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Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage

Antberg, Linn LU ; Cifani, Paolo LU ; Sandin, Marianne LU ; Levander, Fredrik LU and James, Peter LU (2012) In Journal of Proteome Research 11(5). p.2644-2652
Abstract
We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in... (More)
We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
protein and peptide separation, methods comparison, off-gel isoelectric, focusing, GluC, trypsin, GelLC, density gradient cell fractionation, strong cation exchange chromatography, reversed-phase chromatography, proteome coverage
in
Journal of Proteome Research
volume
11
issue
5
pages
2644 - 2652
publisher
The American Chemical Society
external identifiers
  • wos:000303492100003
  • scopus:84860602480
ISSN
1535-3893
DOI
10.1021/pr201257y
project
CREATE Health
language
English
LU publication?
yes
id
094446f9-353a-45eb-94d9-6903c1bde974 (old id 2813018)
date added to LUP
2012-06-25 09:11:46
date last changed
2017-02-05 03:18:10
@article{094446f9-353a-45eb-94d9-6903c1bde974,
  abstract     = {We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.},
  author       = {Antberg, Linn and Cifani, Paolo and Sandin, Marianne and Levander, Fredrik and James, Peter},
  issn         = {1535-3893},
  keyword      = {protein and peptide separation,methods comparison,off-gel isoelectric,focusing,GluC,trypsin,GelLC,density gradient cell fractionation,strong cation exchange chromatography,reversed-phase chromatography,proteome coverage},
  language     = {eng},
  number       = {5},
  pages        = {2644--2652},
  publisher    = {The American Chemical Society},
  series       = {Journal of Proteome Research},
  title        = {Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage},
  url          = {http://dx.doi.org/10.1021/pr201257y},
  volume       = {11},
  year         = {2012},
}