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Perspective: Proteomics—See "Spots" Run

Edison Biotechnology Institute (J.J.K., E.O.L., G.M.O.K., S.O.), School of Physical Therapy and Program in Neuroscience (D.T.K.), Molecular and Cellular Biology Program (J.J.K., L.Q.), Department of Biomedical Sciences, College of Osteopathic Medicine (J.J.K., S.O.), Ohio University, Athens, Ohio 45701

Address all correspondence and requests for reprints to: John J. Kopchick, Edison Biotechnology Institute, Ohio University, 101 Konneker Research Labs, Building 25, The Ridges, Athens, Ohio 45701. E-mail: . kopchick{at}ohio.edu

	Abstract

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 

	Introduction

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
The recent explosion of data obtained from genomic sequencing and the analysis of transcribed RNAs using automated techniques has provided a valuable knowledge base from which every field of biology can benefit. While this information serves as a beginning for the understanding of tissue-specific gene expression, it does not specifically detail the abundance of proteins nor the posttranslational status of proteins in a given tissue. Protein analysis techniques such as two-dimensional (2-D) electrophoresis coupled with mass spectrometry (MS) have provided valuable insight into both the abundance and posttranslational states of proteins. Hence, the field of proteomics has become the premier arena for the analysis of the functional "proteome." The intent of this review is to give endocrinologists a general overview of proteomics. We will highlight recent technical advances and some practical considerations including sample preparation, 2-D gel electrophoresis, protein detection, and protein identification that will help determine the "proteomic state" of a particular cell type or tissue.

With the nucleotide sequencing of several prokaryotic (1, 2, 3, 4) and eukaryotic (5, 6) organisms now complete or nearly complete, the field of genomics is blossoming and has also spawned a $300 million bioinformatics industry (7). Among the data gleaned from these endeavors is the number of genes that can be transcribed into mRNA. While this is instructive, it is important to remember that mRNAs are not the "functional molecules" of the cell, but rather intermediates between expressed genes and proteins. Thus, quantification of mRNA levels in a particular cell or tissue only serves as a first approximation of the actual levels of the corresponding proteins in the sample. In this regard, several studies have shown that the level of a particular mRNA within cells does not necessarily correlate with the amount of cognate protein (8, 9, 10). In addition, there is an ever-growing class of nontranslated RNAs that do not give rise to protein (11). Therefore, a complete catalog of proteins within a given cell or tissue is required for a better understand its genetic potential.

The term "proteome" (12) or the protein complement of the genome has become popular in the vernacular of biology. "Proteomics" is the systematic analysis of proteins whose expression patterns are correlated in time and space. Ultimately the goal of proteomic research is to identify and establish functional protein networks within cells, tissue, and organs of an individual (13, 14, 15).

The International Human Genome Sequencing Consortium estimates that there are approximately 30,000–40,000 protein-encoding genes in humans (11). However, one gene may encode a variety of protein isoforms due to mechanisms such as alternative splicing or mRNA editing. Moreover, many of the protein differences arise from posttranslational modifications such as glycosylation or phosphorylation reactions. Taken together, the number of unique human proteins has been estimated to be as high as 2,000,000 (16).

Therefore, with such large discrepancies between the number of human genes, mRNAs, and proteins, a direct analyses of the entire spectrum of proteins in a given sample is ultimately the most reliable information one can gain concerning the genetic potential of an individual cell or tissue. The purpose of this review is to highlight several issues important in the analyses of proteins in this area of "proteomics." For more comprehensive discussions in this area, the following articles are suggested (17, 18, 19, 20, 21).

	Sample preparation

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
One of the most problematic areas in the field of proteomics involves establishing optimal protein solubilization conditions. Two of the primary considerations when preparing samples are maximizing protein solubility and minimizing protein alterations. Solubilizing buffers for 2-D gel electrophoresis commonly contain urea, thiourea, nonionic and/or zwitterionic detergent(s), a reducing agent, carrier ampholytes, and protease inhibitors. Because protein solubilization is limited to the composition of the solubilizing buffer, it is important to understand the various components found in these buffers. Hence, a brief description of some of the most common components follows.

