- Email: [email protected]
New methods of proteome analysis: multidimensional chromatography and mass spectrometry
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 27
Proteomics: A Trends Guide | Review
24 Fenyö, D. et al. (1998) Protein identification using mass spectrometric information. Electrophoresis 19, 998–1005 25 Wilkins, M.R. et al. (1998) Multiple parameter cross-species protein identification using MultiIdent – a world-wide web accessible tool. Electrophoresis 19, 3199–3206 26 Service, R.F. (2000) Proteomics: can Celera do it again? Science 287, 2136–2138
27 Eriksson, J. et al. (2000) A statistical basis for testing the significance of mass spectrometric protein identification results. Anal. Chem. 72, 999–1005 28 Küster, B. et al. (1999) Identifying proteins in genome databases using mass spectrometry. In Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, pp. 1897–1898, American Society for Mass Spectrometry
New methods of proteome analysis: multidimensional chromatography and mass spectrometry Shortcomings in two-dimensional gel electrophoresis have encouraged the search for new methods of high-throughput proteome analysis. A variety of chromatographic methods can be coupled directly to a mass spectrometer to accomplish this task. However, multidimensional chromatography must be used to achieve the resolving power of two-dimensional gel electrophoresis. Current systems still fall short of the resolving power of two-dimensional gel electrophoresis but they have the potential to improve.
The analysis of a proteome involves the resolution of the proteins in a sample followed by the identification of the resolved proteins. Two-dimensional polyacrylamidegel electrophoresis (2D PAGE) followed by mass spectrometry (MS) is the most widely used method of protein resolution and identification. In 2D PAGE, proteins are separated in one dimension by isoelectric point (pI) and in the other dimension by molecular weight. As a result, a single 2D-PAGE system can resolve more than 1500 proteins. In spite of the contributions of 2D PAGE to proteomics, there are shortcomings to this technology. Highthroughput analysis of proteomes is challenging because each spot from 2D PAGE must be individually extracted, digested and analysed – a time-consuming process. In addition, owing to the limited loading capacity of 2DPAGE gels and the detection limit of staining methods, 2D PAGE currently has an insufficient dynamic range for complete proteome analysis.The dynamic range of a proteome system can be defined as the number of copies per cell of the most-abundant protein identified by a system
Proteomics: A Trends Guide
July 2000
divided by the number of copies per cell of the leastabundant protein identified by a system.These shortcomings of 2D PAGE as a separation medium for proteomics have encouraged the development of alternative methods for protein or peptide separation. A true substitute to 2D-PAGE–MS for proteome analysis should resolve proteins as well as 2D PAGE does and also allow the rapid identification of resolved proteins. This article describes some alternative methods that combine separation and identification of proteins, including one- and two-dimensional (1D and 2D, respectively) chromatography methods using highperformance liquid chromatography (HPLC), capillary isoelectric focusing (CIEF), capillary electrophoresis (CE) or microcapillary chromatography. As with 2D PAGE, MS is the method of choice for identifying proteins resolved by liquid separation methods. By eliminating the steps required to transfer proteins from the separation device to the mass spectrometer, several of the methods described might be better suited to high-throughput analysis.
1471-1931/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1471-1931(00)00005-7
Michael P. Washburn* mwashburn@ u.washington.edu
John R. Yates, III† [email protected] *Department of Molecular Biotechnology, Box 357730, University of Washington, Seattle, WA 98195-7730, USA. †
Department of Cell
Biology, SR11, The Scripps Research Institute, La Jolla, CA 92037, USA.
