A microfluidic reactor for rapid, low-pressure proteolysis with on-chip electrospray ionization (original) (raw)
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Microfluidic systems in proteomics
…, 2003
We present the state-of-the-art in miniaturized sample preparation, immunoassays,one-dimensional and multidimensional analyte separations, and coupling of micro-devices with electrospray ionization-mass spectrometry. Hyphenation of these differ-ent techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are alreadyclose to fulfill the requirements for proteomics, and that miniaturization results at thesame time in a dramatic increase in analysis throughput. Throughout this review,some examples of analytical operations that cannot be achieved without micro-fluidics will be emphasized. Finally, conditions for the spreading of microanalyticalsystems in routine proteomic labs will be discussed.
Molecular & Cellular Proteomics, 2002
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 l 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 C 18 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 ϳ5 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 gelisolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system.
Lab on a chip, 2012
A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic microreaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, nonspecific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.
Analytical Chemistry, 2005
The present investigation describes the analytical performances of a microfluidic device comprising an enrichment column, a reversed-phase separation channel, and a nanoelectrospray emitter embedded altogether in polyimide layers. This configuration minimizes transfer lines and connections and reduces postcolumn peak broadening and dead volumes. This compact and versatile modular nanoLC-chip system was interfaced to both ion trap and time-of-flight mass spectrometers, and its analytical potentials were evaluated in the context of proteomics applications. The figures of merit of this system in terms of peak capacity, reproducibility, sensitivity, and linear dynamic range of peptide detection were determined using tryptic digests of complex protein extracts including albumin-and immunoglobulin-depleted rat plasma samples. The analysis of peak profiles for more than 600 peptide ions reproducibly detected across replicate nanoLC-chip-MS runs (n ) 10) indicated that this system provided good reproducibility of retention time and peak intensity with RSD values of less than 0.5 and 9.1%, respectively. Variation in peptide abundance as low as 2-fold changes was identified for spiked tryptic digests present at levels of 2-5 fmol in plasma samples. Sensitivity measurements were performed on dilution series of protein digests spiked into rat plasma samples and provided a detection limit of 1-5 fmol. The modular concept of the microfluidic systems also facilitated the integration of two-dimensional chromatography (strong cation exchange/C 18 ) thereby increasing the sample loading and selectivity of the nanoLC-chip-MS system. The application of this integrated device was evaluated for complex rat plasma samples to compare the number of protein identifications obtained using one-and twodimensional nanoLC-chip-MS/MS. Zhou, H.; Lin, H.; Roy, S.; Shaler, T. A.; Hill, L. R.; Norton, S.; Kumar, P.; Anderle, M.; Becker, C. H. Anal. Chem. 2003, 75, 4818-4826. (9) Radulovic, D.; Jelveh, S.; Ryu, S.; Hamilton, T. G.; Foss, E.; Mao, Y.; Emili, A. Mol. Cell. Proteomics, in press.
Microfluidics with MALDI analysis for proteomics—A review
Analytica Chimica Acta, 2009
Various microfluidic devices have been developed for proteomic analyses and many of these have been designed specifically for mass spectrometry detection. In this review, we present an overview of chip fabrication, microfluidic components, and the interfacing of these devices to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. These devices can be directly coupled to the mass spectrometer for on-line analysis in real-time, or samples can be analyzed on-chip or deposited onto targets for off-line readout. Several approaches for combining microfluidic devices with analytical functions such as sample cleanup, digestion, and separations with MALDI mass spectrometry are discussed.
Rapid desalting of protein samples for on-line microflow electrospray ionization mass spectrometry
Analytical Biochemistry, 2005
The accurate determination of protein masses is important for many applications, ranging from the veri-Wcation of the expression of recombinant proteins to the detection of posttranslational modiWcations. We describe here a semiautomated and highly reliable method for rapid desalting of protein samples for online microXow electrospray ionization (ESI) 1 mass spectrometry. A homemade reversed-phase trap column was coupled directly to a computer-controlled, 2-position/10port high-performance liquid chromatography (HPLC) valve with a two-solvent delivery system. The trap column gives a minimal backpressure during desalting with aqueous solvent at a relatively high Xow rate (400 l/ min). In addition, the small bed volume of the trap column ensures rapid elution with organic solvent at a much lower Xow rate (20 l/min) optimal for electrospraying. We demonstrate that our setup allows the rapid analysis of proteins of a wide molecular weight range within 2 min with a sensitivity in the low picomole range.
Top-down proteomics on a microfluidic platform
2019
Protein identification and profiling is critical for the advancement of cell and molecular biology as well as medical diagnostics. Although mass spectrometry and protein microarrays are commonly used for protein identification, both methods require extensive experimental steps and long data analysis times. Here we present a microfluidic top down proteomics platform giving multidimensional read outs of the essential amino acids of proteins. We obtain hydrodynamic radius and fluorescence signals relating to the content of tryptophans, tyrosines and lysines of proteins using a combination of diffusional sizing of proteins, label-free detection and on-chip labelling of proteins with a latent fluorophore in the solution phase. We thereby achieve identification of proteins on a single microfluidic chip by separating and mapping proteins in multidimensional space based on their characteristic physical parameters. Our results have significant implications in the development of easy and rapi...
Journal of the American Society for Mass Spectrometry, 2009
An electrospray coupled microfluidic reactor for the measurement of millisecond time-scale, solution phase kinetics is introduced. The device incorporates a simple two-channel design that is etched into polymethyl methacrylate (PMMA) by laser ablation. The outlet of the device is laser cut to a sharp tip, facilitating low dead volume 'on chip' electrospray. Fabrication is fast, straightforward and highly reproducible, supporting rapid prototyping and large-scale reproduction. Device performance is characterized using a cytochrome c unfolding reaction. Unfolding processes with rates in excess of 30 s Ϫ1 are easily measured, including the appearance of a 'native-like' intermediate that is maximally populated 180 ms post reaction initiation. To extract reliable rates from the data, a theoretical framework for the analysis of kinetics acquired under square-channel laminar flow is introduced. (J Am Soc Mass Spectrom 2009, 20, 124 -130)
Journal of Chromatography A, 2003
A membrane-based desalting step integrated in a MS microchip is presented: drugs, peptides and proteins are adsorbed on a hydrophobic poly(vinylidene difluoride) membrane, which allows the washing out of salts. The integration with microfluidics permits a controlled elution of analytes from the membrane and their direct mass spectrometric analysis by electrospray ionisation MS. The desalting process is demonstrated with picomole amounts of propanolol, insulin and cytochrome c. Moreover, this stop-and-go desalting process is tolerant to high concentrations of urea, and to the presence of reductants such as dithiothreitol. This particular feature allowed the chemical tagging of cysteines in b-lactoglobulin A with iodoacetamide. Finally, the integration of chemical tagging, on-chip desalting and MS microchip paves the way for the development of high-throughput analytical procedure for structural proteomics.