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One-step fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays

Ingvarsson, Johan LU ; Lindstedt, Malin LU ; Borrebaeck, Carl LU and Wingren, Christer LU (2006) In Journal of Proteome Research 5(1). p.170-176
Abstract
Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was... (More)
Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was used for evaluation. The proteome was pre-fractionated based on size in a simple one-step procedure using centrifugal filter devices of various molecular weight cutoffs. The results showed that the generation of a nondiluted low molecular weight (LMW) fraction, corresponding to less than 2% of the original protein content, was critical for the successful screening of cytokines in the sub pg/mL range. The reduced complexity of the LMW fraction significantly improved the assay sensitivity, by improving the fluorescent tagging step and/or reducing the nonspecific binding to the substrates. (Less)
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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
polymer substrate, Ni2+-NTA coated substrate, sample handling, sensitivity, fractionation, antibody microarrays, proteome analysis
in
Journal of Proteome Research
volume
5
issue
1
pages
170 - 176
publisher
The American Chemical Society (ACS)
external identifiers
  • wos:000234668300018
  • pmid:16396508
  • scopus:30744463080
ISSN
1535-3893
DOI
10.1021/pr050301d
language
English
LU publication?
yes
id
753fffa8-561c-4153-a861-f53af6eb4610 (old id 419763)
date added to LUP
2016-04-01 12:27:15
date last changed
2020-07-22 01:39:35
@article{753fffa8-561c-4153-a861-f53af6eb4610,
  abstract     = {Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was used for evaluation. The proteome was pre-fractionated based on size in a simple one-step procedure using centrifugal filter devices of various molecular weight cutoffs. The results showed that the generation of a nondiluted low molecular weight (LMW) fraction, corresponding to less than 2% of the original protein content, was critical for the successful screening of cytokines in the sub pg/mL range. The reduced complexity of the LMW fraction significantly improved the assay sensitivity, by improving the fluorescent tagging step and/or reducing the nonspecific binding to the substrates.},
  author       = {Ingvarsson, Johan and Lindstedt, Malin and Borrebaeck, Carl and Wingren, Christer},
  issn         = {1535-3893},
  language     = {eng},
  number       = {1},
  pages        = {170--176},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Journal of Proteome Research},
  title        = {One-step fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays},
  url          = {http://dx.doi.org/10.1021/pr050301d},
  doi          = {10.1021/pr050301d},
  volume       = {5},
  year         = {2006},
}