Microfluidic platform with mass spectrometry detection for the analysis of phosphoproteins

Dawoud, Abdulilah A.; Sarvaiya, Hetal A.; Lazar, Iulia M.

doi:10.1002/elps.200700355

Cited by 17 publications

(16 citation statements)

References 54 publications

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“…LC separation efficiencies were on the average $25 000-35 000 plates/channel (2 cm long separation channel), as calculated by N 5 5.54Ã (t r /W 1/2 ) 2 , where N is the plate number, t r is the analyte retention time, and W 1/2 is the width of the peak at half height. The reproducibility of retention times was demonstrated in earlier work with standard protein mix digests (RSD 5 5-15%) [36].…”

Section: Resultsmentioning

confidence: 91%

Microfluidic chips for protein differential expression profiling

2009

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Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

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Section: Resultsmentioning

confidence: 91%

Microfluidic chips for protein differential expression profiling

2009

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“…Several microfluidic devices have been developed by integrating phosphopeptide enrichment with other functions, such as protein alkylation and reduction [15], protein digestion [16], peptide separation and on-line interface for MS instrument [17][18][19][20], which have shown great potentials in the field of phosphoproteome research. However, in these microchip devices, phosphopeptide enrichment components are usually fabricated by packing functionalized particles, such as TiO 2 or IMAC beads, into microchannels.…”

Section: Introductionmentioning

confidence: 99%

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

Wang

Duan

et al. 2011

J of Separation Science

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To meet the demands of protein phosphorylation study, immobilized zirconium ion affinity chromatography (Zr 41 -IMAC) monolith was prepared by combining UV-initiated polymerization of monolithic support and subsequent photografting in both capillary columns and microchannels. Hydrophilic poly(2-hydroxyethyl methacrylate (HEMA)-coethylene dimethacrylate (EDMA)) monolithic support was prepared under UV irradiation at the wavelength of 365 nm with monomer HEMA, crosslinker EDMA and 2,2-dimethoxy-2-phenylacetophenone as photoinitiator in 1-decanol solution, which provides good biocompatibility and permeability for biomolecule analysis. To introduce chelating ligands, such as phosphate groups, on the pore surface of monolith for metal ion immobilization, photografting of ethylene glycol methacrylate phosphate with benzophenone as the photoinitiator was performed at 254 nm for 300 s. The grafting process and metal ion immobilization can be monitored by measuring the electroosmotic flow produced by the modified monolith, providing a quantitative evaluation of post-modification. This new method for the preparation of Zr 41 -IMAC monolith simplifies the optimization of monolith preparation and avoids the time-consuming chemical modification process. Additionally, advantages include facile preparation in microdevices, easy regenerability and good reproducibility. After optimization, the microchip-based Zr 41 -IMAC monolith was used for phosphopeptide analysis and showed good selectivity in phosphopeptide enrichment with matrix-assisted laser desorption ionization mass spectrometry detection. IntroductionIn recent years, protein phosphorylation has gained much attention because of its significant roles in cellular signal transduction processes [1,2]. However, the amount of phosphorylated proteins in eukaryotic cells often exists in substoichiometry level, which makes the identification of phosphorylation sites challenging [3]. To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.Recently, chip-based system has been introduced as a promising platform for phosphopeptide analysis, due to the benefits of low sample amount, high throughput, and easy integration [14]. Several microfluidic devices have been developed by integrating phosphopeptide enrichment with other functions, such as protein alkylation and reduction [15], protein digestion [16], peptide separation and on-line interface for MS instrument [17][18][19][20], which have shown great potentials in the field of phosphoproteome research. However, in these microchip devices, phosphopeptide enrichment components are usually fabricated by packing functionalized particles, such as TiO 2 or IMAC beads, into m...

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“…Advantages include: (a) high speed due to short separation channels, (b) HT due to parallelization, (c) different injection schemes and miniaturization steps enable the handling of very small sample volumes, and (d) the two spatial dimensions and variable manufacturing possibilities enable the integration of CE separation on-chip combined with enzyme reactions, sample dilution, derivatization, concentration, and purification, etc. [29,81,82]. The ability of multielement microfluid platform for the analysis of phosphoproteins, which will eventually allow detection of PTMs, has recently been demonstrated.…”

Section: Chip-based Ce-msmentioning

confidence: 99%

The role of capillary electrophoresis–mass spectrometry to proteome analysis and biomarker discovery

Ahmed

2009

Journal of Chromatography B

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Microfluidic platform with mass spectrometry detection for the analysis of phosphoproteins

Cited by 17 publications

References 54 publications

Microfluidic chips for protein differential expression profiling

Microfluidic chips for protein differential expression profiling

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

The role of capillary electrophoresis–mass spectrometry to proteome analysis and biomarker discovery

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