- Email: [email protected]
2-D protein gel electrophoresis: an old method with future potential
MISCELLANEA , ,,,,,, . r
The technique of two-dimensional protein electrophoresis (2-DE), with its ability to resolve several thousand proteins, is potentially extremely powerful. For example, a single 2-D gel can separate all the proteins of E. coil or yeast cells and the result can be a beautiful image of the cell's protein machinery. Mammalian cells are likely to produce more proteins than a single 2-D gel can routinely resolve. However, subfractionation of cells prior to 2-D gel analysis should make it possible to identify all the proteins of individual cell types. This approach has been used to document the proteins present in human keratinocytes and has resulted in a 2-D gel database that contains over 4000 proteins ~ (Julio Cells, Aarhus, Denmark). No less impressive is the fact that over 1000 of these proteins have oeen identified by using a range of complementary techniques. Such results are not easy to reproduce and there are arguably three major obstacles to being able to make use of the power of 2-D gel technology. First, we need to be able to produce 2-D gels that can resolve thousands of proteins with quantitative and spatial reproducibility. Next, it becomes important to be able to convert the beautiful (or perhaps not so beautiful) 2-D pattern of proteins into the Identification of the individual proteins on the gels, And finally, the vast amount of information generated by such analyses needs to be catalogued, shared and, perhaps more importantly, Integrated with other protein and DNA sequence databases so that It can be used to good effect. These and other Issueswere addressed at a recent conference on 2-DE*. Towards better 2.D gels There are two predominant applications of 2-DE. A number of investigators use the method to undertake comprehensive analyses of changes in the production or covalent modifi. cation of proteins in disease states or in response to 'activation' of cells and tissues by a wide variety of stimuli. Others have begun the task of generating 2-D gel databases in which all the proteins present on a 2.D gel of specific cells (human liver, keratin. ocytes), tissues (heart) or organisms (E. coil, yeast) are identified. Both groups depend on the ability to produce well-resolved and quantitatively reproducible 2-D gels. As those who have attempted to run 2.D gels will testify, this in itself is no mean feat. It may come as no surprise to those who have tried and failed that, with one
, . . ~
m
IBIHIWII~IF
~
2-D protein gel electrophoresis: an old method with future potential Stephen Pe~r,!~g~on exception, no major breakthroughs have been made in the methodology or technology of 2-D electrophoresis since the landmark publication by O'Farrell in 1975 (Ref. 2). The method is still time consuming and technically demanding: such was the conclusion of a review of past and present methods (Mike Dunn, Harefield, UK). The sole exception has been the development of gels that contain immobilized pH gradients (IPGs) for isoelectric focusing in the first dimension 3. These gels are bonded to a plastic backing, run horizontally, are commercially available and have a number of advantages over the more traditional 'rod' gels in which the pH gradient is established with soluble ampholytes (Angelika Gorg, Munich, Germany). Whether they make 2-DE any more accessible to new users is questionable. Attempts to improve 2-D technology are always being made. New developments In the application of novel polyacrylamide matrices and the regulation of polymerization with photocatalysts are currently under. v..,~', but are unlikely to have immediate impact (Pier Rlghetti, Milan, Italy). Similarly, the possibility of generating re.usable gel matrices that can achieve multiple and automated protein separations (in a manner analogous to the column of an HPLC apparatus) seems attractive and their development has been started, but many problems still have to be overcome (Michael Harrington, Pasadena, USA). One delegate at another recent conference remarked that he knew little about 2-DE and thought the technique was wonderful, while those in his lab who knew a lot about it were certainly less enamoured (approximate transcription). This is probably a ~entiment echoed in many labs, and so not surprisingly attempts are being made to develop a fully automated 2-DE instrument (Hamrington; Leigh Anderson, Rockville, USA). MoJc importantly, such an instrument might promote more widespread use of 2-DE for routine medical diagnosis. The technical difficulties in developing
TRENDS IN CELLBIOLOGYVOL. 4 DECEMBER1994
