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Microfluidic systems for analysis of the proteome with mass spectrometry
Lab-on-a-Chip R.E. Oosterbroek and A. van den Berg (eds.) © 2003 Elsevier B.V. All rights reserved.
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Microfluidic systems for analysis of the proteome with mass spectrometry D. Jed Harrison*, Richard D. Oieschuk^ Pierre Thibault*' ^Dept. of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada ^Dept. of Chemistry, Queens University, Kingston, ON, K7L 3N6, Canada ""Institute for Biological Sciences, 100 Sussex Dr., Ottawa, ON KIA 0R6, Canada
1. INTRODUCTION Proteomics represents a significant challenge to those that would develop new instrumentation methods for protein analysis. It is a tremendous opportunity for the application of microfluidic systems. This chapter will first introduce some of the needs of Proteomics researchers, then explore the role that microfluidics can play in resolving those needs. The Proteome is a term coined to represent all the proteins in an organism, in the same sense that the genome represents all the genes in an organism [1,2]. Researchers in Proteomics have set themselves the task of understanding how the Proteome changes as a result of a stimuli, such as a temperature change, disease, drug, or the passage of time. Proteomics thus represents a "whole biosystem" approach to understanding the response of a cell or organism at a molecular level of detail. Such research will identify important biological pathways, and explore the role or response of proteins to a given event. New pathways, new protein interactions and new biomarkers are the most important outcomes. These new discoveries will ultimately lead to new targets used in screening for potential drugs using now-standard, ultra-high throughput, drug screening tests. Considering that past biomedical researchers may have devoted a large fraction of a career to understanding just one protein or one biochemical pathway, we can see that Proteomics represents an astronomical increase in the effort required if past methodologies continue to dominate the analysis of proteins. The challenge associated with this effort provides an opportunity for the development of new technology to accomplish the goals of Proteomics research with less laborious, more rapid, and more sensitive methods than are currently in use. Microsystem technologies such as microfluidic systems [3] and
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microarrays [4] are well suited to play a valuable role in this world-wide research effort. Success in determining the human genome came about in large part as the result of the new technology of multi-capillary gel electrophoresis being developed and commercialized [5]. This is a highly illustrative example of how a challenging project can spur new technology, as well as showing how crucially success can depend upon one or two major technological breakthroughs. The challenges associated with mapping and understanding the Proteome are probably a factor often greater than those borne by the Human Genome Project. Researchers in Proteomics will require several orders of magnitude improvements in sensitivity, sample size, and speed of analysis over current methods. Proteins are far more diverse in chemistry, structure and function than is DNA, necessitating more specific approaches to their chemical preparation and their analysis. Protein analysis frequently requires powerful separation methods, concentrations can be so low that preconcentration techniques are demanded, and the large range of hydrophobic and hydrophilic properties of proteins and peptides lead to drastically different characteristics that complicate the analysis. Yet, to analyze thousands of samples a day, a relatively generic group of platform methodologies are required. This challenge is significant, and it is anticipated that current methods, such as two-dimensional gel electrophoresis (2-d gel), will need to be replaced or drastically improved in terms of sensitivity, dynamic range, and separation power. Microfluidic systems clearly offer a number of advantages, many listed throughout this monograph, several of which are relevant to Proteomics research. Two important advantages are the reduced sample and reagent volumes required by microfluidic devices, and the increased analysis speed that results from the small dimensions associated with a chip [6]. Perhaps the key value of microfluidic systems in Proteomics will prove to be the integration of many sample processing steps together within one platform. Through incorporation of protein isolation, purification, digestion and separation steps within a chip, protein sample preparation can be done more rapidly, in an automated fashion. Automation can increase sample throughput significantly, and decrease the amount of highly trained manpower required per analysis. Such an advance would contribute significantly to attaining the goal of understanding the Proteome.
2. PROTEIN ANALYSIS METHODOLOGY Mass spectrometry (MS) has come to play a significant role in the analysis of proteins for Proteomics [7,8]. MS is capable of determining large numbers of proteins in a short time, utilizing small quantities of material. Typically, the
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proteins are separated in some fashion, then digested to give peptide fragments. These fragments are then analyzed by the mass spectrometer. Electrospray ionization MS (ESMS) involves sample ionization at the tip of a capillary as it flows towards an atmospheric pressure sampling inlet of a mass spectrometer. This method is well suited to on-line interfacing with a separation technique such as liquid chromatography (LC) or capillary electrophoresis (CE), allowing a final sample clean up and separation step. Often, a triple quadrupole mass spectrometer is used to perform tandem MS (or MS-MS). Coupling LC to ESMS-MS has become a standard approach to analyzing peptide mixtures derived from protein samples. Tandem MS provides protein sequence information, which is critical for the unequivocal identification of a protein. The first quadrupole MS is used to select a specific mass in the sample. The selected mass is then run through a quadrupole collision cell, in which high energy collisions cause it to fragment. These fragments are identified in the third MS. Quadrupole instruments are also paired with a time of flight MS to perform tandem MS, and these offer higher speed, sensitivity and resolving power than do triple quadrupoles. The other major MS method used in Proteomics is matrix assisted laser desorption (MALDI), which is usually coupled with time of flight MS (MALDITOF) for the analysis of samples deposited as spots on solid substrates. This method decouples the sample preparation step from the MS analysis stage, in contrast to on-line LC-MS methods, where the spectrometer waits to be supplied sample by the LC. MALDI plates can be prepared in advance, stored, and analyzed by the MALDI-TOF MS at the instruments optimal sampling rate, making more efficient use of the MS. . Until recently, MALDI-TOF could not offer as high a quality protein analysis as ESMS-MS, but developments in MALDI-TOF instrumentation, such as the addition of a quadrupole MS, mean that tandem MS can now be coupled with MALDI. Since MALDI is a faster method than is ESMS, MALDI-tandem MS can be expected to play an increasing role in Proteomics. The number of proteins and peptides that have been characterized by MS is continuously growing. The large volume of data generated has been pooled into databases such as Swiss-Prot (http://www.expasy.ch/sprot/sprot-top.html), which contain information about the sequence, function, domain structures, variants and post-translational modifications of a specific protein. The advent of protein databases has shifted the focus from "brute force" de novo protein sequencing techniques, such as tandem MS, to identification by comparison using peptide mass fingerprint analysis. This approach involves digesting a purified protein followed by MS analysis of the individual peptide fragments (i.e. the peptide mass fingerprint). The resulting characteristic pattern of peptides is compared with theoretical mass fingerprints, stored within protein databases, yielding possible matches for the identity of the unknown protein.
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Although peptide mass fingerprints can be generated in a matter of seconds to minutes, continuously supplying protein and peptides to the mass spectrometer fast enough becomes difficult. Protein sample preparation from the initial tissue, blood or cell culture represents a major bottleneck in MS analysis of proteins, and would benefit from greater automation. It is in the preparation of samples for the MS that microfluidic systems could play their greatest role. Protein sample preparation requires initial isolation of proteins from a biological sample. The key challenge, though, is the separation of the protein sample into individual proteins or peptides that can ultimately be presented to the MS. The classic approach has been to use the high resolving power of twodimensional (2-d) gel electrophoresis to separate proteins into as many as 2000 individual spots in a gel. Spots are then excised, proteins are digested with trypsin within the gel, salts and buffers are exchanged for optimizing the MS analysis, and the proteins are delivered to the MS. While this is the current workhorse of Proteomics, 2-d gel methods typically identify only 1000 to 2000 proteins, whereas as many as 15,000 to 30,000 may be expressed at any one time in mammals. Thus, the 2-d gel approach needs to be improved upon significantly. One more recent altemative is the shotgun approach, in which protein mixtures are digested directly, and then ion exchange and reverse phase chromatography are used to provide two dimensions of separation to resolve these extraordinarily complex mixtures before delivering them to an ESMS-MS system [9]. Another altemative uses specific isotopic labeling of proteins to provide mass tags that can be used to differentiate between samples prepared under differing conditions, such as cells cultured in the presence and absence of a drug. Aebersold and co-workers [10] have utilized the naturally abundant chlorine isotopes ^^Cl and ^^Cl (3:1 ratio) in a labeling reagent specific to cysteine residues in a peptide sequence. The observed masses, combined with auxiliary constraints, are then searched against sequence databases to identify proteins. The method works even when several proteins are co-migrating in a single spot of a 2-d gel. Regnier's research teams have developed combinations of ^O and *^0 with ^H and ^H isotopic labeling in order to [11] determine changes in abundance of proteins [11]. Such procedures are very promising, as they do not necessarily utilize 2-d gel methods, but they do require alternate separation procedures, such as LC or CE, followed by MS analysis. The need to automate, and to improve the sensitivity of these protein preparation procedures provides an attractive opportunity for microfluidic systems, which has not yet been significantly explored. The initial entree for microfluidic devices in Proteomics was as a sample input system for ESMS, as published independently by B. Karger's [12], M. Ramsey's [13] and R. Abersold's [14] research groups in 1997. They recognized that microfluidics and MS are surprisingly well matched, given that the nL volumes manipulated in microfluidics are similar to those required for sample
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introduction to the MS. These researchers showed that microfluidics could have a future as a sample processing and introduction method for MS, increasing the throughput of these costly instruments. The concept of a turn key proteomics system enabled by microfluidics is attractive, but clearly faces many technical challenges. Figeys [4] has outlined a set of multiple microfluidic stages for protein processing prior to introduction into a mass spectrometer, involving selective peptide capture, preconcentration by SPE and infusion into an ESMS, illustrated in Figure 1.
