Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: mapping the nuclear proteome of human fibroblasts.
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.
Malmström
Johan
J
Cell & Molecular Biology, BMC, C13, University of Lund, Lund, Sweden.
Larsen
Kristoffer
K
Malmström
Lars
L
Tufvesson
Ellen
E
Parker
Ken
K
Marchese
Jason
J
Williamson
Brian
B
Patterson
Dale
D
Martin
Steve
S
Juhasz
Peter
P
Westergren-Thorsson
Gunilla
G
Marko-Varga
György
G
D002467:Cell Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D008168:Lung / Q000196:embryology; D008904:Miniaturization / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin
D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D014357:Trypsin