such a machine are numerous but there is every possibility that one will be produced - eventually! Measuring changes In gene expression by 2-DE Using existing technology, a number of groups continue to apply 2-DE to investigate changes in the production of proteins- most often in the context of changes induced by disease. For example, while 2-DE analysis of cerebrospinal fluid (CSF)from Alzheimer's disease patients has failed to reveal any specific changes in proteins compared with those in other neurological disorders, a protein spot, identified as haptoglobin (ct-1) is more prominent in CS~;from Alzheimer's disease patients than CSF from healthy individuals (Karl Mattila, Tampere, Finland). Similar studies on plasma cells (red blood cell membranes, plateleLs and lymphocytes) and post mortem brain tissue have identified a number of protein spot changes associated with Alzheimer's disease and the 'related' disorder Down syndrome (Mattlla). The identity of the protein~ remains unknown. Others have shown that two fragments of the 13.chain of fibrinogen are present in CSF from patients with multiple sclerosis (Dirk Koerschenhausen, Berg, Germany). It is assumed that their presence in CSF reflects an incre-~se in inflammatory processes. The existence of these protein spots in CSF correlates with the severity of the disease but provides little useful information on its likely course. The seemingly inherent variability in the protein patterns of samples obtained from different individuals was highfighted by a study of heart proteins from patients with dilated cardiomyopathy (Joseph Corbett, Harefield, UK). However, careful analysis of these protein patterns, with the assistance of image.analysis software, has enabled potentially significant qualitative and quantitative changes in a number of individual proteins to be identified in subsets of the disease samples. For example, myosin light
~ 1994ElsevierSac~ceLid 0962.8924/94/S07.00
*2-D Bectrophoresis: from protein mapsto genomes. Siena,Italy; 5-7 September 1994.
~,tephen Penningtonis at the Dept of Human Anatomyand Cell Biology, The University of bverpool, PO Box 147, Uveq~ool,UK L69 3BX 439
|IIRffilIIDiJ~iJUWI~
chain 2 is reduced or absent in some of the heart samples from cardiomyopathic patients. Identification of the other heart proteins that change is being pursued actively. Others at the conference revealed ambitious plans to develop databases that contain correlations of the quantitative and qualitative protein changes in plasma, CSF and urine in disease states. It is intended that this approach will ultimately document the changes in all the 1500-3000 proteins that can be resolvedon a 2-D gel of the clinical samples. This study is currently concentrated on a more modest but nonetheless revealing analysis of acute-phase proteins (Carl Merril, Washington DC, USA). It seems likely that this databaseof 2-D gel data will be linked with a 'Disease Protein' database that is being developed and will contain information, taken from the literature, on changes in specific proteins in disease states (Peter Lemkin, Frederick, USA). A single 2-D gel contains a vast amount of information and analysisof changes in protein levels by comparison of 2-D gels is greatly facilitated by the use of suitable image.analysisand database.construction software. The Quest II softwarefor analysisof 2-D gel images is very powerful, particularly in its ability to match groups of spots automatically by their quantitative relationships across a series of gels, The software also enables collabor. atlve databasesto be constructed over the World Wide Web network and provides linkage to other databases (lerry Latter, Cold Spring Harbour, USA), Quest II will become Increas. tngly important as such shared databases develop,
From protein spot to protein Identification It's all ',ery well to document changes in protein levels or modification in diseasesor in responseto cell activation, but virtually all those who use 2-DE want to be able to identify proteins from the gels. A number of techniques can be used: these include N-terminal or internal sequenceanalysis, peptide mapping by mass spectrometP#, and amino acid composition analysis, The first of these, protein sequence analysis, has been the bedrock for identification of proteins from gels. However, the technique is both expensive and time consuming and a number of speakers noted that the continued use of microsequencing as a first stage in the analysisof a protein from 2-D gels represents a waste of 440
MISCELLANEA
i
sequencertime if, as is often the case, the protein turns out to have been previously characterized. For this reason alone, the development of complemen~aw techniques that are rapid and lessexpe~s~',,~h~s considerable appeal and excitii~g ~ :'vancesare being made in such r~'~ethods.The identification of proteins ~rom 2-D gels by these aitemative methods involves comparing experimentally obtained data on the protein of interest (mass, pl or amino acid composition, etc.) with information in sequence databases to produce a 'match' table. In such ~,ablesthe score of the match gives an indication of the likelihood of the match being correct - the identification is rarely unequivocal. The techniques do however serve~s useful 'first pass' methods in an integrated approach to protein identification. A method that permits accurate automated amino acid composition analysis of proteins recovered from 2-D gels by electroblotting onto PVDFmembranes has been developed (Marc Wilkins, Sydney, Australia). To explore the method's potential for protein identification, 27 proteins were picked randomly from the 2-D map of E. coliand subjected to parallel amino acid composition analysisand microsequencing, Comparison of the data revealed that the amino acid composition analysis correctly identified t 3 proteins, while 11 were identified Incorrectly and three were classifiedas previously unidentified. Each amino acid analysisused a single protein spot and all 27 proteins were analysed in three days. Matrix-assisted laser desorptlon ionization mass spectrometry (MALDI. MS)4 can be used to determine accurately the mass of proteins directly from membrane blots (Christoph Eckerskorn, Martinsried, Germany) or of peptides in mixtures derived from chemical or enzymatic cleavage of proteins (RuedlAebersold, Vancouver, Canada; Scott Patterson, Thousand Oaks, USA). The technique can also provide information on co. and posttranslational modifications. MALDIMS can be extremely sensitive - S0 pmol of protein loaded on a 1-D gel and blotted onto membrane has been used to obtain total mass and cyano. gen bromide fragment ma~es (from -20% of the sample) with the remainder being enzymatically digested. Of this, only 3% was required for deter. mination of peptide masses with the remainder being left for microsequencing if required (Patterson). With the limits of sensitivity being
pushed down by further technical developrr~.~t and the ability to obtain sequence-specific fragment ion data (Patterson), MALDI-MS has become an essential tool for protein identification. A stunning illustration of the potential of MALDI-MS was provided by a description of 'laser scanning' of a blot from a 2-D gel (Eckerskorn). In this approach a laser can be scanned in a track across a 2-D gel blot to give accurate mass information on the proteins encountered even if they are in poorly resolved spots. With a little imagination a machine can be envisaged for scanning an entire 2D gel and providing the accurate masses of all the proteins- or is this too fanciful?
2-D gel protein databases A number of groups have started to generate experimental and theoretical 2-D gel protein databases with the intention of linking these to protein and DNA sequence databases. In the experimental databases, images of 2-D gels are linked to information on the identity of the protein spots. The SWISS-2DPAGEdatabase is accessible on the World Wide Web and it contains reference images of 2-D gels from various samplesincluding human liver and plasma (Denis Hochstrasser, Geneva, Switzerland). Information on the protein spots, including cross. reference to sequence databases, can be obtained by clicking on the spot; alternatively, the theoretical location of any protein in the SWISS.PROT database can be mapped onto any of the reference images. The genome sequence information of yeast (Socchammycescerevisloe)and the literature on yeast proteins is being used to generate a protein database containing information such as the calculated isoelectdc point, molecular weights, calculated codon bias and known modifications of yeast pro. reins, This has been used to prcduce a theoretical 2-D gel map (J lines Garrels, Cold Spring Harbour, LISA). The theoretical map and data~ase are linked to an experimental yeast 2-D protein database and similar approaches are being used for protein databases of other organisms. As the genome sequencing projects proceed apace these 2-D protein databases are likely to provide much information on the expression of the sequenced genes including the subcellular localization, eel! and tissue distribution and charl~es in productinn of their products ,~nder various conditions. The possibility of sharing such
TRENDSIN CELLBIOLOGYVOL 4 DECEMBER1994
MISCELLANEA
database information over international networks generatedexcitement in many at the conference and caution in a few. None, however, is likely to forget the moment when 'live' connection to the World Wide Web was made during presentations held in a beautiful fresco-walled University