^"^ buffer 1
/
/
buffer 2
MS
sample elution affinity module
Fig. 1.
elutJon SPE module
buffer 3
Conceptual scheme illustrating the protein preparation and processing steps that could be performed within a microfluidic system in order to prepare samples for electrospray mass spectrometry (ESMS). Reprinted with permission from [15]. © 2001 Wiley-VCH.
More complex schemes can be envisaged, in which cells are introduced to a chip, lysed, protein is harvested, separated, digested, preconcentrated, separated again and then electrosprayed. This overall scheme, or some similar approach appears far off today. But, within the near future, we expect some subset of these steps will be performed routinely within microfludic devices. A number of the components needed have already been demonstrated. For example cell mobilization, lysis and enzyme analysis was first reported [16] at ^-TAS 98, and could be adapted to proteomics analysis. Immunoassays on bead beds were described by Oleschuk et al. [17] at ^i-TAS 2000 and by Kitamori's group [18], and the designs can be adapted to an affinity capture system for specific protein analysis as discussed below. Such a system could be easily coupled with cell lysis on-chip. Preconcentration elements, electrophoretic and electrochromatographic separations, and on-chip digestion methods are also under development [3,19,20]. A variety of other elements will undoubtedly be needed in the microfluidic toolbox to completely address the challenges of fully automating Proteomics within a microsystem.
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3. COUPLING ON-CHIP CAPILLARY ELECTROPHORESIS WITH ELECTROSPRAY MASS SPECTROMETRY The chip-ESMS interface has proven to be critical to translating on-chip separations to the MS. Several groups proposed chip-ESMS couplings differentiated by two different interface designs [12-14, 21]. These designs can be characterized as interfaces that "spray" directly from a micromachined interface such as an exposed channel at the side of a chip or a microfabricated nozzle, and those that use a capillary attached to the microchip as the electrospray emitter. The latter is similar to those used for capillary electrophoresis [22] and offers several advantages over spraying directly from the chip, the primary one being that good electrospray conditions are more readily achieved. A tapered capillary (end diam <10 ^m) produces small, welldefined droplets and stable, sensitive electrospray ionization [3]. Advances in micromachining, discussed below, should eventually allow comparable performance using nozzles fabricated directly on the chips. A sufficiently simple microfabrication method would probably confer manufacturing advantages compared to inserting capillaries. Dead volume is critical to the performance of the capillary sprayer design if separation is to be performed on-chip. Two methods of coupling developed in Harrison's laboratory [23,24], illustrated in Figure 2, were employed and compared in terms of dead volume and efficiency using laser induced fluorescence detection. Strikingly, only a 0.7 nL dead volume was required to lower the separation efficiency to 16-25 % of the predicted values [23]. However, the low dead volume coupling in Figure 2b produced 98 % of the predicted separation efficiency. The low dead volume connection preserves the separation that occurs on the chip prior to the connection, by preventing the peak (band) broadening associated with the capillary/chip junction. Figeys et al. [25] first demonstrated that the chip can be used to deliver not only simple samples, but also to introduce more complex samples such as tryptic digests of proteins. The combined chip-MS configuration provided sufficient resolution that the observed digestion fragments could be used to search a protein database, identifying the native protein. However, in that work no separation was performed on the chip, instead the resolving power of the mass spectrometer was relied upon. For relatively simple peptide mixtures this type of analysis is suitable. With more complex samples a separation step is required, in order to simplify the mass spectrum obtained enough for protein identification. The first on-chip separations of peptide mixtures coupled with ESMS were performed by Li et al. [24]. Chip-ESMS and tandem-MS analysis of a peptide mixture utilizing integrated CE for separation gave efficiencies as high as 300,000 plates/meter. Resolution in an analysis of a tryptic digest, using only 150 fmol of protein, was sufficient to allow the protein to be identified when
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comparing digestion fragments to a protein database. Chip-ESMS-MS analysis allowed the peptide sequence to be identified with a 45 amol/mL concentration detection limit. A similar design developed by Karger's group was utilized to separate and analyze a simple peptide mixture and a tryptic digest of cytochrome c [26]. These early studies established that Karger, Ramsey, and Aebersold's concept of using microfluidic chips to supply an MS with sample were feasible, and that high quality results could be obtained. This opened the way for integration of even more sample preparation steps, leading towards microfluidic systems that can compete with conventional approaches to Proteomics.
laration channel
capillar^ dead volume approx. 0.7 nl
separation channel capillar)' H
edge of device Fig. 2.
A very low dead volume connection design is needed to interface a microfluidic chip to electrospray ionization source for mass spectrometry (MS), if a separation performed on chip is to be successfully transferred to the MS. The upper image shows a small dead volume of 700 pL, the lower image appears to have zero dead volume. Reprinted with permission from [23]. © 1999 American Chemical Society.
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Although a capillary attached to a chip has proven to be suitable for highresolution separations, it still requires substantial machining to fabricate and prepare the device. A more attractive alternative would be to fabricate the electrospray tip/nozzle within the structure of the microchip using the remarkable capabilities of modem micromaching [21]. Methods to fabricate electrospray tips as part of a chip [21] should eventually provide robust devices for electrospray chip-MS. Research by Craighead and Knapp's groups for example has illustrated the potential for fabricating electrospray nozzles/tips in plastic devices [27,28]. The creative efforts of these and other groups to extend plastic fabrication methods for electrospray microchips is likely to be an important development in moving chip-ESMS towards a commercial reality. 4. MICROCHIP SYSTEMS FOR MALDI-TIME OF FLIGHT MASS SPECTROMETRY While most of the chip-MS couplings have used ESI as the method of ionization. Little et al. [29] were the first to develop a method utilizing matrix assisted laser desorption ionization (MALDI) with samples for eventual MALDI-MS analysis. MALDI-MS has become a workhorse for the proteomics industry providing precise molecular weight information for analyses of proteins and peptides excised from 2-d gels. Advancements such as the quadrupole-TOF instrument mean that MALDI can also be used directly for protein sequencing now. In Little et. al's study, a 100-element array of micro-wells was microfabricatied. DNA samples were administered with a piezoelectric pipette, then each well was subjected to MALDI analysis, yielding 100 mass spectra from a sample device less than I in 2. This design and others [30,31] demonstrate another possible application of microsystem technologies for rapid sample preparation and introduction to mass spectrometers. Because of the ability of MALDI-MS methods to operationally decouple the sample preparation step from the MS analysis stage in the lab, we can expect that the use MALDI will continue to grow in importance. As a consequence, the opportunity for Microsystems technology in this area may become significant. 5. PROTEIN SAMPLE PREPARATION ON MICROFLUIDIC CHIPS Preparation of protein samples involves many different processes, and there will not be one generic method in the future, in contrast to the dominance of just a few methods in DNA analysis. Key steps required in preparing samples involve the initial protein isolation, subsequent separation of proteins, physically as in a gel, or chemically using methods such as isotope labeling, digestion to
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peptides, and adjustment of the solvent and solute conditions to optimize subsequent MS analysis. Many of the specific methods used in sample preparation can be integrated within microfluidic systems. The integration of a CE separation step with ESMS has already been discussed above, and represents a key component of most sample preparation methods. Other methods discussed below include sample preconcentration, by electrokinetic means and by more traditional solid phase extraction techniques, as well as protein digestion, and affinity separation methods. We perceive the integration of each of these methods as an expansion of the microfluidics toolbox available for automating Proteomics on a chip. Preconcentration is particularly important for protein samples that are to be analyzed by MS. Ramsey and co-workers [32] were the first to demonstrate the use of sample stacking on a chip, obtaining concentration enhancements of about 10 times for dye samples. Since then the method has been directed toward protein preparation by Li et al. [33], who utilized large volume sample stacking methods on-chip to preconcentrate peptides and tryptic digests of proteins isolated from 2-d gel separations. Figure 3 illustrates the stacking procedure. buffer
buffer
buffer
sample waste
W^^^
sample
t
to MS
injection Fig. 3.
stacking
separation
Electrokinetic sample stacking can be used to concentrate a sample prior to performing ESMS. This diagram shows the steps required to focus a 10 nL volume into about 0.5 nL, prior to performing capillary electrophoresis (CE) and ESMS. Concentration detection limits were improved by a factor of about 20 in this way. Reprinted with permission from [33]. © 2000 Wiley-VCH.