Urea is a nonionic chaotrope (a chemical agent that denatures proteins) commonly used for protein solubilization (22). It has good overall solubilizing ability, but it is important to note that in aqueous solutions, a small amount of urea is converted to ammonium cyanate. The ammonium cyanate can carbamylate proteins, which interferes with subsequent MS analysis (23, 24). Hence, carrier ampholytes (scavengers of cyanate) are often added to sample preparations to prevent carbamylation, in addition to their role of creating a pH gradient during subsequent isoelectric focusing (IEF). Thiourea is another nonionic chaotrope that is often added to increase the overall denaturing power of the buffer. A concern when using thiourea is that it interferes with the protein alkylation step by scavenging the alkylating agent, iodoacetamide (25). Therefore, when thiourea is present, it is suggested that iodoacetamide be added in crystalline form (25).

Nonionic and zwitterionic detergents also are added for their ability to solubilize hydrophobic proteins. While certain ionic detergents such as SDS are very strong denaturants, their overall charge causes them to migrate to the poles during IEF and interfere with the focusing process. Ionic detergents, therefore, are either not used, or are used only during initial solubilization steps on samples that are difficult to solubilize and are removed before IEF.

Intermolecular and intramolecular disulfide bonds are usually broken by the addition of a reducing agent to the sample buffer. While several reducing agents are commonly used, including dithiothreitol, 2-mercaptoethanol, and tributylphosphine, tributylphosphine is more efficient at reducing disulfide bonds.

Solubilizing sample buffers must guard against protein degradation and changes in the posttranslational modifications. Because 2-D gel electrophoresis resolves proteins in various posttranslational modified states, and because the presence or absence of a single modification (such as a phosphate group) can dictate the difference between an "active" and an "inactive" protein, it is important to prevent protein modifications from occurring during sample preparation. While detergents and chaotropes are added to sample buffers to denature and solubilize proteins, they also conveniently protect the posttranslational modifications of peptides by denaturing modifying enzymes. And while most protein modifying enzymes are inactivated rapidly by detergents and chaotropes, some proteases are extremely resistant to temperature, urea, and SDS (26, 27). Therefore, protease inhibitors are usually included to prevent any residual protease activity in solubilizing buffers.

Following protein solubilization, the samples should be reduced and the sulfhydryl groups on the cysteine residues should undergo an alkylation reaction. According to several recent studies, it was concluded that reduction/alkylation must be performed to prevent intra- and intermolecular disulfide bond formation, which can result in large numbers of artifactual protein spots (25, 28, 29). In addition, it was found that when reduction/alkylation was performed during the equilibration step (between the first and second electrophoretic dimension), the presence of SDS in the equilibration buffer interfered with the alkylation process (28). Therefore, it is strongly suggested that reduction/alkylation be performed before IEF (25, 28, 29).

	2-D gel electrophoresis

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
After solubilization and following reduction/alkylation of the samples, the proteins are ready to be separated by IEF (the first dimension) and then by SDS-PAGE (the second dimension). It is possible to resolve and analyze more than 10,000 proteins using this 2-D system (30). There are currently two methods to create pH gradients for the first dimension. The first method utilizes carrier ampholytes (CA), which are low molecular weight compounds containing both amino and carboxyl groups each with a distinct isoelectric point. The pH gradient is created when an electronic field is applied to the mixture. A CA gel is usually prepared in a glass tube or alternatively as a slab gel that is sliced into thin strips after IEF.

The second method utilizes molecules called immobilines (which are acrylamide derivatives containing both amino and carboxyl groups) that can be copolymerized with the polyacrylamide gel to generate an immobilized pH gradient (IPG). The pH resolution of IPG is superior to CA-IEF as "drifting" (improper migration of CA during the focusing process) is essentially eliminated when IPG strips are used for focusing. Moreover, IPG strips with very narrow ranges (0.1 pH unit) can be prepared or purchased to facilitate further protein separation. IPG strips are also readily available from commercial sources, which saves time and presumably increases reproducibility. The entire process of IEF can be automated and performed overnight by an integrated system. Following IEF, an equilibration step is performed to coat proteins with SDS molecules before separation by SDS-PAGE.

	Dyes used for visualizing proteins in 2-D gels

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
Traditionally, proteins separated by 2-D gel electrophoresis have been visualized using colloidal Coomassie brilliant blue (Coomassie blue) or silver stain. While Coomassie blue is relatively simple to use and exhibits a broad linear range, its sensitivity is limited (31). Silver stain can be up to one hundred times more sensitive than Coomassie blue (32); however, it does not exhibit a linear dynamic range and can bind differentially to proteins depending on posttranslational modifications (33). In addition, both of these staining methods can interfere with subsequent MS and sequencing analysis (31, 33, 34).