Cells on CD Histology Text.stack on CD-ROM Part of the Keyboard Histology
Series, Keyboard Publishing, 1994. Macintosh*/IBM.compatible tormot: $280,00 No area of education is moving faster than the introduction of computeraided learning. As computing power and the ability to store massive amounts of information in cheap and robust formats improves, so the variety of educational resources available to students and teachers is increasing, The Keyboard Histology Series comes In a collection of parts that are bought separately, The core package Is a CDROM and floppy disk with the Histology TextStack, containing the complete text and illustrations from the textbook Basic Histology by Junqueira,Camelro and Kelly(Appleton and Lange publishers). You can add to this the Slice of Life videodisk, which is a collection of some 38 500 medical images of which about 7000 are histology; 1400 of these can be accessed from within the Histology TextStack. The snag is that you need to buy a videodisk player, a colour'lV that usesthe American NTSC standard, and videodisk to use Slice of Life. The Histology TextStockcan be either run directly from the CD.ROM or, if speed is a premium, copied onto a hard disk, where it will take about 50 Mb of space. The application opens with a standard HyperCard window designed to fit the small 9" screen of the earlier Macintoshes. Unfortunately, all the images except two are in black and white. This is a pity,
building in the heart of Siena. The possibility of intemational teleworking from Tuscany crossed more than a few mindd I~fL~l'elnlces 1 CFJJS,J.E.eta/.(1993)Electrophoresis14, 1091-1198
since what histology loses by fixation of biological tissues can in some way be offset by the visual impact of a stained section. Seeing stained sections is especially important in the early stages of learning the subject, and images that are of much worse quality than the originals in the book are a considerable drawback of the soft. ware. Clearly, the program will be really worthwhile only when used with the videodisk and all the supporting equipment. Two questions need to be answered when a software version of a book appears. First, what do you gain from the computer version that is not avail. able in the book form, and second, do the gains merit (in this case) the tenfold increase in the price? The program does allow you to search rapidly through the chapters and to label passages with bookmarks. Unfortunately, little effort seems to have gone Into the software, which still has a very HyperCard feel to It, so much so that windows appear with little or no explanation of what to do with them and the very brief paper Instruction booklet suggests consulting the HyperCard user's manual (which even Apple do not supply with the program). Keyboard Publishing have attached their own 'enhancement' to HyperCard called the Transcriber. This allows you to record a series of steps through the stack and to play them back. Potentially, this nice idea will allow teachers to pick and choose the information they wish the students to
20'FARRELL,P.H. (1975)1.BioLChem. 250,4007-4021 3 GORG,A., POSTEL,W. and GUNTHER,S.(1988)Electrophoresis9, 531- 546 4 PAPPIN,D. J.C., HOJRUP,P.and BLEASBY,A. I. (1993)Curt.Biol.3, 327- 332
browse, but unfortunately I found the Transcriber difficult to use and subject to peculiar little error messages.Sadly, computerization of this book has lost many of the advantages of the printed copy. The question of price is more difficult. The program is networkable, at extra cost, and could be made available to a class of students each with their own computer and monitor. Alternatively, the program might be accessed over a campus network allowing many students 24-hour access. Unfortunately, as the singleuser version of the Histology TextStack stands, the book remains far better value. Although the Histology TextStack might not be state of the art, it is a valuable early step on a very fast. moving road. You can get a taste of the sort of projects that will be appearing in the next few years from browsing the World Wide Web on the Internet. One of the most elegant applications I have seen is the University of Wa~hington's Digital Anatomist project. This uses multimedia technology to bdng together brain images,CT scans and QuickTIme clips In a way that pro. vldes a new method for learning about the structure of the human brain. The beauty of computerized information is the variety of different ways that information can be presented and animated, and the way that the user can interact with the system. A computer isn't a book and it is a mistake to try to make it one.
