Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategy.
We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.
Korecká
Lucie
L
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Strossova 239, Pardubice, Czech Republic.
Jankovicová
Barbora
B
Krenková
Jana
J
Hernychová
Lenka
L
Slováková
Marcela
M
Le-Nell
Anne
A
Chmelik
Josef
J
Foret
Frantisek
F
Viovy
Jean-Louis
JL
Bilková
Zusana
Z
D019149:Bioreactors; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008280:Magnetics; D008666:Metalloendopeptidases / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry; D009496:Neurotensin / Q000032:analysis*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification
D004800:Enzymes, Immobilized; D008024:Ligands; D010455:Peptides; C011661:anhydrotrypsin; D009496:Neurotensin; D012822:Silicon Dioxide; C044499:submandibular proteinase A; D012697:Serine Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase