Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: Mapping the nuclear proteome of human fibroblasts.
(2003) In Electrophoresis 24(21). p.3806-3814- Abstract
- Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized... (More)
- Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/118979
- author
- organization
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Electrospray ionization-tandem mass spectrometry, Fibroblast, Matrix-assisted laser desorption/ionization-tandem mass spectrometry, Miniaturization, Nanocapillary liquid chromatography, Nucleus
- in
- Electrophoresis
- volume
- 24
- issue
- 21
- pages
- 3806 - 3814
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- wos:000186858700032
- pmid:14613209
- scopus:9144228659
- ISSN
- 0173-0835
- DOI
- 10.1002/elps.200305619
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Biomedical Engineering (011200011), Analytical Chemistry (S/LTH) (011001004), Respiratory Medicine and Allergology (013230111), Departments at LTH (011200000), Department of Experimental Medical Science (013210000)
- id
- 5a5613af-2165-4c77-b7ca-91ab52e20753 (old id 118979)
- alternative location
- http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14613209&dopt=Abstract
- date added to LUP
- 2016-04-01 12:01:17
- date last changed
- 2024-01-08 05:16:47
@article{5a5613af-2165-4c77-b7ca-91ab52e20753, abstract = {{Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.}}, author = {{Malmström, Johan and Larsen, Kristoffer and Malmström, Lars and Tufvesson, Ellen and Parker, Ken and Marchese, Jason and Williamson, Brian and Patterson, Dale and Martin, Steve and Juhasz, Peter and Westergren-Thorsson, Gunilla and Marko-Varga, György}}, issn = {{0173-0835}}, keywords = {{Electrospray ionization-tandem mass spectrometry; Fibroblast; Matrix-assisted laser desorption/ionization-tandem mass spectrometry; Miniaturization; Nanocapillary liquid chromatography; Nucleus}}, language = {{eng}}, number = {{21}}, pages = {{3806--3814}}, publisher = {{John Wiley & Sons Inc.}}, series = {{Electrophoresis}}, title = {{Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: Mapping the nuclear proteome of human fibroblasts.}}, url = {{http://dx.doi.org/10.1002/elps.200305619}}, doi = {{10.1002/elps.200305619}}, volume = {{24}}, year = {{2003}}, }