*Thisreviewis basedonthe Madntoshversion (a HyperCardstack).
|eremy Rashbaas Dept of Hlstopathology, Addenbrooke's Hospital, Cambridge,UK CB2 2QQ. Email: jr.l@rnole. bio.cam.ac.uk
LETTERS Trends in Cell Biology welcomes correspondence. Letters to the Editor relating to articles in previous issues, or items of general interest to cell biologists, will be published at the discretion of the Editor. Letters should be addressed to The Editor, Trends in Cell Biology, ElsevierTrends Ioumals, 68 Hills Road, Cambridge, UK CB2 1LA
TRENDSIN CELLBIOLOGYVOL. 4 DECEMBER1994
441
The technique of two-dimensional protein electrophoresis (2-DE), with its ability to resolve several thousand proteins, is potentially extremely powerful. For example, a single 2-D gel can separate all the proteins of E. coil or yeast cells and the result can be a beautiful image of the cell's protein machinery. Mammalian cells are likely to produce more proteins than a single 2-D gel can routinely resolve. However, subfractionation of cells prior to 2-D gel analysis should make it possible to identify all the proteins of individual cell types. This approach has been used to document the proteins present in human keratinocytes and has resulted in a 2-D gel database that contains over 4000 proteins ~ (Julio Cells, Aarhus, Denmark). No less impressive is the fact that over 1000 of these proteins have oeen identified by using a range of complementary techniques. Such results are not easy to reproduce and there are arguably three major obstacles to being able to make use of the power of 2-D gel technology. First, we need to be able to produce 2-D gels that can resolve thousands of proteins with quantitative and spatial reproducibility. Next, it becomes important to be able to convert the beautiful (or perhaps not so beautiful) 2-D pattern of proteins into the Identification of the individual proteins on the gels, And finally, the vast amount of information generated by such analyses needs to be catalogued, shared and, perhaps more importantly, Integrated with other protein and DNA sequence databases so that It can be used to good effect. These and other Issueswere addressed at a recent conference on 2-DE*. Towards better 2.D gels There are two predominant applications of 2-DE. A number of investigators use the method to undertake comprehensive analyses of changes in the production or covalent modifi. cation of proteins in disease states or in response to 'activation' of cells and tissues by a wide variety of stimuli. Others have begun the task of generating 2-D gel databases in which all the proteins present on a 2.D gel of specific cells (human liver, keratin. ocytes), tissues (heart) or organisms (E. coil, yeast) are identified. Both groups depend on the ability to produce well-resolved and quantitatively reproducible 2-D gels. As those who have attempted to run 2.D gels will testify, this in itself is no mean feat. It may come as no surprise to those who have tried and failed that, with one
, . . ~
m
IBIHIWII~IF
~
2-D protein gel electrophoresis: an old method with future potential Stephen Pe~r,!~g~on exception, no major breakthroughs have been made in the methodology or technology of 2-D electrophoresis since the landmark publication by O'Farrell in 1975 (Ref. 2). The method is still time consuming and technically demanding: such was the conclusion of a review of past and present methods (Mike Dunn, Harefield, UK). The sole exception has been the development of gels that contain immobilized pH gradients (IPGs) for isoelectric focusing in the first dimension 3. These gels are bonded to a plastic backing, run horizontally, are commercially available and have a number of advantages over the more traditional 'rod' gels in which the pH gradient is established with soluble ampholytes (Angelika Gorg, Munich, Germany). Whether they make 2-DE any more accessible to new users is questionable. Attempts to improve 2-D technology are always being made. New developments In the application of novel polyacrylamide matrices and the regulation of polymerization with photocatalysts are currently under. v..,~', but are unlikely to have immediate impact (Pier Rlghetti, Milan, Italy). Similarly, the possibility of generating re.usable gel matrices that can achieve multiple and automated protein separations (in a manner analogous to the column of an HPLC apparatus) seems attractive and their development has been started, but many problems still have to be overcome (Michael Harrington, Pasadena, USA). One delegate at another recent conference remarked that he knew little about 2-DE and thought the technique was wonderful, while those in his lab who knew a lot about it were certainly less enamoured (approximate transcription). This is probably a ~entiment echoed in many labs, and so not surprisingly attempts are being made to develop a fully automated 2-DE instrument (Hamrington; Leigh Anderson, Rockville, USA). MoJc importantly, such an instrument might promote more widespread use of 2-DE for routine medical diagnosis. The technical difficulties in developing
