Proteomic expression profiling and identification of serum proteins using immobilized trypsin beads with MALDI-TOF/TOF.
MALDI-TOF mass spectrometry is a widely used technique for serum protein expression profiling and biomarker discovery. Many profiling strategies typically employ chemical affinity beads or surfaces to decrease sample complexity of dynamic fluids such as serum or plasma. However, many of the proteins captured on a particular surface or bead are not resolved in the lower mass ranges where time-of-flight mass spectrometers are most effective. Thus, a majority of reported protein expression profiling studies primarily interrogate the native low molecular mass constituents of the target sample. We report an expression profiling workflow that utilizes immobilized trypsin paramagnetic beads following an initial affinity bead fractionation step, thereby reducing large mass proteins to peptides that are better suited to analysis and sequencing determinations. Our bead-based trypsin approach resulted in more efficient digestion of complex serum protein extracts at short incubation times. This method was reproducible and readily adaptable to robotic sample handling and may be combined in tandem with other bead fractionation surfaces. When weak cationic and weak anionic bead surfaces were used, experimental conditions were optimized for tandem combinations of these beads with the immobilized trypsin step to produce an efficient serum fractionation strategy. A proof-of-concept pilot experiment using pooled human serum samples demonstrating reproducibility is presented, along with the sequence determination of selected tryptic peptides of serum proteins.
Karbassi
Izabela D
ID
George L Wright, Jr. Center for Biomedical Proteomics, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Nyalwidhe
Julius O
JO
Wilkins
Christopher E
CE
Cazares
Lisa H
LH
Lance
Raymond S
RS
Semmes
O John
OJ
Drake
Richard R
RR
D000465:Algorithms; D001798:Blood Proteins / Q000032:analysis*; D002852:Chromatography, Ion Exchange; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D011470:Prostatic Hyperplasia / Q000097:blood; D011471:Prostatic Neoplasms / Q000097:blood; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D001798:Blood Proteins; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D014357:Trypsin