27
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 28
Review | Proteomics: A Trends Guide
1D chromatography and MS The combination of CIEF with MS has been investigated as a method to replace 2D-PAGE–MS. CIEF separates components based on their pI and MS adds a very accurate molecular weight measurement. Initial studies have coupled CIEF to electrospray ionization (ESI) MS (Ref. 1) and Fourier-transform ion-cyclotron-resonance (FTICR) MS (Refs 2, 3). However, in two of these studies, very few proteins were electrophoretically resolved1,2. Several hundred proteins from an Escherichia coli cell lysate were resolved when CIEF was coupled to FTICR MS, but only a few proteins were identified3, a situation that is inadequate for any proteomics study. The direct coupling of CIEF to MS allowed higher-throughput analyses than the spot-byspot identification of 2D PAGE and permitted extremely accurate molecular-weight measurements. The added MS dimension resolved mass:charge values, allowing the molecular weights of many of the unresolved proteins to be calculated. An alternative way to separate intact proteins is to digest the collection of proteins into a peptide mixture and use tandem mass spectrometry (MS–MS) to generate amino-acid-sequence-specific data4. Injecting peptides rather than proteins into mass spectrometers allows superior protein identification. Peptide-mass mapping by matrix-assisted laser desorption–ionization time-of-flight (MALDI–TOF) MS and peptide-fragmentation pattern matching by MS–MS are the two primary methods used to determine protein identities5. Peptides injected into a tandem mass spectrometer can be fragmented in a predictable manner, generating a tandem mass spectrum. Eng et al. have developed a widely used algorithm named SEQUEST to match the observed tandem mass spectrum of a peptide to theoretical mass spectra from protein or DNA databases, which allows the identification of proteins present in mixtures6. Using this search algorithm, McCormack et al. identified proteins from complex protein mixtures by digesting the proteins into peptides and loading the peptide mixture onto a reverse-phase (RP) microcolumn4. Via an HPLC pump, an RP gradient directly eluted peptides into an ESI tandem mass spectrometer and protein identities were determined by SEQUEST.Thus, this method combines the separation and identification steps. Multidimensional chromatography Theoretically, 1D chromatography cannot resolve as many proteins as 2D PAGE. 2D PAGE is a multidimensional system in which proteins are separated by independent methods (i.e. pI and molecular weight).A true multidimensional separation system like 2D PAGE must subject proteins or peptides to two or more independent separation methods
28
and maintain the separation of two components after they have been resolved in one step7. If these requirements are fulfilled, the resolving power of a 2D-separation method can theoretically be the peak capacity (a measure of resolving power) of the first dimension multiplied by the peak capacity of the second dimension7. Moore et al. have described several 2D methods to resolve peptides and proteins by liquid chromatography and CE (Ref. 8). 2D liquid-phase electrophoresis and MS Preparative 2D liquid-phase electrophoresis (2D-LPE) has been used to resolve and to identify proteins from human cerebrospinal fluid9 and human pleural eluate10, both complex protein mixtures dominated by a few highly abundant proteins. Like 2D PAGE, 2D-LPE resolves proteins based on pI and molecular weight, and proteins remain in the liquid phase throughout the analyses9,10, however, 2D-LPE does not require that the proteins are separated from the gel material as 2D PAGE does. Selected fractions were digested with trypsin and protein identities determined by peptide mass mapping using MALDI– TOF MS. Because 2D-LPE has a high loading capacity, low-abundance proteins could be detected and identified in the human cerebrospinal fluid and human pleural eluate. 2D-LPE is a promising method because it allows a high throughput and has a large loading capacity, but it currently lacks the resolving power of 2D PAGE (Ref. 10). 2D HPLC and MS Several proteome analyses have been carried out using 2D HPLC coupled to MS. For example, Raida et al. analysed peptides from human plasma filtrate by cation-exchange chromatography followed by RP HPLC (Ref. 11); the RP effluent was eluted directly into an ESI MS and the peptide masses were recorded11. Even though they detected 3000 distinct peptide masses, they determined the identity of relatively few peptides11. Wall et al. carried out a detailed comparison of 2D PAGE with a 2D-liquid-chromatography system coupled to MS (Ref. 12).They used a human erythroleukemia cellline lysate and collected 20 fractions from an IEF apparatus, then separated these individually by RP HPLC (Ref. 12). Selected proteins were digested and identified by peptide mass mapping on a MALDI–TOF MS (Ref. 12). Even though the resolving power of this method approaches that of 2D PAGE, very few proteins could be identified – only 38 of the ~700 proteins were resolved12. Protein identification using peptide-mass mapping requires almost-complete resolution of protein mixtures, and so this off-line combination offers few advantages over the use of 2D PAGE with MALDI–TOF MS.