A sample is introduced in low ionic strength buffer and directed towards the MS. After the separation channel (about 10 nL) is filled, the flow is
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reversed, and a higher ionic strength buffer is directed back along the separation channel towards sample waste, causing sample stacking to occur within a final volume of about 0.2-0.5 nL. The process is automatically stopped when the current flow reaches about 99% of maximum, indicating the separation channel is nearly full of the more conductive, high ionic strength buffer. The flow direction is reversed again and running buffer is delivered, in order to induce separation as the sample is directed towards the MS. Detection limits for model peptides such as bradykinin and somatotatin could be reduced from about 20 nM to about 0.5 nM. Low intensity spots in 2-d gels could be digested in situ, preconcentrated using sample stacking and measured directly by electrospray MS from the chip. While such large volume sample stacking can be accomplished in a conventional capillary, these procedures are far easier to automate within a multi-channel network in a microfluidic device. Figeys' et al. [34] were the first to use microchip-ESMS systems with solid phase extraction (SPE) on a hydrophobic phase for preconcentration of proteins, demonstrating the value of this step on an on-line processing system. They coupled on-chip generation of solvent elution gradients with off-chip SPE in a capillary located between the chip and the ESMS interface. Li et al. [33] tested the coupling of a pre-chip solid phase extraction device coupled to an entry port of a chip for subsequent CE separation and electrospray. Detection limits with SPE were 2.5-20 nM depending upon the peptide's hydrophobicity. Notably, even though the total volume of solution concentrated (1.5 |iL concentrated into 25 nL elution volume) was many times higher than for the electrokinetic stacking method described above, the detection limits were poorer. This discrepancy arose in part from the mismatch of off-chip on on-chip volumes. They concluded that integration of SPE within the microchip structure should provide considerable improvement in efficiency of sample utilization with SPE if a microfluidic system is also to be used in subsequent steps. More recently, Li et al. [35,36] have integrated a soHd phase extraction bed into a CE-ESMS chip and interfaced that to a tandem MS system and to an autosampler. This design embeds the microfluidic system within a larger laboratory system, illustrating the concept that the chip can play a key role as part of the lab, not act as a full replacement for the lab. (Which is a liberating perspective compared to the misconception that a micro total analysis system must integrate all components needed for analysis, in order to be worthwhile.) In the system they developed protein is first separated in a one or two dimensional gel system. Protein spots are then excised, transferred to a microtitre plate and digested by trypsin within the gel. The released peptides are transferred by capillary to the SPE bed on the chip, eluted by a change in solvent, and introduced to the CE channel on the chip. This stage provides a desalting step to optimize the sample for ESMS, along with concentrating the sample to improve sensitivity. The subsequent CE step separates the peptides.
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allowing the MS a better chance to correctly identify them. When coupled to a tandem MS system, peptide sequencing and unequivocal mass identification is possible. Figure 4 shows a composite diagram illustrating the system. The autosampler-CE-ESMS-MS system was able to process samples at a rate of 2 min/sample, including a washing step.
transfer
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96-well plate
concentrate & elute
mass/charge Fig. 4.
Diagram illustrating a microfluidic chip which performs preconcentration, CE, and ESMS, embedded within a larger protein analysis system. Protein extracts are subjected to 1- or 2-d gel electrophoresis, spots or bands are excised and digested in a microtitre plate, then an autosampler delivers them to the chip for the final analysis steps.
It was successfully tested with a variety of real samples, such as 25 p.g of soluble proteins extracted from N. meningitides, subjected to 1-d gel electrophoresis. The SPE concentration step added 3-5 min/sample for a sample volume of 8-10 |il, and an elution volume of 200 nL. The system was tested with the SPE stage included using 300 (ig of cell lysate from the prostate cancer line, LNCaP, subjected to 2-d electrophoresis. In the latter case over 70 gel spots were analyzed by tandem MS in stand-alone, automated fashion by the system, with identification of over 85 % of the proteins in the primary analysis step. Figure 5 illustrates the results for a few protein spots. This automated system was shown to have a limit of quantitation (signal = 10 times std. dev.
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noise) of 5 nM for leu-enkephalin with a 5 times concentration step (1.0 |iL reduced to 200 nL), and 10 % rsd in peak heights. On-C/7/p Preconcentration ^ MS/MS &CE
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Diagram showing the analysis of 300 ^g of cell lysate from the prostate cancer line, LNCaP, by 2-d gel electrophoresis. Over 70 spots were analyzed in automated fashion by the system shown in Figure 4. The base peak ion (BPI) electropherogram is shown for spot 14, along with a mass survey scan taken at 2.57 min, and a tandem mass spectrum of the m/z ion 506.7. Reprinted with permission from [36]. © American Soc. Biochem. Molec. Biol. Inc.
More selective forms of sample preconcentration can also be thought of as separation methods. Li et al. [36] have demonstrated affinity chromatography on-chip, and integrated it with CE-ESMS, using a bed packed with a selective adsorptive phase. In one example an antibody was attached to beads in a bed within a chip. The antibody was selective for a peptide sequence, GEQKLISEEDLN, generated by the human proto-oncogene loiown as c-myc, which is involved in cellular transformation, apoptosis, and cell-cycle progression. Figure 6 shows the reconstructed ion electropherogram (the signal obtained by the MS as a function of time at a specified mass) and the spectrum for this sequence. The analysis was performed on a human blood serum sample, which was spiked to 50 pg/mL with the peptide then dialyzed against a 100 kDalton membrane to remove endogenous antibodies, before introduction to the
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affinity bed on the microchip. The high quahty of the data illustrates the ability of on-chip affinity methods to produce extremely well cleaned up samples for MS analysis, despite the high protein and salt content of the sample. The same study [36] also explored the performance of an immobilized metal affinity column (IMAC) on-chip. These beds selectively bind phosphorylated peptide fragments; they are used to enrich samples that contain this common posttranslational protein modification. Figure 7 illustrates the enrichment achieved for a 2 picomole a-casein sample. The sample was prepared in a 1-d separation gel, in gel digested, then introduced to the chip. The 2 picomole sample was enough to allow tandem MS analysis, providing unambiguous identification of the site of phosphorylation on the protein. This is similar to the amount required by current methods, indicating competitive performance by the chip system. 0.90
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Analysis of the human proto-oncogene c-myc in a human blood serum sample, performed on a chip like that in Figure 4. An anti-c-myc loaded packed bed for affinity capture and purification of the sample was employed in the entry channel of the chip. A reconstructed ion electropherogram for a m/z value of 687.30 a), and the extracted mass spectrum of the electropherogram peak b) are shown. Adapted with permission from [36]. © American Soc. Biochem. Molec. Biol. Inc.
The embedding of a microfluidic system within a larger automated process in a laboratory is a significant step in demonstrating that microfluidics can meet the challenge presented by the very difficult samples associated with protein analysis. In the work described above the chip system was about twice as fast per sample as current conventional LC-ESMS-MS systems, and the analytical performance was as good as that obtained with current methods. It is important that any new technology not represent a decrease in performance if it is to be adopted. Further, since the work above represents just the initial attempts to introduce microfluidics into automated protein analysis, we anticipate that further improvements in performance and speed of analysis will be possible. Thus, microfluidics appears to be competitive with current
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approaches. The abihty to integrate more protein processing steps into the microfluidics platform will then confer a significant advantage to the microfluidics approach to automating protein analysis. 577.1
a-casein no IMAC selection 621.1 639.
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Mass spectra obtained for 2 pmoles of a-casien with (lower), and without (upper), the use of an immobilized metal affinity column (IMAC) on-chip for selective isolation of phosphorylated peptide fragments (marked with *). Significant enrichment is clearly obtained using the on-chip process. Adapted with permission from [36]. © American Soc. Biochem. Molec. Biol. Inc.
Separation is an important component to protein preparation. Capillary electrophoresis on-chip has been the dominant method explored for subsequent ESMS, as a one-dimensional format. Several of the affinity bed separations discussed above represent beginning steps at adding two dimensional separations to the chip repertoire. However, these do not provide sufficient resolving power for generic analysis of complex protein and peptide samples generated by tryptic digests. Recently the first two-dimensional separations have been performed in a microfluidic manifold with enough resolution to provide a
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separation for peptidic digests. Ramsey and co-workers demonstrated [37] a 2-d separation of TRITC-labeled tryptic peptides of jS-casein, shown in Figure 8. The separation involved both open-channel electrochromatography using a micellar electrokinetic format, and capillary zone electrophoresis. The samples were fluorescently labeled, in order to use fluorescence detection, as the system was not yet interfaced to a mass spectrometer. These separations are not truly orthogonal, but the work demonstrates the feasibility of performing 2-d separations within a microfluidic environment. Further developments in chromatography on chip will increase the ability to perform orthoganol separations on-chip in the future, further enhancing the microfluidics toolbox for Proteomics.
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a) Photograph of a 2-d separation chip, which perforais micellar electrokinetic chromatography in a first stage of separation, followed by capillary zone electrophoresis in a second stage, b) Data traces show the 2-d separation results obtained with fluorescence detection on labeled peptides. Reprinted with permission from [37]. © 2001 American Chemical Society.
6. PROTEIN DIGESTION ON-CHIP Enzymatic digestion of proteins with trypsin is an extremely common step in protein identification or sequencing. Integration of the digestion step into a microfluidic device reduces sample losses and should increase overall sample utilization. The use of a packed trypsin bead bed for digestion reduces problems with auto-digestion of trypsin and consequent sample contamination. Wang et
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al. [38] have reported an on-chip trypsin loaded, packed bed about 2 cm long, 1 mm wide and 0.15 mm deep, into which several |iL of sample are fed by a syringe pump. The products collected are then analyzed downstream by CEESMS. Figure 9 illustrates the separation of cytochrome c digested on-chip. Cytochrome c 4 |jl/min
Cytochrome c
2 |jl/min
1 |jl/min
0.5 Ml/min
time (min) Fig. 9.
