Protein sequencing using microreactors and capillary electrophoresis with thermo-optical absorbance detection

Li, Xing‐Fang; Ren, Hongji; Qi, Ming; Lewis, Darren; Ireland, Ian D.; Waldron, Karen C.; Dovic̀hi, Norman J.

doi:10.1016/s1080-8914(97)80004-x

Cited by 3 publications

(2 citation statements)

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“…Previous attempts at incorporating miniature reactors involved the use of rotary valves and a syringe pump-based system, but performance did not exceed that of a commercial instrument. 31,32,19 Here, we report development of a novel instrument with miniaturized reaction cartridge and conversion flask (each about one-tenth the size of a high-end commercial instrumentsApplied Biosystem's model 494cLC) and reagent/solvent flow paths. Our sequencer allows identification of amino acid derivatives well below 50 fmol, with repetitive yields in the 90-94% range; chemical background is very low.…”

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Microflow-Based Automated Chemistries: Application to Protein Sequencing

Powell

Tempst

2001

Anal. Chem.

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We describe a microflow-based instrument, consisting of multiple rotary valves, capillary tubing, and miniaturized reaction vessels, for the purpose of performing automated chemical and biochemical reations on a very small scale (i.e. submicroliter volumes). The novelty is that close to 100% of the reaction end products are available in a minimal volume (< or = 5 microL) inside a pressurized microvial for subsequent analysis. This makes the system compatible with capillary HPLC and, in principle, with continuous-flow nanoelectrospray mass spectrometry. Total control of flow path combinations and directions, temperatures, and gas pressures enables precise execution of complex biochemical laboratory procedures. Instrument performance was convincingly demonstrated by partially sequencing 100 fmol of an intact protein using classical Edman chemistry in combination with capillary-bore liquid chromatography. To our knowledge, this is the smallest amount of protein ever reported to be successfully analyzed in this way. Additional applications that merge enzymatic and chemical procedures with high-sensitivity analytical devices can be envisioned in the future.

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confidence: 99%

“…This can only be done by further miniaturization of the chemistry. Previous attempts at incorporating miniature reactors involved the use of rotary valves and a syringe pump-based system, but performance did not exceed that of a commercial instrument. ,, …”

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confidence: 99%