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 29
Proteomics: A Trends Guide | Review
Figure 1. Twodimensionalchromatography system coupled to MS–MS
Off-line
S. cerevisiae whole-cell lysate Digestion Complex peptide mixture Loading of packed microcolumn SCX
On-line Waste
HPLC
RP
Attach microcolumn to system
SCX
RP
Database searching
ESI MS–MS
Identification of proteins in original sample
Power supply Proteomics – A Trends Guide
Opiteck et al. gave an early description of a fully automated 2D HPLC MS system13. In this, an E. coli lysate was injected onto a cation-exchange column and proteins eluted in a stepwise fashion onto an RP column13. The RP column eluant was sprayed into an ESI MS and protein masses were recorded13. This method is considered to be ‘on line’ because no sample handling was required once the sample was loaded onto the cationexchange column. Amenable to high-throughput analyses, the shortcoming of this method was again the lack of protein identifications. In a different system, Opiteck et al. coupled size-exclusion and RP HPLC (Ref. 14). Of approximately 450 proteins isolated from an E. coli lysate, the identities of 14 were determined by MS and Edman sequencing14. Although this method has a high throughput with a large protein capacity and is well suited to automation, it lacks a direct interface to the mass spectrometer and cannot perform automated protein identification. Two of the main shortcomings of 2D PAGE are the time needed to identify many spots individually and the limited dynamic range, which makes the identification of low-abundance proteins difficult. Some of the methods described so far might have higher throughputs and might detect low-abundance proteins because larger protein loads can be handled9–14, but few of the resolved proteins were identified. In each case, an additional handling step was needed to determine the identities of the majority of the resolved proteins.
2D chromatography and MS–MS To date, the most successful 2D-chromatography–MS approaches have incorporated 2D methods directly with ESI MS–MS. In MS–MS, peptides form fragments in a predictable manner, yielding amino acid sequence information. In an example of 2D chromatography followed by MS–MS, E. coli periplasmic proteins were fractionated by strong-anion-exchange HPLC and portions of each of 20 fractions were digested with trypsin and analysed by RP microcolumn HPLC (Ref. 15). By eluting peptides directly from the microcolumn into a tandem mass spectrometer, 80 proteins were identified and the tandem mass spectra were analysed by SEQUEST (Ref. 15).The identification of proteins was fully automated with the RP microcolumn HPLC, making this a higher-throughput system than 2D PAGE and the other methods described. In a more integrated system, Tong et al. analysed purified yeast ribosomes (which contain 75 proteins) with an on-line 2D separation method16. A peptide mixture from digested Saccharomyces cerevisiae ribosomes was loaded onto a solid-phase RP extraction cartridge16. The peptide mixture was eluted in a stepwise fashion into a CE column from which peptides were separated and sprayed directly into an ESI tandem mass spectrometer16; 136 unique peptides were sequenced and 66 proteins were identified via the SEQUEST algorithm16. Although the step gradients were loaded manually, no further sample handling was required once a peptide mixture of the sample was prepared and loaded onto the first dimension.
Proteins from a Saccharomyces cerevisiae whole-cell lysate were digested into a complex peptide mixture. The peptide mixture was loaded onto a biphasic microcapillary column packed with strong cation exchange (SCX) and reverse phase (RP) materials. Peptides were first displaced from the SCX resin to the RP resin by a salt gradient. Next, peptides were eluted off the RP resin into the electrospray-ionization tandem mass spectrometry (ESI MS–MS). In an iterative process, the microcolumn was re-equilibrated and an additional salt step of higher concentration displaced more peptides from the SCX resin to the RP resin. Peptides were again eluted by an RP gradient into the ESI MS–MS and the process repeated. Tandem mass spectra generated are correlated to theoretical mass spectra generated from protein or DNA databases by the SEQUEST algorithm. Using this system, Link et al. identified 189 unique proteins from an S. cerevisiae wholecell lysate17. Abbreviation: high performance liquid chromatography, HPLC.
