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| Naji, M. |
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| Motta, Antonella |
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| Aletan, Dirar |
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| Mohamed, Tarek |
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| Ertürk, Emre |
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| Taccardi, Nicola |
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| Kononenko, Denys |
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| Petrov, R. H. | Madrid |
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| Alshaaer, Mazen | Brussels |
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| Bih, L. |
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| Casati, R. |
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| Muller, Hermance |
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| Kočí, Jan | Prague |
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| Šuljagić, Marija |
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| Kalteremidou, Kalliopi-Artemi | Brussels |
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| Azam, Siraj |
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| Ospanova, Alyiya |
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| Blanpain, Bart |
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| Ali, M. A. |
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| Popa, V. |
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| Rančić, M. |
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| Ollier, Nadège |
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| Azevedo, Nuno Monteiro |
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| Landes, Michael |
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| Rignanese, Gian-Marco |
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Wouters, Bert
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Publications (3/3 displayed)
- 2012High-pressure ion chromatography in capillary columns and microfluidic chips
- 2011High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns
- 2011Ultra-high-resolution separations of intact proteins with LC-TOF-MS using polymer monolithic columns
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document
Ultra-high-resolution separations of intact proteins with LC-TOF-MS using polymer monolithic columns
Abstract
Top-down approaches to proteomic analyses are attractive, because they have the potential to supply information about the modification status of proteins, including proteolytic processing, oxidative damage, glycosylation, or other forms of post-translational processing. However, several limitations have prevented top-down workflows from becoming widely adopted, in particular (1) fragmentation of proteins, (2) data analysis of the complex MS/MS spectra, (3) chromatographic separation of mixtures of intact proteins, and (4) sensitivity of detection for the intact proteins. To reduce spectral complexity, and also to minimize ion suppression of low abundant species, a high-efficiency separation method is required prior to ESI-MS analysis.In the present contribution, we discuss the separation of intact proteins and protein isoforms arising from various amino-acid modifications employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometry via electrospray interfacing. Using a 250 mm x 0.2 mm monolithic capillary column constructed by in-situ polymerization we have achieved peak capacities of> 600 with a 2h gradient. Detection on a maXis UHR-Qq-TOF of proteins from 200 fmol per protein on columnwith TFA added as ion pairing agent yielded a high signal to noise ratio (11 < S/N < 322) and excellent resolution (res > 30000). Using FA as ion-pairing agent, a 3-fold increase in signal intensity was obtained. The advantages of the combination of ultra-high-resolution chromatography with high-sensitivity high-accuracy MS is demonstrated with the separation of protein isoforms that differ only in their oxidation and glycosylation states.