The recent development of fluorescent stains has allowed for the detection of proteins with sensitivities approaching that of silver staining, and linear dynamic ranges greater than that achieved with Coomassie blue (31, 34, 35). Three of these types of stains, SYPRO Red, Ruby, and Orange (Molecular Probes, Inc., Eugene, OR) have proven to be very compatible with subsequent analysis by matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF-MS) and sequencing by liquid chromatography (LC)-tandem MS (33). While SYPRO Ruby is the fluorescent dye recommended by the manufacturer for use with 2-D gels, its cost (due to the presence of the metal ruthenium) may limit its use in some laboratories. However, recent studies at Pharmacia Corp. and in our own laboratory indicate that SYPRO Orange staining of 2-D gels using a modified protocol developed by Malone et al. (35) yields similar results to those obtained with SYPRO Ruby. We routinely use this stain in our laboratory.

	Protein identification by MS

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
MS plays a pivotal role in proteomics for the identification of the protein "spots." The isoelectric point and molecular weight of a protein separated by 2-D gel electrophoresis rarely provides enough of the information required to identify a protein with certainty. MS is used for obtaining attributes of the protein including mass fingerprints (the mass spectrum obtained from digested protein fragments) and partial sequences of protein samples. For mass fingerprinting, MALDI-TOF has proven to be the method of choice for rapid identification following 2-D gel separation due to the high sensitivity, unlimited mass range capabilities, and ease of use (36, 37).

MALDI is a method by which nonvolatile biological samples are vaporized and ionized from a solid-state phase directly into the gas phase. First, excised gel spots are digested with trypsin. The peptide fragment mixture is then desalted before MS analysis. Samples for MALDI-TOF are prepared by combining the peptide fragments with small organic compounds (such as sinapinic acid and -cyano-4-hydroxycinnamic acid) referred to as the matrix. The mixture is dried on a target plate. A laser beam then serves as both desorption and ionization sources in MALDI. As the matrix absorbs the laser light energy, the peptides are vaporized and ionized. The charged molecules are transferred electrostatically into a vacuum TOF-MS chamber. Their flight time through the vacuum tube is proportional to the square root of their mass-to-charge ratios. The fragments are analyzed on the basis of these ratios. By comparing the experimental mass values obtained from MS to a set generated by in silico digestion of all possible peptides in a protein and DNA sequence databases, the exact match or similar matches can be determined. If a protein of interest is included in a database (see below), it can be characterized and some of its biochemical properties can be obtained. Alternatively, if the protein is not found in a database, proteins with similar properties can be identified. This method of identification is considerably faster and far more sensitive than automated Edman sequencing, with the detection sensitivity in the low picomole to femtomole range.

Tandom MS systems, such as quadropole-TOF with either MALDI or electrospray ionization (the latter one usually coupled with LC or capillary electrophoresis) or MALDI tandem TOF-MS, are also used to obtain partial peptide sequence information and/or detect posttranslational modifications beyond fingerprinting (38, 39). Different mass analyzer combinations vary with respect to their accuracy, resolution, detection mass range, speed, and other parameters, all with detection sensitivities in the low femtomole range.

	Databases

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
Image analysis software such as Quest (40) and Melanie III (41) interprets complex 2-D gel patterns using novel algorithms for image detection, acquisition, registration, and differential expression calculations. They have specific features to enhance and annotate images and enable quantitative comparisons of proteins found in different gels. Proteomic databases such as the one maintained by the Danish Center for Human Genome Research at the University of Aarhus (42) (http://biobase.dk/cgi-bin/celis) can be used for these comparisons. The Swiss Institute of Bioinformatics at the University of Geneva maintains the ExPASy (Expert Protein Analysis System) proteomics server (http://www.expasy.org/) that provides links to many important mirror and complementary sites. These database interfaces have tools designed to overcome the enormous computational challenges associated with proteome analysis. For example, it is possible to search databases (e.g. SWISS-PROT, TrEMBL) for proteins whose theoretical isoelectric point, molecular weight, amino acid composition, or peptide mass fingerprint match experimentally derived data. There also are tools that predict posttranslational modifications and protein structure. The National Cancer Institute offers a method for comparing images of 2-D protein gels from different Internet sources obtained on a Web browser (43, 44) (http://www-lecb.ncifcrf.gov/flicker/). Using these images as a guide, differentially expressed proteins can be manually or robotically excised from the gels and prepared for analysis by MS and protein sequencing.