Total ion current electropherogram obtained following on-chip digestion of cytochrome c with a trypsin digestion bed, using the chip in Figure 4. Traces show the effect of pumping speed, and thus digestion time, on digestion of 3 ^L of 16 ^M cytochrome c. The parent protein is completely consumed after only 3 min of digestion.. Reprinted with permission from [38]. © 2000 John Wiley & Sons, Ltd.
The digest may also be delivered continuously to the MS, i.e. infused. The digestion time required to fully consume cytochrome c was about 3 min, in contrast to conventional solution phase digestion off chip, which does not fully consume cytochrome c in 2 h. Digestion of the simple peptide, melittin, took only 5 s to reach the same stage as that seen for 15 min of off-chip digestion using trypsin loaded beads. This dramatic increase in digestion speed is the result of the high trypsin concentration that is achieved by loading it onto the beads, and the efficient mass transfer obtained in a flow through packed bed. While these advantages are not specific to a microfluidic chip, the volume of the chips is well matched to that required to the MS, leading to efficient sample utilization. These packed digestion beds could also be integrated with SPE beds on the same chip, in order to enhance sensitivity. Figure 10 illustrates the sensitivity difference observed for cytochrome c when digestion was performed
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on-chip, with and without SPE. Preconcentration recovery values of 90-105 % were attained for most peptide fragments, depending upon the specific hydrophobicity of the peptides. The results demonstrate the ability to integrate an increasing number of the desired protein preparation processes within a microchip system.
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900
1000
m/z Fig. 10. Mass spectra obtained for on-chip digestion of 200 nM cytochrome c with (upper) and without (lower) an on-chip solid phase extraction stage. The chip was similar to that in Figure 4, but contained an extra bed, loaded with solid phase extraction beads, at the sample inlet stage, downstream of the digestion bed. Reprinted with permission from [41]. © 2002 Kluwer Publishing.
7. MULTIPLEXING ON A CHIP FOR MASS SPECTROMETRY Multiplexing is an aspect of chip-MS coupling that has remained mostly unexplored, while holding significant promise for proteomic applications. The ability to handle multiple samples (multiplexing) using microchip devices, either simultaneously or sequentially, is one of the most significant advantages offered by a microfluidic platform. The first example of multiplexing combined with chip-MS involved spraying directly from the edge of the chip, utilizing 6
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independent devices on one wafer, with 6 separate spray orifices [12]. Aebersold and coworkers showed an example of automating the continuous flow analysis of three [14] and nine [34] samples on a single chip using a single electrospray outlet, as shown in Figure 11.
Fig. 11. Layout of a 9-sample multiplexing chip. Reprinted with permission from [25]. © 1998 Am. Chem. Soc.
Voltages applied to each sample reservoir sequentially caused each protein sample to flow into and through the effusion channel and out through a single electrospray needle into the MS. While the three-channel design suffered from cross contamination of samples, caused by flow leakage and siphoning effects, the nine-sample configuration produced much lower cross contamination. Careful consideration of leakage affects should help reduce cross contamination and increase the number of samples that can be placed on a single microfluidic device. Although combining multiplexing with MS detection has so far been limited to directly infusing pre-prepared samples, others have demonstrated complex separations in a multiplexed format. These have included immunoassay on a 6 manifold device[39], as well as the very aggressive incorporation of 96 and 384 individual separation channels on one 4 inch diameter device for DNA separation [40]. These demonstrations illustrate the concept is certainly achievable for chip-MS systems in the future. Taylor et al. [41] have in fact presented a 20-channel protein preparation chip-ESMS design at n-TAS 02. While it has not been fully characterized at this stage, the design shows how a variety of the sample preparation steps discussed above could be integrated into a high throughput, automated microchip system in the future. It is clear that multiplexing capillary gel electrophoresis was a key to rapidly analyzing the human genome [5]. It is likely that significant further effort in multiplexing protein analysis on-chip will be required to truly deliver the promise that microfluidics offers to automate the analysis of thousands of Proteomics samples.
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8. MICROCHIPS FABRICATED IN POLYMER SUBSTRATES The advice given in the movie The Graduate that "the future Ues in plastic" is indeed excellent advice for many applications of microfluidics. To date most devices have been prepared in glass substrates, although plastic devices offer major benefits in terms of cost, particularly for single-use, disposable chips. Polymers can be fabricated using relatively inexpensive protocols such as micromolding, injection molding, laser ablation or embossing [42]. They are available with greatly differing chemical, optical and mechanical properties compared to glass, providing a wide range of properties to draw from. Considerable effort has focused on polydimethylsiloxane (PDMS) as the substrate material [43], because of its remarkable ability to mold features as small as a few nanometers. It is also self-sealing, obviating the use of adhesives to bond the cover layer. However, PDMS devices are best suited for demonstrating the feasibility of using polymers, since the surface of PDMS is extremely hydrophobic, causing it to be incompatible with a wide range of analytes. The solid phase micro-extraction (SPME) literature is replete with studies in which PDMS is used as a sorbent phase for many chemicals [44], leading to problems if it is used for a chip in which surface adsorption is to be avoided. The other two polymers explored extensively for microfluidic devices are poly(methymethacrylate) and polycarbonate [42]. These are likely to be much more suitable for commercially produced devices than PDMS, due to greater solvent and sample compatibility. There is an extensive knowledge base in plastic fabrication of small scale parts, such as micro-lenses and other optical components. However, the tolerances required are not the same as those for microfluidic devices, and a sound basis for manufacturing-friendly microfabrication procedures for microfluidics is only just coming into place. Processes tend to be held as proprietary, and procedures published for bonding plastic materials, stamping, or molding them by other means, may not prove easy to reproduce between labs as yet. One can expect this situation to change in the next five years, as the technology matures. More problematic may be the solvent compatibility issues. Many plastics swell in organic solvents, and they adsorb a variety of chemicals. While adsorption is also a problem with glass surfaces, it has very high solvent compatibility, and procedures for glass fabrication, device bonding, are well known. The factors that control the choice of glass are also reasonably well demonstrated, whereas the difference between two manufacturer's polymer products are much less apparent at present. For this reason, glass chips are likely to continue to be used in the near ftiture for microfluidic devices that analyze multiple samples. But a tidal wave of plastic chips can be expected in the fiiture once some of the above challenges arefrillyaddressed.
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9. SUMMARY The minute sample size required by microfluidic devices, combined with extremely fast analysis times, make these devices ideal for filling the analysis bottleneck afforded by current analytical systems. As discussed above, key protocols required for a turnkey proteomics system have already been demonstrated. Many of the additional components needed to perform more fully integrated Proteomics on a chip have also been demonstrated; that is to say, the toolbox has been greatly expanded over the past few years. Significantly, many of the individual sample processing elements that were developed over the past decade have been integrated together, creating true systems for sample processing. These microfluidic devices have been embedded within a larger laboratory system and used to automate sample preparation. Importantly, the performance of these systems has been as good or better than conventional methods, despite the fact these are still laboratory prototypes. This success provides strong motivation for furthering the effort to use microfluidic systems in Proteomics. Continuing research on two-dimensional separation methods onchip, on integrating increasing numbers of processing steps within the chips, and on manufacturing the chips at lower cost in plastic materials should pave the way to the creation of turn key Proteomics systems on chips that will spur Proteomics research significantly. In 1992 Zelazney and Thomas [45] discussed a microfluidic system that "involved staining and spooling the genetic material into ever-flowing fluid channels which were capillaried into silicon control blocks no wider than a human hair. ... Target (virus or cell) strains no longer grew; they were immediately and continuously manufactured'' This prescient science fiction text in Flare combined proteomics and genetics in a microfluidic device to manufacture organisms and biochemicals directly. In fact, slightly before the publication of Flare^ early papers on microarrays and microfluidic devices [6,19,20] had shown it was possible to synthesize DNA on a chip and to manipulate flowing streams and separate chemicals within capillaries narrower than a hair. This chapter has presented a number of integrated protein processing systems that can play a major role in developing our understanding of biochemistry enough to help realize the remainder of their science fiction vision.
