Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons.
Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.
Department of Biological Chemistry, University of Padova, Italy.
Roberto
R
Stella
Paolo
P
Cifani
Caterina
C
Peggion
Karin
K
Hansson
Cristian
C
Lazzari
Maria
M
Bendz
Fredrik
F
Levander
Maria Catia
MC
Sorgato
Alessandro
A
Bertoli
Peter
P
James
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000032:analysis* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D018621:PrPC Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D008565:Membrane Proteins; D010446:Peptide Fragments; D018621:PrPC Proteins; D020543:Proteome