The Proteomic Reactor:  A Microfluidic Device for Processing Minute Amounts of Protein Prior to Mass Spectrometry Analysis

Ethier, Martin; Hou, Weimin; Duewel, Henry S.; Figeys, Daniel

doi:10.1021/pr060312m

Cited by 83 publications

(105 citation statements)

References 20 publications

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“…The small sample and reagent requirements, rapid analysis times, high throughput processing capabilities, and low operating costs are among the driving forces for the development of these systems [5][6][7][8]. Different microfluidic devices have been applied to specific aspects of protein processing, in particular, protein purification and separation, protein digestion, and protein identification by mass spectrometry [9].…”

mentioning

confidence: 99%

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Lee

Musyimi

Soper

et al. 2008

J. Am. Soc. Mass Spectrom.

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An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 ILm wide, 100 ILm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethy1carbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 ILL/min, producing a reaction time of -24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, Significant progress has been made toward the development of microchip-based technologies for proteomics through the integration of analytical processes into platforms that can provide rapid identification of proteins and the subsequent characterization of various post-translational modifications [1][2][3][4]. The small sample and reagent requirements, rapid analysis times, high throughput processing capabilities, and low operating costs are among the driving forces for the development of these systems [5][6][7][8]. Different microfluidic devices have been applied to specific aspects of protein processing, in particular, protein purification and separation, protein digestion, and protein identification by mass spectrometry [9].There have been a number of approaches to on-line and off-line matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) integrated to chipbased devices [10][11][12][13][14][15][16][17][18]. The primary advantage of the MALDI approach compared with electrospray ionization (ESI) when coupling to microfluidic chips is the potential for multiplexing [19]. The directional control Address reprint requests to Dr. Kermit K. Murray, Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70802, USA. E-mail: kkmurray@!su.edu of the ESI spray can be difficult with a high-density array of spray tips. Furthermore, microfluidic chip interfaces to ESI can suffer from stability problems when sprays are started or stopped [20,21]. This limits the speed of moving from one sample to another and therefore, limits throughput.With recent advances in analytical methods for proteomics, attention has been directed toward development of effic...

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mentioning

confidence: 99%

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Lee

Musyimi

Soper

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…These results suggest that the stability of immobilized proteases is superior to that of free proteases. A typical sample preparation for proteolysis before digestion involves multi-steps including denaturation, reduction of disulfide bond, and alkylation of free thiol group to reduce the conformational stability of protein; steps which are expected to produce enhancement of digestion efficiency (Ma et al, 2008;Li et al, 2007a;Ethier et al, 2006;Lin et al, 2008). However, the multi-step procedure is time-consuming.…”

Section: Proteolysis By the Protease-immobilized Microreactorsmentioning

confidence: 99%

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

Yamaguchi¹,

Miyazaki²,

Maeda³

2012

Integrative Proteomics

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“…However, the serial (unidirectional) nature of channel microfluidics has limited such systems to a single step (with some exceptions noted 47 ). Implementation of multistep reactions with different incubation periods and changes in pH may be challenging for enclosed microchannels.…”

Section: Proteomic Reactions In Channel Microfluidicsmentioning

confidence: 99%

“…Unfortunately, this challenge is typical in proteomics; for example, in shotgun proteomics, samples are subjected to a multiday, multistep procedure including acidification, denaturing, reduction, alkylation, enzymatic digestion (twice), purification, and dilution prior to analysis by separations and mass spectrometry. 13 We acknowledge that microvalves 48 may offer some relief from this problem, but we submit that in general, microchannels are ill-suited for implementing the complicated series of reactions necessary for many proteomic applications (with some exceptions noted 47 Because each droplet is isolated from its surroundings rather than being embedded in a stream of fluid, DMF is a facile method for forming microreactors in which there is no possibility that reagents or samples will diffuse away. Most importantly, because each droplet is controlled individually, multistep methods involving many different reagents can be easily programmed.…”

Section: Introductionmentioning

confidence: 99%

A Digital Microfluidic Approach to Proteomic Sample Processing

2009

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Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL -10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.iii Methods for implementing proteomic multi-step solution-phase processes (reduction, alkylation, and digestion) on DMF were developed. This was accompanied by the development of heterogenous enzymatic microreactors for efficient protein digestion. Strategies for reducing non-specific adsorption were developed. Finally, in proof-of-concept work, a combination of protein extraction and protein processing was demonstrated on DMF devices.iv ACKNOWLEDGEMENTS

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The Proteomic Reactor: A Microfluidic Device for Processing Minute Amounts of Protein Prior to Mass Spectrometry Analysis

Cited by 83 publications

References 20 publications

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

A Digital Microfluidic Approach to Proteomic Sample Processing

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