29
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 30
Review | Proteomics: A Trends Guide
Link et al. carried out the largest proteome analysis by 2D chromatography and MS yet described in a fully integrated system17 (Fig. 1). A peptide mixture from digested S. cerevisiae whole-cell lysate was loaded onto a biphasic 2D microcapillary column packed with strong cation exchange (SCX) and RP materials17. Peptides were displaced iteratively by salt from the SCX onto the RP material; this was followed by an RP gradient, which eluted peptides directly into an ESI MS–MS. After re-equilibrating the RP material, raising the salt concentration displaced more peptides onto the RP material and the process was repeated. In a single experiment, this method resolved and identified 189 unique proteins from an S. cerevisiae whole-cell lysate via SEQUEST, the most proteins resolved and identified in any 2D-chromatography–MS study to date. Conclusion A true substitute for 2D-PAGE–MS as the method of choice for proteome analysis must resolve proteins as well as 2D PAGE and allow the rapid identification of the proteins resolved. None of the alternative methods described in this review have yet attained this goal but we believe that they will in the future. A multidimensional chromatography method is needed to achieve the resolving power of 2D PAGE. Many different types of chromatography (e.g. ion exchange, RP, size exclusion) could be used in tandem as long as they are largely independent and components resolved in one dimension remain resolved in the second dimension7. A high-throughput method is needed that combines resolution and identification, removing all sample-handling steps once the sample is loaded onto the system. In our opinion, the most promising system is that developed by Link et al. – 2D microcolumn SCX–RP followed by MS–MS (Ref. 17). Because a complex peptide mixture was loaded onto the system, rather than a complex protein mixture, MS–MS could identify 189 proteins from the sample17. Future improvements in the other methods described in this review might also allow them to become fully integrated. However, future high-throughput proteome analysis might resolve complex peptide mixtures of entire proteomes using multidimensional chromatography coupled directly to MS–MS for ‘on-line’ protein identification. Combining such a system with methods of protein quantitation by MS (Refs 18,19) might allow the rapid analysis of protein-expression levels in entire proteomes. When separation, quantitation and identification become fully integrated, proteomics should have a similar impact on biology to the global quantitative analysis of gene expression.
30
Acknowledgements We would like to acknowledge funding from the US National Institutes of Health (R33CA81665-01 and RR11823-03). M.P.W. would like to acknowledge support from genome training grant T32HG000035-05. References 1 Tang, W. et al. (1997) Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69, 3177–3182 2 Yang, L. et al. (1998) Capillary isoelectric focusing–electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization. Anal. Chem. 70, 3235–3241 3 Jensen, P.K. et al. (1999) Probing proteomes using capillary isoelectric focusing–electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 71, 2076–2084 4 McCormack, A.L. et al. (1997) Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767–776 5 Yates, J.R., III (2000) Mass spectrometry: from genomics to proteomics. Trends Genet. 16, 5–8 6 Eng, J.K. et al. (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 7 Giddings, J.C. (1987) Concepts and comparisons in multidimensional separation. J. High Res. Chromatogr. 10, 319–323 8 Moore, A.W. et al. (1996) Two-dimensional liquid chromatography–capillary electrophoresis techniques for analysis of proteins and peptides. Methods Enzymol. 270, 401– 419 9 Davidsson, P. et al. (1999) Characterization of proteins from human cerebrospinal fluid by a combination of preparative twodimensional liquid-phase electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 71, 642–647 10 Nilsson, C.L. et al. (1999) Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 20, 860–865 11 Raida, M. et al. (1999) Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate. J. Am. Soc. Mass Spectrom. 10, 45–54 12 Wall, D.B. et al. (2000) Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI–TOF mass spectrometry. Anal. Chem. 72, 1099–1111 13 Opiteck, G.J. et al. (1997) Comprehensive on-line LC/LC/MS of proteins. Anal. Chem. 69, 1518–1524 14 Opiteck, G.J. et al. (1998) Comprehensive two-dimensional highperformance liquid chromatography for the isolation of overexpressed proteins and proteome mapping. Anal. Biochem. 258, 349–361 15 Link, A.J. et al. (1997) A strategy for the identification of proteins localized to subcellular spaces: application to E. coli periplasmic proteins. Int. J. Mass Spectrom. Ion Proc. 160, 303–316 16 Tong, W. et al. (1999) Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry. Anal. Chem. 71, 2270–2278 17 Link, A.J. et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 18 Oda,Y. et al. (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 96, 6591–6596 19 Gygi, S.P. and Aebersold, R. (2000) Using mass spectrometry for quantitative proteomics. In Proteomics: A Trends Guide (Blackstock, W. and Mann, M., eds), pp. 31–36, Elsevier
14/7/00
12:02 pm
Page 27
Proteomics: A Trends Guide | Review
24 Fenyö, D. et al. (1998) Protein identification using mass spectrometric information. Electrophoresis 19, 998–1005 25 Wilkins, M.R. et al. (1998) Multiple parameter cross-species protein identification using MultiIdent – a world-wide web accessible tool. Electrophoresis 19, 3199–3206 26 Service, R.F. (2000) Proteomics: can Celera do it again? Science 287, 2136–2138
27 Eriksson, J. et al. (2000) A statistical basis for testing the significance of mass spectrometric protein identification results. Anal. Chem. 72, 999–1005 28 Küster, B. et al. (1999) Identifying proteins in genome databases using mass spectrometry. In Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, pp. 1897–1898, American Society for Mass Spectrometry
New methods of proteome analysis: multidimensional chromatography and mass spectrometry Shortcomings in two-dimensional gel electrophoresis have encouraged the search for new methods of high-throughput proteome analysis. A variety of chromatographic methods can be coupled directly to a mass spectrometer to accomplish this task. However, multidimensional chromatography must be used to achieve the resolving power of two-dimensional gel electrophoresis. Current systems still fall short of the resolving power of two-dimensional gel electrophoresis but they have the potential to improve.
