High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers.
An enzymatic microreactor with a volume of 470 nL has been prepared by immobilizing trypsin on a 10 cm long reactive porous polymer monolith located in a 100 microm i.d. fused silica capillary. This reactor affords suitable degrees of digestion of proteins even after very short residence times of less than 1 min. The performance is demonstrated with the digestion of eight proteins ranging in molecular mass from 2848 to 77 754. The digests were analyzed using mass spectrometry in two modes: off-line MALDI and in-line nanoelectrospray ionization. The large numbers of identified peptides enable a high degree of sequence coverage and positive identification of the proteins. The extent of sequence coverage decreases as the molecular mass of the digested protein increases.
Peterson
Dominic S
DS
E. O. Lawrence Berkeley National Laboratory, Materials Sciences Division, Berkeley, California 94720, USA.
Rohr
Thomas
T
Svec
Frantisek
F
Fréchet
Jean M J
JM
D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*
D011506:Proteins; D014357:Trypsin