	Future perspectives

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 
2-D gel electrophoresis achieves very high separation efficacy. However, the technique involves many steps of manual handling such as gel preparations, sample applications, and protein visualization. Analysis is time consuming, and sophisticated image analysis is essential to extract meaningful data. Due to these limitations, alternatives are being devised. These techniques include 2-D liquid chromatography (cation-exchange/reversed-phase chromatography or size-exclusion/reversed-phase chromatography) coupled to MS, capillary isoelectric focusing coupled to MS, and preparative isoelectric focusing followed by size-exclusion chromatography with MS (45). Capillary isoelectric focusing coupled to MS and preparative isoelectric focusing followed by size- exclusion chromatography with MS are based on IEF and mass-dependent separation; therefore, it may be possible to link databases with these 2-D gel approaches. Chromatography coupled to MS can be fully automated, thus achieving high throughput.

DNA array technology is beginning to yield important functional data in genome-wide transcript profiling. However, protein profiling arrays are far more difficult to establish (46). First, it is necessary to find appropriate protein recognition molecules to bind specific proteins. Then the molecules need to be attached to the surface of array chip while keeping their binding capability intact. Finally, there is a need for quantitative detection methods to measure the bound proteins. Moreover, unlike the DNA-DNA annealing interaction, protein binding to protein recognition molecules may not possess relatively uniform binding affinities. One recent approach to the problem was to create protein microarrays by the use of covalent mRNA-protein fusion, hence using the existing DNA microarray technology. A mRNA-protein fusion product was produced by in vitro translation using puromycin-conjugated mRNA (the PROfusion process), and the array was produced by DNA-RNA hybridization using DNA probes immobilized to the surface (47). It was demonstrated that subattomole amounts of protein could be detected by this hybrid array. Other protein array systems will certainly be discovered/developed in the near future.

With the coming of the genomics and proteomic age, many fields of biology are set to "blast off" in terms of data accumulation and analyses. The field of endocrinology is no exception. The ability to "see spots run" and to identify these protein "spots" in a variety of tissue under distinct environmental conditions is truly awesome. For example, proteins from the brains of a male and female mouse were resolved by 2-D gel electrophoresis and visualized with SYPRO Ruby (Fig. 1). Striking differences in the protein "spots" are readily observed. Extension of these types of protein profiling of tissue from an animal treated with a hormone vs. an untreated individual will yield "volumes" of data. Various databases can be used to analyze and compare data. This overall ability to identify proteins in all tissue as it relates to the areas of reproduction, growth, metabolism, or any other endocrine related field will certainly lead to the a better understanding of the "molecular" behavior of a cell or tissue under a selected set of circumstances. Also, the discovery of these protein profile changes or novel proteins will result in new approaches for the discovery and development of diagnostics, therapeutics, and therapeutic targets.

View larger version (66K): [in this window] [in a new window]   Figure 1. The protein profile from the cortex of male and female mice. Adult (5 months) C57/BL6 mice were killed by cervical dislocation, and their brains were quickly removed and rinsed in ice-cold PBS. The cortex was carefully dissected and dounce-homogenized in buffer containing containing 0.25 M sucrose, 50 mM Tris-HCl (pH 7.6), 25 mM KCl, 5 mM MgCl2, and protease inhibitor cocktail (Sigma, St. Louis, MO) at a concentration of 50 µl/g of brain tissue (wet weight). Crude brain homogenates were then separated into subcellular fractions using differential gradient ultracentrifugation as previously described (48 ) to yield fractions enriched in cytoplasmic-free nuclei, heavy membrane, light membrane/polysomes and soluble cytoplasm. Proteins (200 µg) from the soluble cytoplasmic were separated by 2-D gel electrophoresis using the IEF cell and Protean II (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s specifications employing active rehydration of IPG strips and visualized on a Fuji Photo Film Co., Ltd. (Tokyo, Japan) FLA 3000G laser scanning device using the SYPRO Ruby fluorescent protein gel stain.

	Acknowledgments

 

	Footnotes

 
This work was supported, in part, by the state of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll and by DiAthegen, LLC.

Abbreviations: CA, Carrier ampholytes; 2-D, two-dimensional; IEF, isoelectric focusing; IPG, immobilized pH gradient; LC, liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-MS; MS, mass spectrometry.

