Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith: integrating solid-phase extraction and enzymatic digestion for peptide mass mapping.
Microfluidic devices with a dual function containing both a solid-phase extractor and an enzymatic microreactor have been prepared, and their operation has been demonstrated. The devices were fabricated from a 25-mm-long porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith prepared within a 50-microm-i.d. capillary. This capillary with a pulled 9-12-microm needle tip was used as a nanoelectrospray emitter coupling the device to a mass spectrometer. Photografting with irradiation through a mask was then used to selectively functionalize a 20-mm-long portion of the monolith, introducing reactive poly(2-vinyl-4,4-dimethylazlactone) chains to enable the subsequent attachment of trypsin, thereby creating an enzymatic microreactor with high proteolytic activity. The other 5 mm of unmodified hydrophobic monolith served as micro solid-phase extractor (microSPE). The dual-function devices were used in two different flow directions; concentration of myoglobin that was absorbed from its dilute solution, followed by elution and digestion or digestion, followed by concentration. Operations in both directions afforded equal sequence coverage. Different volumes of myoglobin solution ranging from 2 to 20 microL were loaded on the device. Very high sequence coverages of almost 80% were achieved for the highest loading. Despite the very short length of the extractor unit, the device operated in the digest-solid-phase extraction direction also enabled the separation of peaks that mostly contained undigested protein and peptides.
Peterson
Dominic S
DS
Materials Sciences Division, E.O. Lawrence Berkeley National Laboratory, Berkeley, California 94720-8139, USA.
Rohr
Thomas
T
Svec
Frantisek
F
Fréchet
Jean M J
JM
D001896:Boron Compounds / Q000737:chemistry; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000295:instrumentation*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008689:Methacrylates / Q000737:chemistry; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D044085:Microfluidics / Q000295:instrumentation; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods; D010777:Photochemistry; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002364:Caseins; D004800:Enzymes, Immobilized; D008689:Methacrylates; D009211:Myoglobin; D010446:Peptide Fragments; D011108:Polymers; D011506:Proteins; C005044:hydroxyethyl methacrylate; C004919:ethylene dimethacrylate; D014357:Trypsin; C016284:butyl methacrylate