Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator.
The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.
Apffel
A
A
Biomeasurements Group, Hewlett-Packard Laboratories, Palo Alto, CA 94304, USA.
Chakel
J A
JA
Hancock
W S
WS
Souders
C
C
M'Timkulu
T
T
Pungor
E
E
D000581:Amidohydrolases / Q000737:chemistry; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010960:Plasminogen Activators / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase