On-Chip Proteolytic Digestion and Analysis Using “Wrong-Way-Round” Electrospray Time-of-Flight Mass Spectrometry

Lazar, Iulia M.; Ramsey, and Roswitha S.; Ramsey, J. Michael

doi:10.1021/ac001420+

Cited by 111 publications

(88 citation statements)

References 53 publications

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“…Although the digestion efficiency of this bioreactor was high, not all of the peptides were identified, which may be due to incomplete digestion for protease-inaccessible proteins, solubility of digested peptides, and lack of a denaturation step in the bioreactor protocol [39]. Sequence coverage ranging from 18% to 95% have been reported for in-solution digestion of cytochrome c, which requires 15 min to 24 h reaction time [25,29,40].…”

Section: Maldi Analysis Of Solid-phase Bioreactor Digested Cytochrome Cmentioning

confidence: 99%

“…When a relatively low enzyme-tosubstrate molar ratio is employed, the enzymatic reaction has low efficiency [40,46]; however, this low molar ratio is necessary to avoid excessive autodigestion of trypsin. Lazar et al [40] discussed the effects of trypsin concentration on the rate of digestion in solution. In their work, in-solution digestion of various proteins was accomplished using enzyme-to-substrate molar ratios of 1:1 to 1:20 for 15 min to 24 h of incubation.…”

Section: Digestion Capacity Of the Solid-phase Open Channel Bioreactormentioning

confidence: 99%

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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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Section: Maldi Analysis Of Solid-phase Bioreactor Digested Cytochrome Cmentioning

confidence: 99%

Section: Digestion Capacity Of the Solid-phase Open Channel Bioreactormentioning

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.

View full text Add to dashboard Cite

show abstract

“…First, microfabrication robustness and reproducibility make available reliable micro-and nanospray emitters, with automation possibilities [21]. Second, the design flexibility provided by microfabrication allows the integration of different analytical processes on the same microfluidic device prior to delivery to the mass spectrometer, such as sample preparation [22][23][24], analyte separation [25][26][27], proteolytic digestion [9,28], or even integration of the whole process [29].…”

mentioning

confidence: 99%

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry

Roussel

Dayon

Lion

et al. 2004

J. Am. Soc. Mass Spectrom.

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We present herein a review of our work on the on-line electrochemical generation of mass tags toward cysteine residues in peptides and proteins. Taking advantage of the inherent electrochemical nature of electrospray generated from a microfabricated microspray emitter, selective probes for cysteine were developed and tested for on-line nonquantitative mass tagging of peptides and proteins. The nonquantitative aspect of the covalent tagging thus allows direct counting of free cysteines in the mass spectrum of a biomolecule through additional adduct peaks. Several substituted hydroquinones were investigated in terms of electrochemical properties, and their usefulness for on-line mass tagging during microspray experiments were assessed with L-cysteine, peptides, and intact proteins. Complementarily, numerical simulations were performed to properly understand the respective roles of mass transport, kinetics of electrochemical-chemical reactions, and design of the microspray emitter in the mass tagging overall efficiency. Finally, the on-line electrochemical tagging of cysteine residues was applied to the analysis of tryptic peptides of purified model proteins for protein identification through peptide mass fingerprinting. (J Am Soc Mass Spectrom 2004, 15, 1767-1779

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“…The ESI-TOF-MS technique has been considered to be particularly suitable for the analysis of large species of biological interest and the determination of various organic compounds. [68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] A combination of ESI with an ion storage TOF-MS was proposed as a feasible method for a rapid determination of low and high molecular compounds. 68 The described instrument allowed the routine detection of low picomole and sub-picomole quantities of multiply charge molecules with improved resolution and duty cycle.…”

Section: An Overview Of Analytical Applications Of Tof-ms Analyzersmentioning

confidence: 99%

“…The analysis of human hemoglobin by using a hybrid microchip nanospray device and TOF-MS has been described. 79 Inorganic and organic antimony compounds have been investigated by ESI-TOF-MS. 81 Electrospray TOF-MS has been found to be an excellent detector (analysis in the low femtomol range) for fast liquid chromatography 77,82,83 and capillary electrophoresis 70,71,[73][74][75][76][77]80 used for the identification and quantification of low and high molecular-weight species. The combination of electrospray-TOF-MS with on-line liquid chromatography for estimating the precision of the exact mass 84 Soon after the development of ESI-TOF-MS, a tandem (hybrid) technique incorporating a quadrupole as a first mass analyzer and orthogonal TOF-MS to record the product ions was developed.…”

Section: An Overview Of Analytical Applications Of Tof-ms Analyzersmentioning

confidence: 99%

An Overview of Analytical Applications of Time of Flight-Mass Spectrometric (TOF-MS) Analyzers and an Inductively Coupled Plasma-TOF-MS Technique

Balcerzak

2003

ANAL. SCI.

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On-Chip Proteolytic Digestion and Analysis Using “Wrong-Way-Round” Electrospray Time-of-Flight Mass Spectrometry

Cited by 111 publications

References 53 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

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry

An Overview of Analytical Applications of Time of Flight-Mass Spectrometric (TOF-MS) Analyzers and an Inductively Coupled Plasma-TOF-MS Technique

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