Advanced

Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis

Nissum, Mikkel; Kuhfuss, Sabine; Hauptmann, Monika; Obermaier, Christian; Sukop, Ute; Wildgruber, Robert; Weber, Gerhard; Eckerskorn, Christoph and Malmström, Johan LU (2007) In Proteomics 7(23). p.27-4218
Abstract

Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the... (More)

Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the plasma proteins into specific FFE fractions. Tryptic digests of the separated proteins were generated and subsequently separated using FFE. The protein separation medium was optimized to segregate albumin into specific fractions containing only few other proteins. An optimization of throughput for the protein separation reduced the separation time of 1 mL of plasma to approximately 3 h providing sufficient material for digestion and the subsequent peptide separation. Our approach revealed low-abundant proteins (e.g., L-selectin at 17 ng/mL and vascular endothelial-cadherin precursor at 30 ng/mL) and several tissue leakage products, thus providing a powerful orthogonal separation step in the proteomics workflow.

(Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
keywords
Blood Protein Electrophoresis, Blood Proteins, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, Humans, Hydrogen-Ion Concentration, Isoelectric Focusing, Isoelectric Point, Peptide Fragments, Proteome, Proteomics, Serum Albumin, Tandem Mass Spectrometry, Trypsin, Journal Article
in
Proteomics
volume
7
issue
23
pages
10 pages
publisher
John Wiley & Sons
external identifiers
  • scopus:37049003330
ISSN
1615-9853
DOI
10.1002/pmic.200700166
language
English
LU publication?
no
id
44ca15f9-e7d8-40de-add5-1bdc011cdc06
date added to LUP
2017-09-04 17:21:48
date last changed
2017-09-10 05:23:43
@article{44ca15f9-e7d8-40de-add5-1bdc011cdc06,
  abstract     = {<p>Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the plasma proteins into specific FFE fractions. Tryptic digests of the separated proteins were generated and subsequently separated using FFE. The protein separation medium was optimized to segregate albumin into specific fractions containing only few other proteins. An optimization of throughput for the protein separation reduced the separation time of 1 mL of plasma to approximately 3 h providing sufficient material for digestion and the subsequent peptide separation. Our approach revealed low-abundant proteins (e.g., L-selectin at 17 ng/mL and vascular endothelial-cadherin precursor at 30 ng/mL) and several tissue leakage products, thus providing a powerful orthogonal separation step in the proteomics workflow.</p>},
  author       = {Nissum, Mikkel and Kuhfuss, Sabine and Hauptmann, Monika and Obermaier, Christian and Sukop, Ute and Wildgruber, Robert and Weber, Gerhard and Eckerskorn, Christoph and Malmström, Johan},
  issn         = {1615-9853},
  keyword      = {Blood Protein Electrophoresis,Blood Proteins,Electrophoresis, Gel, Two-Dimensional,Electrophoresis, Polyacrylamide Gel,Humans,Hydrogen-Ion Concentration,Isoelectric Focusing,Isoelectric Point,Peptide Fragments,Proteome,Proteomics,Serum Albumin,Tandem Mass Spectrometry,Trypsin,Journal Article},
  language     = {eng},
  number       = {23},
  pages        = {27--4218},
  publisher    = {John Wiley & Sons},
  series       = {Proteomics},
  title        = {Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis},
  url          = {http://dx.doi.org/10.1002/pmic.200700166},
  volume       = {7},
  year         = {2007},
}