Serum Peptide profiling bymagnetic Particle-Assisted,Automated Sample Processing and MALDI-TOF Mass Spectrometry

摘要

Humanserum contians a complex array of proteolyticallyderived peptides(serum peptidome)that may provide a correlate of biologicla 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 noveol,automated techonology platform for the simulple,scalable,and generates highly reproducible pattterns.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 ona detailed investigaton of serum handlign,RP ligand and eluant seleciton,smallj-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 poly-peptides (0.8-15-kDa range)in a single droplet(approx 50 muL) of serum,and almost 2000 unique peptides in larger sample sets,which canthen be analyzed using ocmmon microarray data analysis solftware.A pilot study indicatedthat sera from brain tumor patients can be distinguished from controls base dona pattern of 274 peptide masses.This,in turn,served to careate a learnign algorithm that correctly predicted 96.4(percent) of the samples as wigher normar o rdiseased.