In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy.
The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.
Geiser
Laurent
L
Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
Eeltink
Sebastiaan
S
Svec
Frantisek
F
Fréchet
Jean M J
JM
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006454:Hemoglobins / Q000302:isolation & purification; D007783:Lactones; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009211:Myoglobin / Q000302:isolation & purification; D036103:Nanotechnology / Q000295:instrumentation*; D009862:Online Systems; D010434:Pepsin A / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry*; D016062:Porosity; D011506:Proteins / Q000302:isolation & purification*; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004800:Enzymes, Immobilized; D006454:Hemoglobins; D007783:Lactones; D009211:Myoglobin; D010455:Peptides; D011108:Polymers; D011506:Proteins; D012996:Solutions; D010434:Pepsin A