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Microfluidic systems for analysis of the proteome with mass spectrometry D. Jed Harrison*, Richard D. Oieschuk^ Pierre Thibault*' ^Dept. of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada ^Dept. of Chemistry, Queens University, Kingston, ON, K7L 3N6, Canada ""Institute for Biological Sciences, 100 Sussex Dr., Ottawa, ON KIA 0R6, Canada
1. INTRODUCTION Proteomics represents a significant challenge to those that would develop new instrumentation methods for protein analysis. It is a tremendous opportunity for the application of microfluidic systems. This chapter will first introduce some of the needs of Proteomics researchers, then explore the role that microfluidics can play in resolving those needs. The Proteome is a term coined to represent all the proteins in an organism, in the same sense that the genome represents all the genes in an organism [1,2]. Researchers in Proteomics have set themselves the task of understanding how the Proteome changes as a result of a stimuli, such as a temperature change, disease, drug, or the passage of time. Proteomics thus represents a "whole biosystem" approach to understanding the response of a cell or organism at a molecular level of detail. Such research will identify important biological pathways, and explore the role or response of proteins to a given event. New pathways, new protein interactions and new biomarkers are the most important outcomes. These new discoveries will ultimately lead to new targets used in screening for potential drugs using now-standard, ultra-high throughput, drug screening tests. Considering that past biomedical researchers may have devoted a large fraction of a career to understanding just one protein or one biochemical pathway, we can see that Proteomics represents an astronomical increase in the effort required if past methodologies continue to dominate the analysis of proteins. The challenge associated with this effort provides an opportunity for the development of new technology to accomplish the goals of Proteomics research with less laborious, more rapid, and more sensitive methods than are currently in use. Microsystem technologies such as microfluidic systems [3] and
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microarrays [4] are well suited to play a valuable role in this world-wide research effort. Success in determining the human genome came about in large part as the result of the new technology of multi-capillary gel electrophoresis being developed and commercialized [5]. This is a highly illustrative example of how a challenging project can spur new technology, as well as showing how crucially success can depend upon one or two major technological breakthroughs. The challenges associated with mapping and understanding the Proteome are probably a factor often greater than those borne by the Human Genome Project. Researchers in Proteomics will require several orders of magnitude improvements in sensitivity, sample size, and speed of analysis over current methods. Proteins are far more diverse in chemistry, structure and function than is DNA, necessitating more specific approaches to their chemical preparation and their analysis. Protein analysis frequently requires powerful separation methods, concentrations can be so low that preconcentration techniques are demanded, and the large range of hydrophobic and hydrophilic properties of proteins and peptides lead to drastically different characteristics that complicate the analysis. Yet, to analyze thousands of samples a day, a relatively generic group of platform methodologies are required. This challenge is significant, and it is anticipated that current methods, such as two-dimensional gel electrophoresis (2-d gel), will need to be replaced or drastically improved in terms of sensitivity, dynamic range, and separation power. Microfluidic systems clearly offer a number of advantages, many listed throughout this monograph, several of which are relevant to Proteomics research. Two important advantages are the reduced sample and reagent volumes required by microfluidic devices, and the increased analysis speed that results from the small dimensions associated with a chip [6]. Perhaps the key value of microfluidic systems in Proteomics will prove to be the integration of many sample processing steps together within one platform. Through incorporation of protein isolation, purification, digestion and separation steps within a chip, protein sample preparation can be done more rapidly, in an automated fashion. Automation can increase sample throughput significantly, and decrease the amount of highly trained manpower required per analysis. Such an advance would contribute significantly to attaining the goal of understanding the Proteome.
2. PROTEIN ANALYSIS METHODOLOGY Mass spectrometry (MS) has come to play a significant role in the analysis of proteins for Proteomics [7,8]. MS is capable of determining large numbers of proteins in a short time, utilizing small quantities of material. Typically, the
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proteins are separated in some fashion, then digested to give peptide fragments. These fragments are then analyzed by the mass spectrometer. Electrospray ionization MS (ESMS) involves sample ionization at the tip of a capillary as it flows towards an atmospheric pressure sampling inlet of a mass spectrometer. This method is well suited to on-line interfacing with a separation technique such as liquid chromatography (LC) or capillary electrophoresis (CE), allowing a final sample clean up and separation step. Often, a triple quadrupole mass spectrometer is used to perform tandem MS (or MS-MS). Coupling LC to ESMS-MS has become a standard approach to analyzing peptide mixtures derived from protein samples. Tandem MS provides protein sequence information, which is critical for the unequivocal identification of a protein. The first quadrupole MS is used to select a specific mass in the sample. The selected mass is then run through a quadrupole collision cell, in which high energy collisions cause it to fragment. These fragments are identified in the third MS. Quadrupole instruments are also paired with a time of flight MS to perform tandem MS, and these offer higher speed, sensitivity and resolving power than do triple quadrupoles. The other major MS method used in Proteomics is matrix assisted laser desorption (MALDI), which is usually coupled with time of flight MS (MALDITOF) for the analysis of samples deposited as spots on solid substrates. This method decouples the sample preparation step from the MS analysis stage, in contrast to on-line LC-MS methods, where the spectrometer waits to be supplied sample by the LC. MALDI plates can be prepared in advance, stored, and analyzed by the MALDI-TOF MS at the instruments optimal sampling rate, making more efficient use of the MS. . Until recently, MALDI-TOF could not offer as high a quality protein analysis as ESMS-MS, but developments in MALDI-TOF instrumentation, such as the addition of a quadrupole MS, mean that tandem MS can now be coupled with MALDI. Since MALDI is a faster method than is ESMS, MALDI-tandem MS can be expected to play an increasing role in Proteomics. The number of proteins and peptides that have been characterized by MS is continuously growing. The large volume of data generated has been pooled into databases such as Swiss-Prot (http://www.expasy.ch/sprot/sprot-top.html), which contain information about the sequence, function, domain structures, variants and post-translational modifications of a specific protein. The advent of protein databases has shifted the focus from "brute force" de novo protein sequencing techniques, such as tandem MS, to identification by comparison using peptide mass fingerprint analysis. This approach involves digesting a purified protein followed by MS analysis of the individual peptide fragments (i.e. the peptide mass fingerprint). The resulting characteristic pattern of peptides is compared with theoretical mass fingerprints, stored within protein databases, yielding possible matches for the identity of the unknown protein.
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Although peptide mass fingerprints can be generated in a matter of seconds to minutes, continuously supplying protein and peptides to the mass spectrometer fast enough becomes difficult. Protein sample preparation from the initial tissue, blood or cell culture represents a major bottleneck in MS analysis of proteins, and would benefit from greater automation. It is in the preparation of samples for the MS that microfluidic systems could play their greatest role. Protein sample preparation requires initial isolation of proteins from a biological sample. The key challenge, though, is the separation of the protein sample into individual proteins or peptides that can ultimately be presented to the MS. The classic approach has been to use the high resolving power of twodimensional (2-d) gel electrophoresis to separate proteins into as many as 2000 individual spots in a gel. Spots are then excised, proteins are digested with trypsin within the gel, salts and buffers are exchanged for optimizing the MS analysis, and the proteins are delivered to the MS. While this is the current workhorse of Proteomics, 2-d gel methods typically identify only 1000 to 2000 proteins, whereas as many as 15,000 to 30,000 may be expressed at any one time in mammals. Thus, the 2-d gel approach needs to be improved upon significantly. One more recent altemative is the shotgun approach, in which protein mixtures are digested directly, and then ion exchange and reverse phase chromatography are used to provide two dimensions of separation to resolve these extraordinarily complex mixtures before delivering them to an ESMS-MS system [9]. Another altemative uses specific isotopic labeling of proteins to provide mass tags that can be used to differentiate between samples prepared under differing conditions, such as cells cultured in the presence and absence of a drug. Aebersold and co-workers [10] have utilized the naturally abundant chlorine isotopes ^^Cl and ^^Cl (3:1 ratio) in a labeling reagent specific to cysteine residues in a peptide sequence. The observed masses, combined with auxiliary constraints, are then searched against sequence databases to identify proteins. The method works even when several proteins are co-migrating in a single spot of a 2-d gel. Regnier's research teams have developed combinations of ^O and *^0 with ^H and ^H isotopic labeling in order to [11] determine changes in abundance of proteins [11]. Such procedures are very promising, as they do not necessarily utilize 2-d gel methods, but they do require alternate separation procedures, such as LC or CE, followed by MS analysis. The need to automate, and to improve the sensitivity of these protein preparation procedures provides an attractive opportunity for microfluidic systems, which has not yet been significantly explored. The initial entree for microfluidic devices in Proteomics was as a sample input system for ESMS, as published independently by B. Karger's [12], M. Ramsey's [13] and R. Abersold's [14] research groups in 1997. They recognized that microfluidics and MS are surprisingly well matched, given that the nL volumes manipulated in microfluidics are similar to those required for sample
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introduction to the MS. These researchers showed that microfluidics could have a future as a sample processing and introduction method for MS, increasing the throughput of these costly instruments. The concept of a turn key proteomics system enabled by microfluidics is attractive, but clearly faces many technical challenges. Figeys [4] has outlined a set of multiple microfluidic stages for protein processing prior to introduction into a mass spectrometer, involving selective peptide capture, preconcentration by SPE and infusion into an ESMS, illustrated in Figure 1.
^"^ buffer 1
/
/
buffer 2
MS
sample elution affinity module
Fig. 1.
elutJon SPE module
buffer 3
Conceptual scheme illustrating the protein preparation and processing steps that could be performed within a microfluidic system in order to prepare samples for electrospray mass spectrometry (ESMS). Reprinted with permission from [15]. © 2001 Wiley-VCH.
