g., a MAb) may trap only a single protein and some of its variants. Although the MALDI-TOF analysis of the intact proteins does not offer definitive identification, the analysis of changing "patterns" of proteins captured can provide a basis for more in-depth investigation.

Another way that arrays of protein recognition elements will impact proteomics is as an alternative to MS analysis, rather than as a frontend to capture proteins for MS. For example, arrays containing many different antibodies may be used to capture a diverse collection of proteins to which the antibodies are targeted. Use of non-MS detection strategies (e.g., secondary antibody labeling with fluorescent tags) can provide a very sensitive, high-throughput screen for the presence of specific proteins. Of course, the success of this approach depends on the specificity and affinity of the antibodies for their protein targets, on the effect of the antibody-attachment chemistry on antibody efficiency, and on the stringency of conditions under which the antibodies bind their protein targets. Related approaches also are evolving. For example, highly specific aptamer arrays can be envisioned, as the technology for aptamer generation and characterization is improving. The development of a high-throughput means of generating aptamers directed against specific proteins, peptides, or their modified variants could enable the construction of printed ologonucleotide arrays that are used for large-scale proteome analyses.

Another aspect of array approaches for proteomics is that they may serve as tools for more than mining proteomes. Arrays of specific proteins provide the opportunity to perform highly parallel studies of protein-protein interactions and how these interactions are affected by drugs and other chemical or physical factors. In this way, arrays of proteins printed on glass slides or in multiwell plates could be used to study protein-protein or protein-drug interactions in well-defined environments. Subsequent analyses of members of complexes or of protein modifications on individual elements of the arrays could then be performed with the MS tools described earlier in this book.

Suggested Reading

Baldwin, M. A., Medzihradsky, K. F., Lock, C. M., Fisher, B., Settineri, T. A., and Burlingame, A. L. (2001) Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification and structural analysis. Anal. Chem. 73, 1707-1720.

Chaurand, P., Stoeckli, M., and Caprioli, R. M. (1999) Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry. Anal. Chem. 71, 5263-5270.

Conrads, T. P., Alving, K., Veenstra, T. D., Belov, M. E., Anderson, G. A., Anderson, D. J., et al. (2001) Quantitative proteome analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling. Anal. Chem. 73, 2132-2139.

Figeys, D. and Pinto, D. (2000) Lab-on-a-chip: a revolution in biological and medical sciences. Anal. Chem. 72, 330A-335A.

Lopez, M. F. (2000) Better approaches to finding the needle in a haystack: optimizing proteome analysis through automation. Electrophoresis 21, 1082-1093.

MacBeth, G. and Schreiber, S. (2001) Printing proteins as microarrays for high-throughput function determination. Science 289, 1760-1763.

Medzihradsky, K. F., Campbell, J. M., Baldwin, M. A., Falick, A. M., Juhasz, P., Vestal, M. L., and Burlingame, A. L. (2000) The characteristics of peptide collision-induced dissociation using a high performance MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 72, 552-558.

Zhu, H., Biolgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R. A., Gerstein, M. and Snyder, M. (2001) Global analysis of protein activities using proteome chips. Science 293, 2101-2105.

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