Microflow-based automated chemistries: application to protein sequencing.
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.
Protein Center and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
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D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D007782:Lactoglobulins / Q000737:chemistry; D013058:Mass Spectrometry; D020539:Sequence Analysis, Protein / Q000295:instrumentation / Q000379:methods*
D007202:Indicators and Reagents; D007782:Lactoglobulins