TRENDS IN CELLBIOLOGYVOL. 4 DECEMBER1994
such a machine are numerous but there is every possibility that one will be produced - eventually! Measuring changes In gene expression by 2-DE Using existing technology, a number of groups continue to apply 2-DE to investigate changes in the production of proteins- most often in the context of changes induced by disease. For example, while 2-DE analysis of cerebrospinal fluid (CSF)from Alzheimer's disease patients has failed to reveal any specific changes in proteins compared with those in other neurological disorders, a protein spot, identified as haptoglobin (ct-1) is more prominent in CS~;from Alzheimer's disease patients than CSF from healthy individuals (Karl Mattila, Tampere, Finland). Similar studies on plasma cells (red blood cell membranes, plateleLs and lymphocytes) and post mortem brain tissue have identified a number of protein spot changes associated with Alzheimer's disease and the 'related' disorder Down syndrome (Mattlla). The identity of the protein~ remains unknown. Others have shown that two fragments of the 13.chain of fibrinogen are present in CSF from patients with multiple sclerosis (Dirk Koerschenhausen, Berg, Germany). It is assumed that their presence in CSF reflects an incre-~se in inflammatory processes. The existence of these protein spots in CSF correlates with the severity of the disease but provides little useful information on its likely course. The seemingly inherent variability in the protein patterns of samples obtained from different individuals was highfighted by a study of heart proteins from patients with dilated cardiomyopathy (Joseph Corbett, Harefield, UK). However, careful analysis of these protein patterns, with the assistance of image.analysis software, has enabled potentially significant qualitative and quantitative changes in a number of individual proteins to be identified in subsets of the disease samples. For example, myosin light
~ 1994ElsevierSac~ceLid 0962.8924/94/S07.00
*2-D Bectrophoresis: from protein mapsto genomes. Siena,Italy; 5-7 September 1994.
~,tephen Penningtonis at the Dept of Human Anatomyand Cell Biology, The University of bverpool, PO Box 147, Uveq~ool,UK L69 3BX 439
|IIRffilIIDiJ~iJUWI~
chain 2 is reduced or absent in some of the heart samples from cardiomyopathic patients. Identification of the other heart proteins that change is being pursued actively. Others at the conference revealed ambitious plans to develop databases that contain correlations of the quantitative and qualitative protein changes in plasma, CSF and urine in disease states. It is intended that this approach will ultimately document the changes in all the 1500-3000 proteins that can be resolvedon a 2-D gel of the clinical samples. This study is currently concentrated on a more modest but nonetheless revealing analysis of acute-phase proteins (Carl Merril, Washington DC, USA). It seems likely that this databaseof 2-D gel data will be linked with a 'Disease Protein' database that is being developed and will contain information, taken from the literature, on changes in specific proteins in disease states (Peter Lemkin, Frederick, USA). A single 2-D gel contains a vast amount of information and analysisof changes in protein levels by comparison of 2-D gels is greatly facilitated by the use of suitable image.analysisand database.construction software. The Quest II softwarefor analysisof 2-D gel images is very powerful, particularly in its ability to match groups of spots automatically by their quantitative relationships across a series of gels, The software also enables collabor. atlve databasesto be constructed over the World Wide Web network and provides linkage to other databases (lerry Latter, Cold Spring Harbour, USA), Quest II will become Increas. tngly important as such shared databases develop,
From protein spot to protein Identification It's all ',ery well to document changes in protein levels or modification in diseasesor in responseto cell activation, but virtually all those who use 2-DE want to be able to identify proteins from the gels. A number of techniques can be used: these include N-terminal or internal sequenceanalysis, peptide mapping by mass spectrometP#, and amino acid composition analysis, The first of these, protein sequence analysis, has been the bedrock for identification of proteins from gels. However, the technique is both expensive and time consuming and a number of speakers noted that the continued use of microsequencing as a first stage in the analysisof a protein from 2-D gels represents a waste of 440
MISCELLANEA
i
