Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry.
Human serum contains a complex array of proteolytically derived peptides (serum peptidome) that may provide a correlate of biological events occurring in the entire organism; for instance, as a diagnostic for solid tumors (Petricoin, E. F.; Ardekani, A. M.; Hitt, B. A.; Levine, P. J.; Fusaro, V. A.; Steinberg, S. M.; Mills, G. B.; Simone, C.; Fishman, D. A.; Kohn, E. C.; Liotta, L. Lancet 2002, 359, 572-577). Here, we describe a novel, automated technology platform for the simultaneous measurement of serum peptides that is simple, scalable, and generates highly reproducible patterns. Peptides are captured and concentrated using reversed-phase (RP) batch processing in a magnetic particle-based format, automated on a liquid handling robot, and followed by a MALDI TOF mass spectrometric readout. The protocol is based on a detailed investigation of serum handling, RP ligand and eluant selection, small-volume robotics design, an optimized spectral acquisition program, and consistent peak extraction plus binning across a study set. The improved sensitivity and resolution allowed detection of 400 polypeptides (0.8-15-kDa range) in a single droplet (approximately 50 microL) of serum, and almost 2000 unique peptides in larger sample sets, which can then be analyzed using common microarray data analysis software. A pilot study indicated that sera from brain tumor patients can be distinguished from controls based on a pattern of 274 peptide masses. This, in turn, served to create a learning algorithm that correctly predicted 96.4% of the samples as either normal or diseased.
Villanueva
Josep
J
Protein Center, Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Philip
John
J
Entenberg
David
D
Chaparro
Carlos A
CA
Tanwar
Meena K
MK
Holland
Eric C
EC
Tempst
Paul
P
D000465:Algorithms; D001331:Automation; D014408:Biomarkers, Tumor / Q000097:blood; D001798:Blood Proteins / Q000032:analysis*; D001932:Brain Neoplasms / Q000097:blood* / Q000175:diagnosis; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D005909:Glioblastoma / Q000097:blood* / Q000175:diagnosis; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008280:Magnetics; D010455:Peptides / Q000097:blood*; D010865:Pilot Projects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D014408:Biomarkers, Tumor; D001798:Blood Proteins; D020099:Coated Materials, Biocompatible; D010455:Peptides