Serum Peptide Profiling by Magnetic Particle-Assisted, Automated Sample Processing and MALDI-TOF Mass Spectrometry

Josep Villanueva, John Philip, David R. Entenberg, Carlos A. Chaparro, Meena K. Tanwar, Eric C. Holland, Paul Tempst

Research output: Contribution to journalArticle

421 Citations (Scopus)

Abstract

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 (∼50 μL) 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.

Original languageEnglish (US)
Pages (from-to)1560-1570
Number of pages11
JournalAnalytical Chemistry
Volume76
Issue number6
DOIs
StatePublished - Mar 15 2004
Externally publishedYes

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Mass spectrometry
Tumors
Peptides
Microarrays
Processing
Learning algorithms
Brain
Robotics
Robots
Ligands
Liquids

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Serum Peptide Profiling by Magnetic Particle-Assisted, Automated Sample Processing and MALDI-TOF Mass Spectrometry. / Villanueva, Josep; Philip, John; Entenberg, David R.; Chaparro, Carlos A.; Tanwar, Meena K.; Holland, Eric C.; Tempst, Paul.

In: Analytical Chemistry, Vol. 76, No. 6, 15.03.2004, p. 1560-1570.

Research output: Contribution to journalArticle

Villanueva, Josep ; Philip, John ; Entenberg, David R. ; Chaparro, Carlos A. ; Tanwar, Meena K. ; Holland, Eric C. ; Tempst, Paul. / Serum Peptide Profiling by Magnetic Particle-Assisted, Automated Sample Processing and MALDI-TOF Mass Spectrometry. In: Analytical Chemistry. 2004 ; Vol. 76, No. 6. pp. 1560-1570.
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