sequencertime if, as is often the case, the protein turns out to have been previously characterized. For this reason alone, the development of complemen~aw techniques that are rapid and lessexpe~s~',,~h~s considerable appeal and excitii~g ~ :'vancesare being made in such r~'~ethods.The identification of proteins ~rom 2-D gels by these aitemative methods involves comparing experimentally obtained data on the protein of interest (mass, pl or amino acid composition, etc.) with information in sequence databases to produce a 'match' table. In such ~,ablesthe score of the match gives an indication of the likelihood of the match being correct - the identification is rarely unequivocal. The techniques do however serve~s useful 'first pass' methods in an integrated approach to protein identification. A method that permits accurate automated amino acid composition analysis of proteins recovered from 2-D gels by electroblotting onto PVDFmembranes has been developed (Marc Wilkins, Sydney, Australia). To explore the method's potential for protein identification, 27 proteins were picked randomly from the 2-D map of E. coliand subjected to parallel amino acid composition analysisand microsequencing, Comparison of the data revealed that the amino acid composition analysis correctly identified t 3 proteins, while 11 were identified Incorrectly and three were classifiedas previously unidentified. Each amino acid analysisused a single protein spot and all 27 proteins were analysed in three days. Matrix-assisted laser desorptlon ionization mass spectrometry (MALDI. MS)4 can be used to determine accurately the mass of proteins directly from membrane blots (Christoph Eckerskorn, Martinsried, Germany) or of peptides in mixtures derived from chemical or enzymatic cleavage of proteins (RuedlAebersold, Vancouver, Canada; Scott Patterson, Thousand Oaks, USA). The technique can also provide information on co. and posttranslational modifications. MALDIMS can be extremely sensitive - S0 pmol of protein loaded on a 1-D gel and blotted onto membrane has been used to obtain total mass and cyano. gen bromide fragment ma~es (from -20% of the sample) with the remainder being enzymatically digested. Of this, only 3% was required for deter. mination of peptide masses with the remainder being left for microsequencing if required (Patterson). With the limits of sensitivity being
pushed down by further technical developrr~.~t and the ability to obtain sequence-specific fragment ion data (Patterson), MALDI-MS has become an essential tool for protein identification. A stunning illustration of the potential of MALDI-MS was provided by a description of 'laser scanning' of a blot from a 2-D gel (Eckerskorn). In this approach a laser can be scanned in a track across a 2-D gel blot to give accurate mass information on the proteins encountered even if they are in poorly resolved spots. With a little imagination a machine can be envisaged for scanning an entire 2D gel and providing the accurate masses of all the proteins- or is this too fanciful?
2-D gel protein databases A number of groups have started to generate experimental and theoretical 2-D gel protein databases with the intention of linking these to protein and DNA sequence databases. In the experimental databases, images of 2-D gels are linked to information on the identity of the protein spots. The SWISS-2DPAGEdatabase is accessible on the World Wide Web and it contains reference images of 2-D gels from various samplesincluding human liver and plasma (Denis Hochstrasser, Geneva, Switzerland). Information on the protein spots, including cross. reference to sequence databases, can be obtained by clicking on the spot; alternatively, the theoretical location of any protein in the SWISS.PROT database can be mapped onto any of the reference images. The genome sequence information of yeast (Socchammycescerevisloe)and the literature on yeast proteins is being used to generate a protein database containing information such as the calculated isoelectdc point, molecular weights, calculated codon bias and known modifications of yeast pro. reins, This has been used to prcduce a theoretical 2-D gel map (J lines Garrels, Cold Spring Harbour, LISA). The theoretical map and data~ase are linked to an experimental yeast 2-D protein database and similar approaches are being used for protein databases of other organisms. As the genome sequencing projects proceed apace these 2-D protein databases are likely to provide much information on the expression of the sequenced genes including the subcellular localization, eel! and tissue distribution and charl~es in productinn of their products ,~nder various conditions. The possibility of sharing such
TRENDSIN CELLBIOLOGYVOL 4 DECEMBER1994
MISCELLANEA
database information over international networks generatedexcitement in many at the conference and caution in a few. None, however, is likely to forget the moment when 'live' connection to the World Wide Web was made during presentations held in a beautiful fresco-walled University
