Direct cellular peptidomics of supraoptic magnocellular and hippocampal neurons in low-density co-cultures.
Genomic and proteomic studies of brain regions of specialized function provide evidence that communication among neurons is mediated by systems of diverse chemical messengers. These analyses are largely tissue- or population-based, whereas the actual communication is from cell-to-cell. To understand the complement of intercellular signals produced by individual neurons, new methods are required. We have developed a novel neuron-to-neuron, serum-free, co-culture approach that was used to determine the higher-level cellular peptidome of individual primary mammalian neurons. We isolated magnocellular neurons from the supraoptic nucleus of early postnatal rat and maintained them in serum-free low density cultures without glial support layers; under these conditions they required low-density co-cultured neurons. Co-culturing magnocellular neurons with hippocampal neurons permitted local access to individual neurons within the culture for mass spectrometry. Using direct sampling, peptide profiles were obtained for spatially distinct, identifiable neurons within the co-culture. We repeatedly detected 10 peaks that we assign to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enables the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides.
Millet
Larry J
LJ
Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Bora
Adriana
A
Sweedler
Jonathan V
JV
Gillette
Martha U
MU
Physiol Rev. 2001 Jul;81(3):1197-267
11427695
Front Neuroendocrinol. 2003 Jan;24(1):27-61
12609499
Neuroscience. 1981;6(10):1953-60
7029345
Annu Rev Anal Chem (Palo Alto Calif). 2008;1:451-83
20636086
J Proteome Res. 2007 Dec;6(12):4667-76
17979226
J Neurosci Res. 1987;18(3):432-8
3437466
Anal Chem. 2008 May 1;80(9):3379-86
18321135
J Biomater Sci Polym Ed. 2007;18(8):1091-100
17706000
Biochem Soc Trans. 2007 Nov;35(Pt 5):1247-51
17956323
Neuroscience. 1989;32(2):435-9
2586758
Neuroscience. 2003;116(3):761-73
12573718
Neuropeptides. 2005 Aug;39(4):439-43
15885775
J Proteome Res. 2006 Dec;5(12):3368-75
17137338
J Comp Neurol. 1988 Jun 1;272(1):90-113
2454979
J Neurochem. 2008 Dec;107(6):1596-613
19014391
Lab Chip. 2007 Nov;7(11):1454-60
17960271
J Neuroendocrinol. 2008 Jun;20(6):677-86
18601689
Ann N Y Acad Sci. 2003 Jun;994:218-24
12851319
J Neurosci Methods. 1997 Feb;71(2):143-55
9128149
Mass Spectrom Rev. 2006 Jan-Feb;25(1):77-98
15937922
Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3972-7
9520477
Lab Chip. 2007 Aug;7(8):987-94
17653340
Mol Cell Proteomics. 2008 Jun;7(6):1067-76
18256212
J Neurosci Methods. 1996 Jan;64(1):13-8
8869479
Anal Chem. 2006 Oct 15;78(20):7267-72
17037931
Mol Cell Proteomics. 2005 Sep;4(9):1391-405
15970582
Trends Biotechnol. 2000 Apr;18(4):151-60
10740261
Mol Cell Proteomics. 2006 Jun;5(6):998-1005
16501280
Brain Res. 1989 Aug 7;494(1):65-74
2765923
J Neurosci Res. 1990 Jan;25(1):94-102
2319625
J Neurosci Res. 1989 Nov;24(3):445-50
2480456
J Neurosci Res. 1995 Dec;42(5):674-83
8600300
J Neuroendocrinol. 1999 Feb;11(2):145-52
10048470
Cell Tissue Res. 2006 Jan;323(1):43-51
16160861
Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W267-72
16845008
Brain Res. 1984 Jun;316(2):179-83
6380649
J Proteome Res. 2006 May;5(5):1162-7
16674105
Peptides. 2004 Mar;25(3):403-11
15134863
J Neurosci Methods. 1999 Jul 1;89(1):57-67
10476684
J Neurosci Res. 1993 Aug 1;35(5):567-76
8377226
Nat Protoc. 2007;2(8):1987-97
17703210
Mamm Genome. 2008 Feb;19(2):106-20
18213482
J Mass Spectrom. 2005 Feb;40(2):227-37
15706630
J Proteome Res. 2008 Nov;7(11):4992-5003
18816085