The analysis of a proteome involves the resolution of the proteins in a sample followed by the identification of the resolved proteins. Two-dimensional polyacrylamidegel electrophoresis (2D PAGE) followed by mass spectrometry (MS) is the most widely used method of protein resolution and identification. In 2D PAGE, proteins are separated in one dimension by isoelectric point (pI) and in the other dimension by molecular weight. As a result, a single 2D-PAGE system can resolve more than 1500 proteins. In spite of the contributions of 2D PAGE to proteomics, there are shortcomings to this technology. Highthroughput analysis of proteomes is challenging because each spot from 2D PAGE must be individually extracted, digested and analysed – a time-consuming process. In addition, owing to the limited loading capacity of 2DPAGE gels and the detection limit of staining methods, 2D PAGE currently has an insufficient dynamic range for complete proteome analysis.The dynamic range of a proteome system can be defined as the number of copies per cell of the most-abundant protein identified by a system
Proteomics: A Trends Guide
July 2000
divided by the number of copies per cell of the leastabundant protein identified by a system.These shortcomings of 2D PAGE as a separation medium for proteomics have encouraged the development of alternative methods for protein or peptide separation. A true substitute to 2D-PAGE–MS for proteome analysis should resolve proteins as well as 2D PAGE does and also allow the rapid identification of resolved proteins. This article describes some alternative methods that combine separation and identification of proteins, including one- and two-dimensional (1D and 2D, respectively) chromatography methods using highperformance liquid chromatography (HPLC), capillary isoelectric focusing (CIEF), capillary electrophoresis (CE) or microcapillary chromatography. As with 2D PAGE, MS is the method of choice for identifying proteins resolved by liquid separation methods. By eliminating the steps required to transfer proteins from the separation device to the mass spectrometer, several of the methods described might be better suited to high-throughput analysis.
1471-1931/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1471-1931(00)00005-7
Michael P. Washburn* mwashburn@ u.washington.edu
John R. Yates, III† [email protected] *Department of Molecular Biotechnology, Box 357730, University of Washington, Seattle, WA 98195-7730, USA. †
Department of Cell
Biology, SR11, The Scripps Research Institute, La Jolla, CA 92037, USA.
27
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 28
Review | Proteomics: A Trends Guide
1D chromatography and MS The combination of CIEF with MS has been investigated as a method to replace 2D-PAGE–MS. CIEF separates components based on their pI and MS adds a very accurate molecular weight measurement. Initial studies have coupled CIEF to electrospray ionization (ESI) MS (Ref. 1) and Fourier-transform ion-cyclotron-resonance (FTICR) MS (Refs 2, 3). However, in two of these studies, very few proteins were electrophoretically resolved1,2. Several hundred proteins from an Escherichia coli cell lysate were resolved when CIEF was coupled to FTICR MS, but only a few proteins were identified3, a situation that is inadequate for any proteomics study. The direct coupling of CIEF to MS allowed higher-throughput analyses than the spot-byspot identification of 2D PAGE and permitted extremely accurate molecular-weight measurements. The added MS dimension resolved mass:charge values, allowing the molecular weights of many of the unresolved proteins to be calculated. An alternative way to separate intact proteins is to digest the collection of proteins into a peptide mixture and use tandem mass spectrometry (MS–MS) to generate amino-acid-sequence-specific data4. Injecting peptides rather than proteins into mass spectrometers allows superior protein identification. Peptide-mass mapping by matrix-assisted laser desorption–ionization time-of-flight (MALDI–TOF) MS and peptide-fragmentation pattern matching by MS–MS are the two primary methods used to determine protein identities5. Peptides injected into a tandem mass spectrometer can be fragmented in a predictable manner, generating a tandem mass spectrum. Eng et al. have developed a widely used algorithm named SEQUEST to match the observed tandem mass spectrum of a peptide to theoretical mass spectra from protein or DNA databases, which allows the identification of proteins present in mixtures6. Using this search algorithm, McCormack et al. identified proteins from complex protein mixtures by digesting the proteins into peptides and loading the peptide mixture onto a reverse-phase (RP) microcolumn4. Via an HPLC pump, an RP gradient directly eluted peptides into an ESI tandem mass spectrometer and protein identities were determined by SEQUEST.Thus, this method combines the separation and identification steps. Multidimensional chromatography Theoretically, 1D chromatography cannot resolve as many proteins as 2D PAGE. 2D PAGE is a multidimensional system in which proteins are separated by independent methods (i.e. pI and molecular weight).A true multidimensional separation system like 2D PAGE must subject proteins or peptides to two or more independent separation methods
28
and maintain the separation of two components after they have been resolved in one step7. If these requirements are fulfilled, the resolving power of a 2D-separation method can theoretically be the peak capacity (a measure of resolving power) of the first dimension multiplied by the peak capacity of the second dimension7. Moore et al. have described several 2D methods to resolve peptides and proteins by liquid chromatography and CE (Ref. 8). 2D liquid-phase electrophoresis and MS Preparative 2D liquid-phase electrophoresis (2D-LPE) has been used to resolve and to identify proteins from human cerebrospinal fluid9 and human pleural eluate10, both complex protein mixtures dominated by a few highly abundant proteins. Like 2D PAGE, 2D-LPE resolves proteins based on pI and molecular weight, and proteins remain in the liquid phase throughout the analyses9,10, however, 2D-LPE does not require that the proteins are separated from the gel material as 2D PAGE does. Selected fractions were digested with trypsin and protein identities determined by peptide mass mapping using MALDI– TOF MS. Because 2D-LPE has a high loading capacity, low-abundance proteins could be detected and identified in the human cerebrospinal fluid and human pleural eluate. 2D-LPE is a promising method because it allows a high throughput and has a large loading capacity, but it currently lacks the resolving power of 2D PAGE (Ref. 10). 2D HPLC and MS Several proteome analyses have been carried out using 2D HPLC coupled to MS. For example, Raida et al. analysed peptides from human plasma filtrate by cation-exchange chromatography followed by RP HPLC (Ref. 11); the RP effluent was eluted directly into an ESI MS and the peptide masses were recorded11. Even though they detected 3000 distinct peptide masses, they determined the identity of relatively few peptides11. Wall et al. carried out a detailed comparison of 2D PAGE with a 2D-liquid-chromatography system coupled to MS (Ref. 12).They used a human erythroleukemia cellline lysate and collected 20 fractions from an IEF apparatus, then separated these individually by RP HPLC (Ref. 12). Selected proteins were digested and identified by peptide mass mapping on a MALDI–TOF MS (Ref. 12). Even though the resolving power of this method approaches that of 2D PAGE, very few proteins could be identified – only 38 of the ~700 proteins were resolved12. Protein identification using peptide-mass mapping requires almost-complete resolution of protein mixtures, and so this off-line combination offers few advantages over the use of 2D PAGE with MALDI–TOF MS.