Cells on CD Histology Text.stack on CD-ROM Part of the Keyboard Histology
Series, Keyboard Publishing, 1994. Macintosh*/IBM.compatible tormot: $280,00 No area of education is moving faster than the introduction of computeraided learning. As computing power and the ability to store massive amounts of information in cheap and robust formats improves, so the variety of educational resources available to students and teachers is increasing, The Keyboard Histology Series comes In a collection of parts that are bought separately, The core package Is a CDROM and floppy disk with the Histology TextStack, containing the complete text and illustrations from the textbook Basic Histology by Junqueira,Camelro and Kelly(Appleton and Lange publishers). You can add to this the Slice of Life videodisk, which is a collection of some 38 500 medical images of which about 7000 are histology; 1400 of these can be accessed from within the Histology TextStack. The snag is that you need to buy a videodisk player, a colour'lV that usesthe American NTSC standard, and videodisk to use Slice of Life. The Histology TextStockcan be either run directly from the CD.ROM or, if speed is a premium, copied onto a hard disk, where it will take about 50 Mb of space. The application opens with a standard HyperCard window designed to fit the small 9" screen of the earlier Macintoshes. Unfortunately, all the images except two are in black and white. This is a pity,
building in the heart of Siena. The possibility of intemational teleworking from Tuscany crossed more than a few mindd I~fL~l'elnlces 1 CFJJS,J.E.eta/.(1993)Electrophoresis14, 1091-1198
since what histology loses by fixation of biological tissues can in some way be offset by the visual impact of a stained section. Seeing stained sections is especially important in the early stages of learning the subject, and images that are of much worse quality than the originals in the book are a considerable drawback of the soft. ware. Clearly, the program will be really worthwhile only when used with the videodisk and all the supporting equipment. Two questions need to be answered when a software version of a book appears. First, what do you gain from the computer version that is not avail. able in the book form, and second, do the gains merit (in this case) the tenfold increase in the price? The program does allow you to search rapidly through the chapters and to label passages with bookmarks. Unfortunately, little effort seems to have gone Into the software, which still has a very HyperCard feel to It, so much so that windows appear with little or no explanation of what to do with them and the very brief paper Instruction booklet suggests consulting the HyperCard user's manual (which even Apple do not supply with the program). Keyboard Publishing have attached their own 'enhancement' to HyperCard called the Transcriber. This allows you to record a series of steps through the stack and to play them back. Potentially, this nice idea will allow teachers to pick and choose the information they wish the students to
20'FARRELL,P.H. (1975)1.BioLChem. 250,4007-4021 3 GORG,A., POSTEL,W. and GUNTHER,S.(1988)Electrophoresis9, 531- 546 4 PAPPIN,D. J.C., HOJRUP,P.and BLEASBY,A. I. (1993)Curt.Biol.3, 327- 332
browse, but unfortunately I found the Transcriber difficult to use and subject to peculiar little error messages.Sadly, computerization of this book has lost many of the advantages of the printed copy. The question of price is more difficult. The program is networkable, at extra cost, and could be made available to a class of students each with their own computer and monitor. Alternatively, the program might be accessed over a campus network allowing many students 24-hour access. Unfortunately, as the singleuser version of the Histology TextStack stands, the book remains far better value. Although the Histology TextStack might not be state of the art, it is a valuable early step on a very fast. moving road. You can get a taste of the sort of projects that will be appearing in the next few years from browsing the World Wide Web on the Internet. One of the most elegant applications I have seen is the University of Wa~hington's Digital Anatomist project. This uses multimedia technology to bdng together brain images,CT scans and QuickTIme clips In a way that pro. vldes a new method for learning about the structure of the human brain. The beauty of computerized information is the variety of different ways that information can be presented and animated, and the way that the user can interact with the system. A computer isn't a book and it is a mistake to try to make it one.
*Thisreviewis basedonthe Madntoshversion (a HyperCardstack).
|eremy Rashbaas Dept of Hlstopathology, Addenbrooke's Hospital, Cambridge,UK CB2 2QQ. Email: jr.l@rnole. bio.cam.ac.uk
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