Analysis of the human serum proteome |
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Authors: | King C Chan David A Lucas Denise Hise Carl F Schaefer Zhen Xiao George M Janini Kenneth H Buetow Haleem J Issaq Timothy D Veenstra Thomas P Conrads |
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Institution: | (1) Laboratory of Proteomics and Analytical Technologies, National Cancer Institute at Frederick, SAIC-Frederick, Inc, PO Box B, 21702 Frederick, MD;(2) Center for Bioinformatics, National Cancer Institute, 20892 Bethesda, MD |
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Abstract: | Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers.
Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize.
Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed,
yet such depletion results in removal of important low abundant proteins.
A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry
(MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20
fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong
cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled
online with MS/MS analysis.
This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes,
cellular localization, and abundance levels were identified.
This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species
by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum
is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy
combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed
a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate
future investigations of the vast archive of pathophysiological content in serum.
These authors contributed equally to this work. |
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Keywords: | Serum proteomics mass spectrometry multi-dimensional separation isoelectric focusing biomarker |
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