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 29
Proteomics: A Trends Guide | Review
Figure 1. Twodimensionalchromatography system coupled to MS–MS
Off-line
S. cerevisiae whole-cell lysate Digestion Complex peptide mixture Loading of packed microcolumn SCX
On-line Waste
HPLC
RP
Attach microcolumn to system
SCX
RP
Database searching
ESI MS–MS
Identification of proteins in original sample
Power supply Proteomics – A Trends Guide
Opiteck et al. gave an early description of a fully automated 2D HPLC MS system13. In this, an E. coli lysate was injected onto a cation-exchange column and proteins eluted in a stepwise fashion onto an RP column13. The RP column eluant was sprayed into an ESI MS and protein masses were recorded13. This method is considered to be ‘on line’ because no sample handling was required once the sample was loaded onto the cationexchange column. Amenable to high-throughput analyses, the shortcoming of this method was again the lack of protein identifications. In a different system, Opiteck et al. coupled size-exclusion and RP HPLC (Ref. 14). Of approximately 450 proteins isolated from an E. coli lysate, the identities of 14 were determined by MS and Edman sequencing14. Although this method has a high throughput with a large protein capacity and is well suited to automation, it lacks a direct interface to the mass spectrometer and cannot perform automated protein identification. Two of the main shortcomings of 2D PAGE are the time needed to identify many spots individually and the limited dynamic range, which makes the identification of low-abundance proteins difficult. Some of the methods described so far might have higher throughputs and might detect low-abundance proteins because larger protein loads can be handled9–14, but few of the resolved proteins were identified. In each case, an additional handling step was needed to determine the identities of the majority of the resolved proteins.
2D chromatography and MS–MS To date, the most successful 2D-chromatography–MS approaches have incorporated 2D methods directly with ESI MS–MS. In MS–MS, peptides form fragments in a predictable manner, yielding amino acid sequence information. In an example of 2D chromatography followed by MS–MS, E. coli periplasmic proteins were fractionated by strong-anion-exchange HPLC and portions of each of 20 fractions were digested with trypsin and analysed by RP microcolumn HPLC (Ref. 15). By eluting peptides directly from the microcolumn into a tandem mass spectrometer, 80 proteins were identified and the tandem mass spectra were analysed by SEQUEST (Ref. 15).The identification of proteins was fully automated with the RP microcolumn HPLC, making this a higher-throughput system than 2D PAGE and the other methods described. In a more integrated system, Tong et al. analysed purified yeast ribosomes (which contain 75 proteins) with an on-line 2D separation method16. A peptide mixture from digested Saccharomyces cerevisiae ribosomes was loaded onto a solid-phase RP extraction cartridge16. The peptide mixture was eluted in a stepwise fashion into a CE column from which peptides were separated and sprayed directly into an ESI tandem mass spectrometer16; 136 unique peptides were sequenced and 66 proteins were identified via the SEQUEST algorithm16. Although the step gradients were loaded manually, no further sample handling was required once a peptide mixture of the sample was prepared and loaded onto the first dimension.
Proteins from a Saccharomyces cerevisiae whole-cell lysate were digested into a complex peptide mixture. The peptide mixture was loaded onto a biphasic microcapillary column packed with strong cation exchange (SCX) and reverse phase (RP) materials. Peptides were first displaced from the SCX resin to the RP resin by a salt gradient. Next, peptides were eluted off the RP resin into the electrospray-ionization tandem mass spectrometry (ESI MS–MS). In an iterative process, the microcolumn was re-equilibrated and an additional salt step of higher concentration displaced more peptides from the SCX resin to the RP resin. Peptides were again eluted by an RP gradient into the ESI MS–MS and the process repeated. Tandem mass spectra generated are correlated to theoretical mass spectra generated from protein or DNA databases by the SEQUEST algorithm. Using this system, Link et al. identified 189 unique proteins from an S. cerevisiae wholecell lysate17. Abbreviation: high performance liquid chromatography, HPLC.
29
-PROTEOMICS supp. [final]
14/7/00
12:02 pm
Page 30
Review | Proteomics: A Trends Guide
Link et al. carried out the largest proteome analysis by 2D chromatography and MS yet described in a fully integrated system17 (Fig. 1). A peptide mixture from digested S. cerevisiae whole-cell lysate was loaded onto a biphasic 2D microcapillary column packed with strong cation exchange (SCX) and RP materials17. Peptides were displaced iteratively by salt from the SCX onto the RP material; this was followed by an RP gradient, which eluted peptides directly into an ESI MS–MS. After re-equilibrating the RP material, raising the salt concentration displaced more peptides onto the RP material and the process was repeated. In a single experiment, this method resolved and identified 189 unique proteins from an S. cerevisiae whole-cell lysate via SEQUEST, the most proteins resolved and identified in any 2D-chromatography–MS study to date. Conclusion A true substitute for 2D-PAGE–MS as the method of choice for proteome analysis must resolve proteins as well as 2D PAGE and allow the rapid identification of the proteins resolved. None of the alternative methods described in this review have yet attained this goal but we believe that they will in the future. A multidimensional chromatography method is needed to achieve the resolving power of 2D PAGE. Many different types of chromatography (e.g. ion exchange, RP, size exclusion) could be used in tandem as long as they are largely independent and components resolved in one dimension remain resolved in the second dimension7. A high-throughput method is needed that combines resolution and identification, removing all sample-handling steps once the sample is loaded onto the system. In our opinion, the most promising system is that developed by Link et al. – 2D microcolumn SCX–RP followed by MS–MS (Ref. 17). Because a complex peptide mixture was loaded onto the system, rather than a complex protein mixture, MS–MS could identify 189 proteins from the sample17. Future improvements in the other methods described in this review might also allow them to become fully integrated. However, future high-throughput proteome analysis might resolve complex peptide mixtures of entire proteomes using multidimensional chromatography coupled directly to MS–MS for ‘on-line’ protein identification. Combining such a system with methods of protein quantitation by MS (Refs 18,19) might allow the rapid analysis of protein-expression levels in entire proteomes. When separation, quantitation and identification become fully integrated, proteomics should have a similar impact on biology to the global quantitative analysis of gene expression.
30
Acknowledgements We would like to acknowledge funding from the US National Institutes of Health (R33CA81665-01 and RR11823-03). M.P.W. would like to acknowledge support from genome training grant T32HG000035-05. References 1 Tang, W. et al. (1997) Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69, 3177–3182 2 Yang, L. et al. (1998) Capillary isoelectric focusing–electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization. Anal. Chem. 70, 3235–3241 3 Jensen, P.K. et al. (1999) Probing proteomes using capillary isoelectric focusing–electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 71, 2076–2084 4 McCormack, A.L. et al. (1997) Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767–776 5 Yates, J.R., III (2000) Mass spectrometry: from genomics to proteomics. Trends Genet. 16, 5–8 6 Eng, J.K. et al. (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 7 Giddings, J.C. (1987) Concepts and comparisons in multidimensional separation. J. High Res. Chromatogr. 10, 319–323 8 Moore, A.W. et al. (1996) Two-dimensional liquid chromatography–capillary electrophoresis techniques for analysis of proteins and peptides. Methods Enzymol. 270, 401– 419 9 Davidsson, P. et al. (1999) Characterization of proteins from human cerebrospinal fluid by a combination of preparative twodimensional liquid-phase electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 71, 642–647 10 Nilsson, C.L. et al. (1999) Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 20, 860–865 11 Raida, M. et al. (1999) Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate. J. Am. Soc. Mass Spectrom. 10, 45–54 12 Wall, D.B. et al. (2000) Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI–TOF mass spectrometry. Anal. Chem. 72, 1099–1111 13 Opiteck, G.J. et al. (1997) Comprehensive on-line LC/LC/MS of proteins. Anal. Chem. 69, 1518–1524 14 Opiteck, G.J. et al. (1998) Comprehensive two-dimensional highperformance liquid chromatography for the isolation of overexpressed proteins and proteome mapping. Anal. Biochem. 258, 349–361 15 Link, A.J. et al. (1997) A strategy for the identification of proteins localized to subcellular spaces: application to E. coli periplasmic proteins. Int. J. Mass Spectrom. Ion Proc. 160, 303–316 16 Tong, W. et al. (1999) Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry. Anal. Chem. 71, 2270–2278 17 Link, A.J. et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 18 Oda,Y. et al. (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 96, 6591–6596 19 Gygi, S.P. and Aebersold, R. (2000) Using mass spectrometry for quantitative proteomics. In Proteomics: A Trends Guide (Blackstock, W. and Mann, M., eds), pp. 31–36, Elsevier