More complex schemes can be envisaged, in which cells are introduced to a chip, lysed, protein is harvested, separated, digested, preconcentrated, separated again and then electrosprayed. This overall scheme, or some similar approach appears far off today. But, within the near future, we expect some subset of these steps will be performed routinely within microfludic devices. A number of the components needed have already been demonstrated. For example cell mobilization, lysis and enzyme analysis was first reported [16] at ^-TAS 98, and could be adapted to proteomics analysis. Immunoassays on bead beds were described by Oleschuk et al. [17] at ^i-TAS 2000 and by Kitamori's group [18], and the designs can be adapted to an affinity capture system for specific protein analysis as discussed below. Such a system could be easily coupled with cell lysis on-chip. Preconcentration elements, electrophoretic and electrochromatographic separations, and on-chip digestion methods are also under development [3,19,20]. A variety of other elements will undoubtedly be needed in the microfluidic toolbox to completely address the challenges of fully automating Proteomics within a microsystem.
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3. COUPLING ON-CHIP CAPILLARY ELECTROPHORESIS WITH ELECTROSPRAY MASS SPECTROMETRY The chip-ESMS interface has proven to be critical to translating on-chip separations to the MS. Several groups proposed chip-ESMS couplings differentiated by two different interface designs [12-14, 21]. These designs can be characterized as interfaces that "spray" directly from a micromachined interface such as an exposed channel at the side of a chip or a microfabricated nozzle, and those that use a capillary attached to the microchip as the electrospray emitter. The latter is similar to those used for capillary electrophoresis [22] and offers several advantages over spraying directly from the chip, the primary one being that good electrospray conditions are more readily achieved. A tapered capillary (end diam <10 ^m) produces small, welldefined droplets and stable, sensitive electrospray ionization [3]. Advances in micromachining, discussed below, should eventually allow comparable performance using nozzles fabricated directly on the chips. A sufficiently simple microfabrication method would probably confer manufacturing advantages compared to inserting capillaries. Dead volume is critical to the performance of the capillary sprayer design if separation is to be performed on-chip. Two methods of coupling developed in Harrison's laboratory [23,24], illustrated in Figure 2, were employed and compared in terms of dead volume and efficiency using laser induced fluorescence detection. Strikingly, only a 0.7 nL dead volume was required to lower the separation efficiency to 16-25 % of the predicted values [23]. However, the low dead volume coupling in Figure 2b produced 98 % of the predicted separation efficiency. The low dead volume connection preserves the separation that occurs on the chip prior to the connection, by preventing the peak (band) broadening associated with the capillary/chip junction. Figeys et al. [25] first demonstrated that the chip can be used to deliver not only simple samples, but also to introduce more complex samples such as tryptic digests of proteins. The combined chip-MS configuration provided sufficient resolution that the observed digestion fragments could be used to search a protein database, identifying the native protein. However, in that work no separation was performed on the chip, instead the resolving power of the mass spectrometer was relied upon. For relatively simple peptide mixtures this type of analysis is suitable. With more complex samples a separation step is required, in order to simplify the mass spectrum obtained enough for protein identification. The first on-chip separations of peptide mixtures coupled with ESMS were performed by Li et al. [24]. Chip-ESMS and tandem-MS analysis of a peptide mixture utilizing integrated CE for separation gave efficiencies as high as 300,000 plates/meter. Resolution in an analysis of a tryptic digest, using only 150 fmol of protein, was sufficient to allow the protein to be identified when
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comparing digestion fragments to a protein database. Chip-ESMS-MS analysis allowed the peptide sequence to be identified with a 45 amol/mL concentration detection limit. A similar design developed by Karger's group was utilized to separate and analyze a simple peptide mixture and a tryptic digest of cytochrome c [26]. These early studies established that Karger, Ramsey, and Aebersold's concept of using microfluidic chips to supply an MS with sample were feasible, and that high quality results could be obtained. This opened the way for integration of even more sample preparation steps, leading towards microfluidic systems that can compete with conventional approaches to Proteomics.
laration channel
capillar^ dead volume approx. 0.7 nl
separation channel capillar)' H
edge of device Fig. 2.
A very low dead volume connection design is needed to interface a microfluidic chip to electrospray ionization source for mass spectrometry (MS), if a separation performed on chip is to be successfully transferred to the MS. The upper image shows a small dead volume of 700 pL, the lower image appears to have zero dead volume. Reprinted with permission from [23]. © 1999 American Chemical Society.
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Although a capillary attached to a chip has proven to be suitable for highresolution separations, it still requires substantial machining to fabricate and prepare the device. A more attractive alternative would be to fabricate the electrospray tip/nozzle within the structure of the microchip using the remarkable capabilities of modem micromaching [21]. Methods to fabricate electrospray tips as part of a chip [21] should eventually provide robust devices for electrospray chip-MS. Research by Craighead and Knapp's groups for example has illustrated the potential for fabricating electrospray nozzles/tips in plastic devices [27,28]. The creative efforts of these and other groups to extend plastic fabrication methods for electrospray microchips is likely to be an important development in moving chip-ESMS towards a commercial reality. 4. MICROCHIP SYSTEMS FOR MALDI-TIME OF FLIGHT MASS SPECTROMETRY While most of the chip-MS couplings have used ESI as the method of ionization. Little et al. [29] were the first to develop a method utilizing matrix assisted laser desorption ionization (MALDI) with samples for eventual MALDI-MS analysis. MALDI-MS has become a workhorse for the proteomics industry providing precise molecular weight information for analyses of proteins and peptides excised from 2-d gels. Advancements such as the quadrupole-TOF instrument mean that MALDI can also be used directly for protein sequencing now. In Little et. al's study, a 100-element array of micro-wells was microfabricatied. DNA samples were administered with a piezoelectric pipette, then each well was subjected to MALDI analysis, yielding 100 mass spectra from a sample device less than I in 2. This design and others [30,31] demonstrate another possible application of microsystem technologies for rapid sample preparation and introduction to mass spectrometers. Because of the ability of MALDI-MS methods to operationally decouple the sample preparation step from the MS analysis stage in the lab, we can expect that the use MALDI will continue to grow in importance. As a consequence, the opportunity for Microsystems technology in this area may become significant. 5. PROTEIN SAMPLE PREPARATION ON MICROFLUIDIC CHIPS Preparation of protein samples involves many different processes, and there will not be one generic method in the future, in contrast to the dominance of just a few methods in DNA analysis. Key steps required in preparing samples involve the initial protein isolation, subsequent separation of proteins, physically as in a gel, or chemically using methods such as isotope labeling, digestion to
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peptides, and adjustment of the solvent and solute conditions to optimize subsequent MS analysis. Many of the specific methods used in sample preparation can be integrated within microfluidic systems. The integration of a CE separation step with ESMS has already been discussed above, and represents a key component of most sample preparation methods. Other methods discussed below include sample preconcentration, by electrokinetic means and by more traditional solid phase extraction techniques, as well as protein digestion, and affinity separation methods. We perceive the integration of each of these methods as an expansion of the microfluidics toolbox available for automating Proteomics on a chip. Preconcentration is particularly important for protein samples that are to be analyzed by MS. Ramsey and co-workers [32] were the first to demonstrate the use of sample stacking on a chip, obtaining concentration enhancements of about 10 times for dye samples. Since then the method has been directed toward protein preparation by Li et al. [33], who utilized large volume sample stacking methods on-chip to preconcentrate peptides and tryptic digests of proteins isolated from 2-d gel separations. Figure 3 illustrates the stacking procedure. buffer
buffer
buffer
sample waste
W^^^
sample
t
to MS
injection Fig. 3.
stacking
separation
Electrokinetic sample stacking can be used to concentrate a sample prior to performing ESMS. This diagram shows the steps required to focus a 10 nL volume into about 0.5 nL, prior to performing capillary electrophoresis (CE) and ESMS. Concentration detection limits were improved by a factor of about 20 in this way. Reprinted with permission from [33]. © 2000 Wiley-VCH.
A sample is introduced in low ionic strength buffer and directed towards the MS. After the separation channel (about 10 nL) is filled, the flow is
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reversed, and a higher ionic strength buffer is directed back along the separation channel towards sample waste, causing sample stacking to occur within a final volume of about 0.2-0.5 nL. The process is automatically stopped when the current flow reaches about 99% of maximum, indicating the separation channel is nearly full of the more conductive, high ionic strength buffer. The flow direction is reversed again and running buffer is delivered, in order to induce separation as the sample is directed towards the MS. Detection limits for model peptides such as bradykinin and somatotatin could be reduced from about 20 nM to about 0.5 nM. Low intensity spots in 2-d gels could be digested in situ, preconcentrated using sample stacking and measured directly by electrospray MS from the chip. While such large volume sample stacking can be accomplished in a conventional capillary, these procedures are far easier to automate within a multi-channel network in a microfluidic device. Figeys' et al. [34] were the first to use microchip-ESMS systems with solid phase extraction (SPE) on a hydrophobic phase for preconcentration of proteins, demonstrating the value of this step on an on-line processing system. They coupled on-chip generation of solvent elution gradients with off-chip SPE in a capillary located between the chip and the ESMS interface. Li et al. [33] tested the coupling of a pre-chip solid phase extraction device coupled to an entry port of a chip for subsequent CE separation and electrospray. Detection limits with SPE were 2.5-20 nM depending upon the peptide's hydrophobicity. Notably, even though the total volume of solution concentrated (1.5 |iL concentrated into 25 nL elution volume) was many times higher than for the electrokinetic stacking method described above, the detection limits were poorer. This discrepancy arose in part from the mismatch of off-chip on on-chip volumes. They concluded that integration of SPE within the microchip structure should provide considerable improvement in efficiency of sample utilization with SPE if a microfluidic system is also to be used in subsequent steps. More recently, Li et al. [35,36] have integrated a soHd phase extraction bed into a CE-ESMS chip and interfaced that to a tandem MS system and to an autosampler. This design embeds the microfluidic system within a larger laboratory system, illustrating the concept that the chip can play a key role as part of the lab, not act as a full replacement for the lab. (Which is a liberating perspective compared to the misconception that a micro total analysis system must integrate all components needed for analysis, in order to be worthwhile.) In the system they developed protein is first separated in a one or two dimensional gel system. Protein spots are then excised, transferred to a microtitre plate and digested by trypsin within the gel. The released peptides are transferred by capillary to the SPE bed on the chip, eluted by a change in solvent, and introduced to the CE channel on the chip. This stage provides a desalting step to optimize the sample for ESMS, along with concentrating the sample to improve sensitivity. The subsequent CE step separates the peptides.
