PepS: An Innovative Microfluidic Device for Bedside Whole Blood Processing before Plasma Proteomics Analyses (original) (raw)
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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.
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.
Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids
Clinical Chemistry, 2021
Background Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. Methods Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for...
Microfluidics for Peptidomics, Proteomics, and Cell Analysis
Nanomaterials
Microfluidics is the advanced microtechnology of fluid manipulation in channels with at least one dimension in the range of 1–100 microns. Microfluidic technology offers a growing number of tools for manipulating small volumes of fluid to control chemical, biological, and physical processes relevant to separation, analysis, and detection. Currently, microfluidic devices play an important role in many biological, chemical, physical, biotechnological and engineering applications. There are numerous ways to fabricate the necessary microchannels and integrate them into microfluidic platforms. In peptidomics and proteomics, microfluidics is often used in combination with mass spectrometric (MS) analysis. This review provides an overview of using microfluidic systems for peptidomics, proteomics and cell analysis. The application of microfluidics in combination with MS detection and other novel techniques to answer clinical questions is also discussed in the context of disease diagnosis an...
Microfluidic Liquid Chromatography System for Proteomic Applications and Biomarker Screening
Analytical Chemistry, 2006
A microfluidic liquid chromatography (LC) system for proteomic investigations that integrates all the necessary components for stand-alone operation, i.e., pump, valve, separation column, and electrospray interface, is described in this paper. The overall size of the LC device is small enough to enable the integration of two fully functional separation systems on a 3 in. × 1 in. glass microchip. A multichannel architecture that uses electroosmotic pumping principles provides the necessary functionality for eluent propulsion and sample valving. The flow rates generated within these chips are fully consistent with the requirements of nano-LC platforms that are routinely used in proteomic applications. The microfluidic device was evaluated for the analysis of a protein digest obtained from the MCF7 breast cancer cell line. The cytosolic protein extract was processed according to a shotgun protocol, and after tryptic digestion and prefractionation using strong cation exchange chromatography (SCX), selected sample subfractions were analyzed with conventional and microfluidic LC platforms. Using similar experimental conditions, the performance of the microchip LC was comparable to that obtained with benchtop instrumentation, providing an overlap of 75% in proteins that were identified by more than two unique peptides. The microfluidic LC analysis of a protein-rich SCX fraction enabled the confident identification of 77 proteins by using conventional data filtering parameters, of 39 proteins with p < 0.001, and of 5 proteins that are known to be cancer-specific biomarkers, demonstrating thus the potential applicability of these chips for future high-throughput biomarker screening applications. Advanced mass spectrometry (MS) detection enables a comprehensive characterization of the proteomic molecular cell profile. Relevant answers can be provided for questions that relate to the identity and expression level of the existing proteins, the nature, site, and number of the posttranslational modifications, and the specific functions associated with these proteins. The generated information is essential for differentiating normal versus diseased
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.
Digital Microfluidics with In-Line Sample Purification for Proteomics Analyses with MALDI-MS
Analytical Chemistry, 2005
An in-line sample purification method for MALDI-MS, which relies on the electrowetting-on-dielectric (EWOD)based technique for digital microfluidics, is reported. In this method, a droplet containing peptides and impurities is moved by EWOD and deposited onto a Teflon-AF surface. A droplet of water is subsequently moved over the spot, where it dissolves and removes the impurities. A droplet containing MALDI matrix is then moved to the spot, which is analyzed by MALDI-MS. This purification method reduces the number of salt adduct peaks caused by low concentrations of impurities (e.g., 20 mM sodium phosphate), and reduces or eliminates the catastrophic effects of high concentrations of impurities (e.g., 8 M urea). The method was used to purify spots made by depositing multiple droplets of contaminated peptides. Spectra from the purified spots showed an increase in the S/N ratio as a function of the number of droplets deposited; when not purified, the S/N ratio remained constant regardless of the number of droplets. Finally, the method was used to purify protein digests for peptide mass fragment (PMF) searches, and was shown to be more efficient than the conventional method of purification with reversed-phase-packed pipet tips. We anticipate this new, in-line sample purification technique for EWOD-MALDI-MS will enable development of integrated high-throughput proteomics analysis methodologies.
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...
Molecular & Cellular Proteomics, 2006
The advances in proteomic technologies provide tremendous opportunities for applying these technologies in biomarker-related clinical applications; however, the unique characteristics of human biofluids such as high dynamic range in protein abundances and extreme complexity of human proteomes present tremendous challenges for current analytical technologies. In this review, we focus on summarizing the recent advances in LC-MS based proteomic profiling and
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.