Application of Microfluidic Devices to Proteomics Research

This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 μl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of ∼5 nm (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2–1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system. This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 μl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of ∼5 nm (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2–1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system. Proteomics research entails the global characterization of proteins expressed in cells under defined conditions. Such studies are particularly important in view of the conflicting evidence regarding the correlation between the abundance of expressed proteins and gene-expression levels obtained from mRNA microarrays (1Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar, 2Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar). The monitoring of protein expression profiles remains a very challenging task because of the wide dynamic range of expressed proteins and the variability of gene products (splicing variants, N- and C-terminal truncations, co- and post-translational modifications, etc.), which may change between and within the tissues of an organism (3Harry J.L. Wilkins M.R. Herbert B.R. Packer N.H. Gooley A.A. Williams K.L. Proteomics: capacity versus utility.Electrophoresis. 2000; 21: 1071-1081Google Scholar). The traditional approach to isolating and characterizing proteins from biological samples has been separation by two-dimensional gel electrophoresis (2-D 1The abbreviations used are: 2-D, two-dimensional; 2-D gel, 2-D gel electrophoresis; MS-MS, tandem mass spectrometry; LC, liquid chromatography; BCQ, [(acryloylamino)propyl]trimethylammonium chloride; TEMED, N,N,N′,N′-tetramethylethylenediamine; DMP, dimethyl pimelimidate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RIE, reconstructed ion electrophoregram(s); IMAC, immobilized metal affinity chromatography; CE, capillary electrophoresis; nESMS, nanoelectrospray mass spectrometry. 1The abbreviations used are: 2-D, two-dimensional; 2-D gel, 2-D gel electrophoresis; MS-MS, tandem mass spectrometry; LC, liquid chromatography; BCQ, [(acryloylamino)propyl]trimethylammonium chloride; TEMED, N,N,N′,N′-tetramethylethylenediamine; DMP, dimethyl pimelimidate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RIE, reconstructed ion electrophoregram(s); IMAC, immobilized metal affinity chromatography; CE, capillary electrophoresis; nESMS, nanoelectrospray mass spectrometry. gel), followed by identification of protein spots using sensitive mass spectrometry techniques and data base searching (3Harry J.L. Wilkins M.R. Herbert B.R. Packer N.H. Gooley A.A. Williams K.L. Proteomics: capacity versus utility.Electrophoresis. 2000; 21: 1071-1081Google Scholar, 4Abbott A. A post-genomic challenge: learning to read patterns of protein synthesis.Nature. 1999; 401: 715-720Google Scholar, 5Quadroni M. James P. Proteomics and automation.Electrophoresis. 1999; 20: 664-677Google Scholar). An alternate approach to 2-D gel includes a comprehensive chromatographic separation of the proteolytic fragments derived from intact proteins, followed by mass spectral identification and data base searching. This can be achieved using a two-dimensional liquid chromatography approach whereby peptides are fractionated on a strong cation exchange column, followed by an extended gradient elution on a C18 reverse phase column (6Opitek G.J. Lewis K.C. Jorgensen J.W. Moseley M.A. Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.Anal. Biochem. 1998; 258: 349-361Google Scholar, 7Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar). This two-dimensional chromatography approach has been described recently for the comprehensive identification of the yeast proteome (6,113 proteins) and provided the identification of 1484 proteins among which were 131 proteins with three or more predicted transmembrane domains (8Washburn M.P. Wolters D. Yates III, J.R. Large scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Google Scholar). Although questions remain concerning the ability of the 2-D gel approach to analyze hydrophobic proteins and to quantitate and characterize the full dynamic range of protein expression from a given genome (9Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional gel electrophoresis based proteome analysis technology.Proc. Nat. Acad. Sci. U. S. A. 1999; 97: 9390-9395Google Scholar), protein identification via 2-D gel and mass spectrometry is still widely used in numerous proteomics core facilities. This approach enables the visualization of a very large number of proteins simultaneously, and in contrast to 2-D chromatographic separation it facilitates the identification of post-translational modifications and proteolytic processing in a convenient reference format. Furthermore, differential protein expression profiles can be compared easily for large data sets and for protein extracts obtained under different growth conditions, biological perturbations, or following pre-fractionation through organelle-enrichment methods. Unambiguous identification of gel-isolated proteins typically relies on sensitive tandem mass spectrometry (MS-MS) techniques to obtain partial amino acid sequences, which in combination with the mass of the precursor ion and that of the unidentified N- and C-terminal segments, can be used to search protein data bases (10Eng J.K. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass spectra data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Google Scholar, 11Mann M. Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags.Anal. Chem. 1994; 66: 4390-4399Google Scholar, 12Mørtz E. O'Connor P.B. Roepstorff P. Kelleher N. Wood T.D. M. identification of intact proteins by tandem mass spectral data sequence data Nat. Acad. Sci. U. S. A. Scholar). The combination of column to nanoelectrospray has also a widely used for proteomics the identification of fmol of digests with a of T.D. protein identification using a on an ion mass Am. Soc. Mass Spectrom. 1998; Scholar, and R. in of the for Mass Scholar). The of microfluidic to mass spectrometry also an of liquid separation and sensitive detection on of microfabricated or liquid chromatography as the analysis in the of and separation The interface to mass spectrometry has been achieved by a channel to the nanoelectrospray using capillary D. Aebersold R. gradient from a microfabricated device for protein by mass Chem. 1998; Scholar), the for capillary mass Chem. 1999; Scholar), Zhang P. of with an array of for mass Chem. 2000; Scholar), and N.H. P. to with dead Chem. 1999; Scholar, J. P. N.H. of microfabricated to capillary mass spectrometry using a low dead volume application to rapid analyses of proteolytic Chem. 1999; Scholar). on microfluidic been demonstrated using S. nanoelectrospray with mass spectrometry Chem. 1999; Scholar, J. P. separation of peptides from gel-isolated proteins using a microfabricated device to a quadrupole/time-of-flight mass Chem. 2000; Scholar, D.M. Y. D. for an microfluidic to an mass for protein 2000; 21: and ion D. Aebersold R. gradient from a microfabricated device for protein by mass Chem. 1998; Scholar, for capillary mass Chem. 1999; mass for D. Aebersold R. gradient from a microfabricated device for protein by mass Chem. 1998; Scholar, Zhang P. of with an array of for mass Chem. 2000; Scholar, J. P. separation of peptides from gel-isolated proteins using a microfabricated device to a quadrupole/time-of-flight mass Chem. 2000; Scholar). or can also be prior to electrophoretic separation to J. 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Although μl of was to the large channel the of this approach was by the volume on the device this in low and was of for spots obtained from 2-D gel electrophoresis where of protein is typically the of the C18 beads were the large also a to a thus with used for and processing J. and P. in of the for Mass Scholar). This which is in enables the and of more μl of tryptic via a large channel of low to by the and protein in 20 μl of volume thus providing for injections this be indicated in is an channel of that the channel D. 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G.L. Roepstorff P. of the of and differential mass peptide mass for in proteins separated by gel 2001; indicated that comprehensive typically low levels of gel-isolated proteins using affinity on were using both and as A was obtained on phosphopeptides using of was achieved using with studies P. affinity chromatography of Chem. 1999; Scholar). 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The also demonstrated the application of beads for the selection of phosphopeptides from Such studies a level of sequence compared with protein identification using peptide mass or and low levels of tryptic digests are typically to obtain M.R. G.L. Roepstorff P. of the of and differential mass peptide mass for in proteins separated by gel 2001; Scholar). Although demonstrated the of beads on microfluidic for the affinity of phosphopeptides from of of the approach be of application in view of the low abundance of from extracts and variability in this are affinity using of followed by the of on the A. and P. in of the for Mass Scholar). The microfluidic a number of in of and of of media for preconcentration, on-line R. J. P. of immobilized for protein within a microfluidic capillary electrophoresis and an mass spectrometry Mass Spectrom. 2000; Scholar), or of target through affinity Although the device was to a of human prostatic it that the device to a quadrupole/time-of-flight device can a in the of protein identification in proteomics The of modular on the microfluidic device also to a where the different injection, and can be in a comprehensive for for in the of prostatic for in the of the and J. R. for providing the

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