259
allowing the MS a better chance to correctly identify them. When coupled to a tandem MS system, peptide sequencing and unequivocal mass identification is possible. Figure 4 shows a composite diagram illustrating the system. The autosampler-CE-ESMS-MS system was able to process samples at a rate of 2 min/sample, including a washing step.
transfer
.j„nn
jiMR
96-well plate
concentrate & elute
mass/charge Fig. 4.
Diagram illustrating a microfluidic chip which performs preconcentration, CE, and ESMS, embedded within a larger protein analysis system. Protein extracts are subjected to 1- or 2-d gel electrophoresis, spots or bands are excised and digested in a microtitre plate, then an autosampler delivers them to the chip for the final analysis steps.
It was successfully tested with a variety of real samples, such as 25 p.g of soluble proteins extracted from N. meningitides, subjected to 1-d gel electrophoresis. The SPE concentration step added 3-5 min/sample for a sample volume of 8-10 |il, and an elution volume of 200 nL. The system was tested with the SPE stage included using 300 (ig of cell lysate from the prostate cancer line, LNCaP, subjected to 2-d electrophoresis. In the latter case over 70 gel spots were analyzed by tandem MS in stand-alone, automated fashion by the system, with identification of over 85 % of the proteins in the primary analysis step. Figure 5 illustrates the results for a few protein spots. This automated system was shown to have a limit of quantitation (signal = 10 times std. dev.
260
noise) of 5 nM for leu-enkephalin with a 5 times concentration step (1.0 |iL reduced to 200 nL), and 10 % rsd in peak heights. On-C/7/p Preconcentration ^ MS/MS &CE
ExcisQ
21>gel of total cell lysate from LNCaP cells Mr (kDa)
(300 pg)
BPI(m/2 450-1400)
250^ 150i» 0.4 64 ~ V ^
0.8 ""^
1.2
1.6 2.0 Time, min
2.4
2.8
iiiiiillii I III
2 5 V»
7-* gM^e 25% Sd^^
. . ^14
*^'^r
500 600 700 \ 800 900 Ubiquinol'Cytochrome ^ 816 36 C reductase (47985) 701.331 213.09
1000
457.21 554.25 913.44
f*
•rt-T-iir-ri-^
100 200 300 400 500 600 700 800 900 1000
Fig. 5.
m/z
Diagram showing the analysis of 300 ^g of cell lysate from the prostate cancer line, LNCaP, by 2-d gel electrophoresis. Over 70 spots were analyzed in automated fashion by the system shown in Figure 4. The base peak ion (BPI) electropherogram is shown for spot 14, along with a mass survey scan taken at 2.57 min, and a tandem mass spectrum of the m/z ion 506.7. Reprinted with permission from [36]. © American Soc. Biochem. Molec. Biol. Inc.
More selective forms of sample preconcentration can also be thought of as separation methods. Li et al. [36] have demonstrated affinity chromatography on-chip, and integrated it with CE-ESMS, using a bed packed with a selective adsorptive phase. In one example an antibody was attached to beads in a bed within a chip. The antibody was selective for a peptide sequence, GEQKLISEEDLN, generated by the human proto-oncogene loiown as c-myc, which is involved in cellular transformation, apoptosis, and cell-cycle progression. Figure 6 shows the reconstructed ion electropherogram (the signal obtained by the MS as a function of time at a specified mass) and the spectrum for this sequence. The analysis was performed on a human blood serum sample, which was spiked to 50 pg/mL with the peptide then dialyzed against a 100 kDalton membrane to remove endogenous antibodies, before introduction to the
261
affinity bed on the microchip. The high quahty of the data illustrates the ability of on-chip affinity methods to produce extremely well cleaned up samples for MS analysis, despite the high protein and salt content of the sample. The same study [36] also explored the performance of an immobilized metal affinity column (IMAC) on-chip. These beds selectively bind phosphorylated peptide fragments; they are used to enrich samples that contain this common posttranslational protein modification. Figure 7 illustrates the enrichment achieved for a 2 picomole a-casein sample. The sample was prepared in a 1-d separation gel, in gel digested, then introduced to the chip. The 2 picomole sample was enough to allow tandem MS analysis, providing unambiguous identification of the site of phosphorylation on the protein. This is similar to the amount required by current methods, indicating competitive performance by the chip system. 0.90
687.30
iiUi
— I —
0.2
1.0
0.6
time (min)
a Fig. 6.
1.4
600
800
1000
1200
m/z
b
Analysis of the human proto-oncogene c-myc in a human blood serum sample, performed on a chip like that in Figure 4. An anti-c-myc loaded packed bed for affinity capture and purification of the sample was employed in the entry channel of the chip. A reconstructed ion electropherogram for a m/z value of 687.30 a), and the extracted mass spectrum of the electropherogram peak b) are shown. Adapted with permission from [36]. © American Soc. Biochem. Molec. Biol. Inc.
The embedding of a microfluidic system within a larger automated process in a laboratory is a significant step in demonstrating that microfluidics can meet the challenge presented by the very difficult samples associated with protein analysis. In the work described above the chip system was about twice as fast per sample as current conventional LC-ESMS-MS systems, and the analytical performance was as good as that obtained with current methods. It is important that any new technology not represent a decrease in performance if it is to be adopted. Further, since the work above represents just the initial attempts to introduce microfluidics into automated protein analysis, we anticipate that further improvements in performance and speed of analysis will be possible. Thus, microfluidics appears to be competitive with current
262
approaches. The abihty to integrate more protein processing steps into the microfluidics platform will then confer a significant advantage to the microfluidics approach to automating protein analysis. 577.1
a-casein no IMAC selection 621.1 639.
831.1
595.1
787.1 769.1
656.1 *^<<..,.iiii.,i
Li iLllBk 1iLiiii I till iiii..i,U 600
500
• 813-11 i,L»b 111
700
919.\ ii.
*
I
800
900
T14-15(1P)
T7 (2P)
964.3
T14-15 (IP)
T15(1P)
770.3
••u^LAi.j...Lii.^.Lf.^,.|. 500
1000
_ 972.3 • ^ 977.0
651.3
with IMAC selection
\
600
830.9 .. L.J.J.L.ij
il 700
800
900
li
1000
m/z Fig. 7.
Mass spectra obtained for 2 pmoles of a-casien with (lower), and without (upper), the use of an immobilized metal affinity column (IMAC) on-chip for selective isolation of phosphorylated peptide fragments (marked with *). Significant enrichment is clearly obtained using the on-chip process. Adapted with permission from [36]. © American Soc. Biochem. Molec. Biol. Inc.
Separation is an important component to protein preparation. Capillary electrophoresis on-chip has been the dominant method explored for subsequent ESMS, as a one-dimensional format. Several of the affinity bed separations discussed above represent beginning steps at adding two dimensional separations to the chip repertoire. However, these do not provide sufficient resolving power for generic analysis of complex protein and peptide samples generated by tryptic digests. Recently the first two-dimensional separations have been performed in a microfluidic manifold with enough resolution to provide a
263
separation for peptidic digests. Ramsey and co-workers demonstrated [37] a 2-d separation of TRITC-labeled tryptic peptides of jS-casein, shown in Figure 8. The separation involved both open-channel electrochromatography using a micellar electrokinetic format, and capillary zone electrophoresis. The samples were fluorescently labeled, in order to use fluorescence detection, as the system was not yet interfaced to a mass spectrometer. These separations are not truly orthogonal, but the work demonstrates the feasibility of performing 2-d separations within a microfluidic environment. Further developments in chromatography on chip will increase the ability to perform orthoganol separations on-chip in the future, further enhancing the microfluidics toolbox for Proteomics.
OCEC
•^
I
700
2D
0)
E
® O O O
500-1
ll
300-L
SW1 1.0
a Fig. 8.
1.4
1.8
CE miration time [s]
5 cm
b
a) Photograph of a 2-d separation chip, which perforais micellar electrokinetic chromatography in a first stage of separation, followed by capillary zone electrophoresis in a second stage, b) Data traces show the 2-d separation results obtained with fluorescence detection on labeled peptides. Reprinted with permission from [37]. © 2001 American Chemical Society.
