Casein phosphoproteome: identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis.
We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis.
Mamone
Gianfranco
G
Istituto di Scienze dell'Alimentazione del C.N.R., I-83100 Avellino, Italy. mamone@isa.av.cnr.it
Caira
Simonetta
S
Garro
Giuseppina
G
Nicolai
Adalgisa
A
Ferranti
Pasquale
P
Picariello
Gianluca
G
Malorni
Antonio
A
Chianese
Lina
L
Addeo
Francesco
F
D000818:Animals; D002240:Carbohydrate Sequence; D002364:Caseins / Q000032:analysis* / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D009844:Oligosaccharides / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D017421:Sequence Analysis; D012756:Sheep; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D002364:Caseins; D006023:Glycoproteins; D009844:Oligosaccharides; D010750:Phosphoproteins