An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins.
Matrix-assissted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses of complete proteolytic digests are often hampered by contaminations and the complexity of the sample. This results in suppression effects and the formation of adducts which are difficult to assign, thus leading to low scores in database searches. In particular, signals of post-translationally modified peptides such as glycopeptides are often of low intensity or completely suppressed. Online liquid chromatography electrospray ionization mass spectrometry (ESI-MS) can, in part, overcome this problem, but the analytes are completely consumed during the run. Coupling of nano-flow HPLC (nano-LC), microfractionation and MALDI-TOF-MS combines separation and high-sensitivity UV detection with the possibility of collecting fractionated peptides and preserving the sample for detailed mass spectrometric analyses. Here we report on an optimized protocol for nano-LC-MALDI-TOF-MS analyses of glycoproteins. This protocol improves spectral quality, resulting in better protein identification scores in database searches. Furthermore, post-translationally modified peptides could be detected with higher sensitivity by changing the experimental conditions, allowing assignment, localization and characterization of the respective carbohydrate substituents.
Lochnit
Günter
G
Institute of Biochemistry, Medical Faculty, Justus-Liebig University, Friedrichstrasse 24, D-35392 Giessen, Germany.
Geyer
Rudolf
R
D001212:Asialoglycoproteins / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D060748:Fetuins; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000737:chemistry / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D006023:Glycoproteins; D010446:Peptide Fragments; D000509:alpha-Fetoproteins; C017029:asialofetuin; D010447:Peptide Hydrolases; D014357:Trypsin