6. PROTEIN DIGESTION ON-CHIP Enzymatic digestion of proteins with trypsin is an extremely common step in protein identification or sequencing. Integration of the digestion step into a microfluidic device reduces sample losses and should increase overall sample utilization. The use of a packed trypsin bead bed for digestion reduces problems with auto-digestion of trypsin and consequent sample contamination. Wang et
264
al. [38] have reported an on-chip trypsin loaded, packed bed about 2 cm long, 1 mm wide and 0.15 mm deep, into which several |iL of sample are fed by a syringe pump. The products collected are then analyzed downstream by CEESMS. Figure 9 illustrates the separation of cytochrome c digested on-chip. Cytochrome c 4 |jl/min
Cytochrome c
2 |jl/min
1 |jl/min
0.5 Ml/min
time (min) Fig. 9.
Total ion current electropherogram obtained following on-chip digestion of cytochrome c with a trypsin digestion bed, using the chip in Figure 4. Traces show the effect of pumping speed, and thus digestion time, on digestion of 3 ^L of 16 ^M cytochrome c. The parent protein is completely consumed after only 3 min of digestion.. Reprinted with permission from [38]. © 2000 John Wiley & Sons, Ltd.
The digest may also be delivered continuously to the MS, i.e. infused. The digestion time required to fully consume cytochrome c was about 3 min, in contrast to conventional solution phase digestion off chip, which does not fully consume cytochrome c in 2 h. Digestion of the simple peptide, melittin, took only 5 s to reach the same stage as that seen for 15 min of off-chip digestion using trypsin loaded beads. This dramatic increase in digestion speed is the result of the high trypsin concentration that is achieved by loading it onto the beads, and the efficient mass transfer obtained in a flow through packed bed. While these advantages are not specific to a microfluidic chip, the volume of the chips is well matched to that required to the MS, leading to efficient sample utilization. These packed digestion beds could also be integrated with SPE beds on the same chip, in order to enhance sensitivity. Figure 10 illustrates the sensitivity difference observed for cytochrome c when digestion was performed
265
on-chip, with and without SPE. Preconcentration recovery values of 90-105 % were attained for most peptide fragments, depending upon the specific hydrophobicity of the peptides. The results demonstrate the ability to integrate an increasing number of the desired protein preparation processes within a microchip system.
T
• 200 nM cytochrome c + trypsin D polymer bonding
0.2
1 o
With SPE
•4-i«
c
D
O o
_J
\m
Wjv^HvviwJlk* without SPE
wJySllJ 600
700
800
900
1000
m/z Fig. 10. Mass spectra obtained for on-chip digestion of 200 nM cytochrome c with (upper) and without (lower) an on-chip solid phase extraction stage. The chip was similar to that in Figure 4, but contained an extra bed, loaded with solid phase extraction beads, at the sample inlet stage, downstream of the digestion bed. Reprinted with permission from [41]. © 2002 Kluwer Publishing.
7. MULTIPLEXING ON A CHIP FOR MASS SPECTROMETRY Multiplexing is an aspect of chip-MS coupling that has remained mostly unexplored, while holding significant promise for proteomic applications. The ability to handle multiple samples (multiplexing) using microchip devices, either simultaneously or sequentially, is one of the most significant advantages offered by a microfluidic platform. The first example of multiplexing combined with chip-MS involved spraying directly from the edge of the chip, utilizing 6
266
independent devices on one wafer, with 6 separate spray orifices [12]. Aebersold and coworkers showed an example of automating the continuous flow analysis of three [14] and nine [34] samples on a single chip using a single electrospray outlet, as shown in Figure 11.
Fig. 11. Layout of a 9-sample multiplexing chip. Reprinted with permission from [25]. © 1998 Am. Chem. Soc.
Voltages applied to each sample reservoir sequentially caused each protein sample to flow into and through the effusion channel and out through a single electrospray needle into the MS. While the three-channel design suffered from cross contamination of samples, caused by flow leakage and siphoning effects, the nine-sample configuration produced much lower cross contamination. Careful consideration of leakage affects should help reduce cross contamination and increase the number of samples that can be placed on a single microfluidic device. Although combining multiplexing with MS detection has so far been limited to directly infusing pre-prepared samples, others have demonstrated complex separations in a multiplexed format. These have included immunoassay on a 6 manifold device[39], as well as the very aggressive incorporation of 96 and 384 individual separation channels on one 4 inch diameter device for DNA separation [40]. These demonstrations illustrate the concept is certainly achievable for chip-MS systems in the future. Taylor et al. [41] have in fact presented a 20-channel protein preparation chip-ESMS design at n-TAS 02. While it has not been fully characterized at this stage, the design shows how a variety of the sample preparation steps discussed above could be integrated into a high throughput, automated microchip system in the future. It is clear that multiplexing capillary gel electrophoresis was a key to rapidly analyzing the human genome [5]. It is likely that significant further effort in multiplexing protein analysis on-chip will be required to truly deliver the promise that microfluidics offers to automate the analysis of thousands of Proteomics samples.
267
8. MICROCHIPS FABRICATED IN POLYMER SUBSTRATES The advice given in the movie The Graduate that "the future Ues in plastic" is indeed excellent advice for many applications of microfluidics. To date most devices have been prepared in glass substrates, although plastic devices offer major benefits in terms of cost, particularly for single-use, disposable chips. Polymers can be fabricated using relatively inexpensive protocols such as micromolding, injection molding, laser ablation or embossing [42]. They are available with greatly differing chemical, optical and mechanical properties compared to glass, providing a wide range of properties to draw from. Considerable effort has focused on polydimethylsiloxane (PDMS) as the substrate material [43], because of its remarkable ability to mold features as small as a few nanometers. It is also self-sealing, obviating the use of adhesives to bond the cover layer. However, PDMS devices are best suited for demonstrating the feasibility of using polymers, since the surface of PDMS is extremely hydrophobic, causing it to be incompatible with a wide range of analytes. The solid phase micro-extraction (SPME) literature is replete with studies in which PDMS is used as a sorbent phase for many chemicals [44], leading to problems if it is used for a chip in which surface adsorption is to be avoided. The other two polymers explored extensively for microfluidic devices are poly(methymethacrylate) and polycarbonate [42]. These are likely to be much more suitable for commercially produced devices than PDMS, due to greater solvent and sample compatibility. There is an extensive knowledge base in plastic fabrication of small scale parts, such as micro-lenses and other optical components. However, the tolerances required are not the same as those for microfluidic devices, and a sound basis for manufacturing-friendly microfabrication procedures for microfluidics is only just coming into place. Processes tend to be held as proprietary, and procedures published for bonding plastic materials, stamping, or molding them by other means, may not prove easy to reproduce between labs as yet. One can expect this situation to change in the next five years, as the technology matures. More problematic may be the solvent compatibility issues. Many plastics swell in organic solvents, and they adsorb a variety of chemicals. While adsorption is also a problem with glass surfaces, it has very high solvent compatibility, and procedures for glass fabrication, device bonding, are well known. The factors that control the choice of glass are also reasonably well demonstrated, whereas the difference between two manufacturer's polymer products are much less apparent at present. For this reason, glass chips are likely to continue to be used in the near ftiture for microfluidic devices that analyze multiple samples. But a tidal wave of plastic chips can be expected in the fiiture once some of the above challenges arefrillyaddressed.
268
9. SUMMARY The minute sample size required by microfluidic devices, combined with extremely fast analysis times, make these devices ideal for filling the analysis bottleneck afforded by current analytical systems. As discussed above, key protocols required for a turnkey proteomics system have already been demonstrated. Many of the additional components needed to perform more fully integrated Proteomics on a chip have also been demonstrated; that is to say, the toolbox has been greatly expanded over the past few years. Significantly, many of the individual sample processing elements that were developed over the past decade have been integrated together, creating true systems for sample processing. These microfluidic devices have been embedded within a larger laboratory system and used to automate sample preparation. Importantly, the performance of these systems has been as good or better than conventional methods, despite the fact these are still laboratory prototypes. This success provides strong motivation for furthering the effort to use microfluidic systems in Proteomics. Continuing research on two-dimensional separation methods onchip, on integrating increasing numbers of processing steps within the chips, and on manufacturing the chips at lower cost in plastic materials should pave the way to the creation of turn key Proteomics systems on chips that will spur Proteomics research significantly. In 1992 Zelazney and Thomas [45] discussed a microfluidic system that "involved staining and spooling the genetic material into ever-flowing fluid channels which were capillaried into silicon control blocks no wider than a human hair. ... Target (virus or cell) strains no longer grew; they were immediately and continuously manufactured'' This prescient science fiction text in Flare combined proteomics and genetics in a microfluidic device to manufacture organisms and biochemicals directly. In fact, slightly before the publication of Flare^ early papers on microarrays and microfluidic devices [6,19,20] had shown it was possible to synthesize DNA on a chip and to manipulate flowing streams and separate chemicals within capillaries narrower than a hair. This chapter has presented a number of integrated protein processing systems that can play a major role in developing our understanding of biochemistry enough to help realize the remainder of their science fiction vision.
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