Profiling signaling peptides in single mammalian cells using mass spectrometry.
The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and nonenzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than 15 peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and postsource decay sequencing allows identification of 14 of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types.
Rubakhin
Stanislav S
SS
Department of Chemistry, Beckman Institute, University of Illinois, Urbana, Illinois 61801, USA.
Churchill
James D
JD
Greenough
William T
WT
Sweedler
Jonathan V
JV
D000818:Animals; D002477:Cells / Q000378:metabolism*; D005260:Female; D006728:Hormones / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D009474:Neurons / Q000737:chemistry; D010902:Pituitary Gland / Q000166:cytology; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D015398:Signal Transduction
D006728:Hormones; D011498:Protein Precursors; D011506:Proteins