Received February 28, 2002.

Accepted for publication March 25, 2002.

	References

Top Abstract Introduction Sample preparation 2-D gel electrophoresis Dyes used for visualizing... Protein identification by MS Databases Future perspectives References

 

1. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb J-F, Dougherty BA, Merrick JM, McKenney K, Sutton G, FitzHugh W, Fields C, Gocyne JD, Scott D, Shirley R, Liu L-I, Glodek A, Kelley JM, et al. 1995 Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512[Abstract/Free Full Text]
2. Himmelreich R, Hilbert H, Plagens H, Pirkl E, Li BC, Herrmann R 1996 Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res 24:4420–4449[Abstract/Free Full Text]
3. Blattner FR, Plunkett 3rd G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y 1997 The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474[Abstract/Free Full Text]
4. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessières P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, et al. 1997 The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256[CrossRef][Medline]
5. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG 1996 Life with 6000 genes. Science 274:546, 563–567[Abstract/Free Full Text]
6. The C. elegans Sequencing Consortium. 1998 Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2018[Abstract/Free Full Text]
7. Howard K 2000 The bioinformatics gold rush. Sci Am 283:58–63
8. Khochbin S, Gorka C, Lawrence JJ 1991 Multiple control level governing H10 mRNA and protein accumulation. FEBS Lett 283:65–67[CrossRef][Medline]
9. Rousseau D, Khochbin S, Gorka C, Lawrence JJ 1992 Induction of H1(0)-gene expression in B16 murine melanoma cells. Eur J Biochem 208:775–779[Medline]
10. Anderson L, Seilhamer J 1997 A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18:533–537[CrossRef][Medline]
11. Baltimore D 2001 Our genome unveiled. Nature 409:814–816[CrossRef][Medline]
12. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humphery-Smith I 1995 Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16:1090–1094[CrossRef][Medline]
13. Niehrs C, Pollet N 1999 Synexpression groups in eukaryotes. Nature 402:483–487[CrossRef][Medline]
14. Futcher B, Latter GI, Monardo P, McLaughlin CS, Garrels JI 1999 A sampling of the yeast proteome. Mol Cell Biol 19:7357–7368[Abstract/Free Full Text]
15. Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G 2002 Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147[CrossRef][Medline]
16. Service RF 2001 Proteomics. High-speed biologists search for gold in proteins. Science 294:2074–2077[Free Full Text]
17. Rabilloud T, ed. 2000 Poteome research: two-dimensional gel electrophoresis and identification methods. Berlin: Springer
18. Banks RE, Dunn MJ, Hochstrasser DF, Sanchez JC, Blackstock W, Pappin DJ, Selby PJ 2000 Proteomics: new perspectives, new biomedical opportunities. Lancet 356:1749–1756[CrossRef][Medline]
19. Eisenberg D, Marcotte EM, Xenarios I, Yeates TO 2000 Protein function in the post-genomic era. Nature 405:823–826[CrossRef][Medline]
20. Pandey A, Mann M 2000 Proteomics to study genes and genomes. Nature 405:837–846[CrossRef][Medline]
21. Naaby-Hansen S, Waterfield MD, Cramer R 2001 Proteomics—post-genomic cartography to understand gene function. Trends Pharmacol Sci 22:376–384[CrossRef][Medline]
22. Rabilloud T, Chevallet M 2000 Solubilization of proteins in 2D electrophoresis. In: Rabilloud T, ed. Proteome research: two-dimensional gel electrophoresis and identification methods. Berlin: Springer; 9–30
23. Cejka J, Vodrazka Z, Salak J 1968 Carbamylation of globin in electrophoresis and chromatography in the presence of urea. Biochim Biophys Acta 154:589–591[Medline]
24. Lippincott J, Apostol I 1999 Carbamylation of cysteine: a potential artifact in peptide mapping of hemoglobins in the presence of urea. Anal Biochem 267:57–64[CrossRef][Medline]
25. Galvani M, Rovatti L, Hamdan M, Herbert B, Righetti PG 2001 Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation. Electrophoresis 22:2066–2074[CrossRef][Medline]
26. Blumentals II, Robinson AS, Kelly RM 1990 Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol 56:1992–1998[Abstract/Free Full Text]
27. Hanner M, Redl B, Stoffler G 1990 Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochim Biophys Acta 1033:148–153[Medline]
28. Herbert B, Galvani M, Hamdan M, Olivieri E, MacCarthy J, Pedersen S, Righetti PG 2001 Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how? Electrophoresis 22:2046–2057[CrossRef][Medline]
29. Galvani M, Hamdan M, Herbert B, Righetti PG 2001 Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation. Electrophoresis 22:2058–2065[CrossRef][Medline]
30. Monribot C, Boucherie H 2000 Two-dimensional electrophoresis with carrier ampholytes. In: Rabilloud T, ed. Proteome research: two-dimensional gel electrophoresis and identification methods. Berlin: Springer; 31–56
31. Lopez MF, Berggren K, Chernokalskaya E, Lazarev A, Robinson M, Patton WF 2000 A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 21:3673–3683[CrossRef][Medline]
32. Switzer 3rd RC, Merril CR, Shifrin S 1979 A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231–237[CrossRef][Medline]
33. Lauber WM, Carroll JA, Dufield DR, Kiesel JR, Radabaugh MR, Malone JP 2001 Mass spectrometry compatibility of two-dimensional gel protein stains. Electrophoresis 22:906–918[CrossRef][Medline]
34. Patton WF 2000 A thousand points of light: the application of fluorescence technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21:1123–1144[CrossRef][Medline]
35. Malone JP, Radabaugh MR, Leimgruber RM, Gerstenecker GS 2001 Practical aspects of fluorescent staining for proteomic applications. Electrophoresis 22:919–932[CrossRef][Medline]
36. Mann M, Hendrickson RC, Pandey A 2001 Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 70:437–473[CrossRef][Medline]
37. Bonk T, Humeny A 2001 MALDI-TOF-MS analysis of protein and DNA. Neuroscientist 7:6–12[Abstract]
38. Poutanen M, Salusjarvi L, Ruohonen L, Penttila M, Kalkkinen N 2001 Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis. Rapid Commun Mass Spectrom 15:1685–1692[CrossRef][Medline]
39. Griffin TJ, Aebersold R 2001 Advances in proteome analysis by mass spectrometry. J Biol Chem 276:45497–45500[Free Full Text]
40. Garrels JI 1989 The QUEST system for quantitative analysis of two-dimensional gels. J Biol Chem 264:5269–5282[Abstract/Free Full Text]
41. Appel RD, Palagi PM, Walther D, Vargas JR, Sanchez JC, Ravier F, Pasquali C, Hochstrasser DF 1997 Melanie II—a third-generation software package for analysis of two-dimensional electrophoresis images: I. Features and user interface. Electrophoresis 18:2724–2734[CrossRef][Medline]
42. Celis JE, Gromov P, Ostergaard M, Madsen P, Honore B, Dejgaard K, Olsen E, Vorum H, Kristensen DB, Gromova I, Haunso A, Van Damme J, Puype M, Vandekerckhove J, Rasmussen HH 1996 Human 2-D PAGE databases for proteome analysis in health and disease: http://biobase.dk/cgi-bin/celis. FEBS Lett 398:129–134[CrossRef][Medline]
43. Lemkin PF, Thornwall G 1999 Flicker image comparison of 2-D gel images for putative protein identification using the 2DWG meta-database. Mol Biotechnol 12:159–172[CrossRef][Medline]
44. Lemkin PF 1999 Comparing 2-D electrophoretic gels across Internet databases. Methods Mol Biol 112:393–410[Medline]
45. Hille JM, Freed AL, Watzig H 2001 Possibilities to improve automation, speed and precision of proteome analysis: a comparison of two-dimensional electrophoresis and alternatives. Electrophoresis 22:4035–4052[CrossRef][Medline]
46. Jenkins RE, Pennington SR 2001 Arrays for protein expression profiling: towards a viable alternative to two-dimensional gel electrophoresis? Proteomics 1:13–29[CrossRef][Medline]
47. Weng S, Gu K, Hammond PW, Lohse P, Rise C, Wagner RW, Wright MC, Kuimelis RG 2002 Generating addressable protein microarrays with PROfusiontrade mark covalent mRNA-protein fusion technology. Proteomics 2:48–57[CrossRef][Medline]
48. Feng Y, Gutekunst CA, Eberhart DE, Yi H, Warren ST, Hersch SM 1997 Fragile X mental retardation protein: nucleocytoplasmic shuttling and association with somatodendritic ribosomes. J Neurosci 17:1539–1547[Abstract/Free Full Text]

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