Complete map and identification of the phosphorylation site of bovine lens major intrinsic protein.
OBJECTIVE
To determine the complete primary structure, including posttranslational modifications, of bovine lens major intrinsic protein (MIP) using a recently developed combination of liquid chromatography and mass spectrometry.


METHODS
The MIP was isolated from bovine lenses by sucrose gradient centrifugation and was cleaved with cyanogen bromide (CNBr). A high-performance liquid chromatographic system, developed for hydrophobic protein analysis, was used to separate the cleavage fragments. Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry were employed to obtain molecular weight and sequence data from bovine MIP CNBr fragments, directly or after subsequent digestion with trypsin.


RESULTS
The complete sequence of bovine MIP was mapped by molecular weight measurements of CNBr fragments, confirming the reported DNA sequence. The C-terminal peptide (177 to 263) was fully sequenced and the major site of phosphorylation was determined to be at serine 235 rather than at the previously reported serine 243. The level of phosphorylation in the native protein was determined to be 25%. No other posttranslational derivatizations were observed with the exception of the previously detected deamidation of asparagine 246.


CONCLUSIONS
These results represent the first complete MIP sequence map at the amino acid level and identify the single major phosphorylation site at serine 235.
Schey
K L
KL
Department of Pharmacology, Medical University of South Carolina, Charleston 29425, USA.
Fowler
J G
JG
Schwartz
J C
JC
Busman
M
M
Dillon
J
J
Crouch
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification; D007908:Lens, Crystalline / Q000737:chemistry*; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010766:Phosphorylation; D017421:Sequence Analysis; D012694:Serine / Q000737:chemistry
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C030328:aquaporin 0; D012694:Serine