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An optimized protocol for nano‐LC‐MALDI‐TOF‐MS coupling for the analysis of proteolytic digests of glycoproteins
Author(s) -
Lochnit Günter,
Geyer Rudolf
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.399
Subject(s) - chemistry , chromatography , mass spectrometry , electrospray ionization , matrix assisted laser desorption/ionization , protein mass spectrometry , database search engine , glycopeptide , time of flight mass spectrometry , electrospray , analytical chemistry (journal) , ionization , desorption , biochemistry , ion , antibiotics , organic chemistry , adsorption , computer science , information retrieval , search engine
Matrix‐assissted laser desorption ionization time‐of‐ight 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 difcult to assign, thus leading to low scores in database searches. In particular, signals of post‐translationally modied 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‐ow 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 identication scores in database searches. Furthermore, post‐translationally modied peptides could be detected with higher sensitivity by changing the experimental conditions, allowing assignment, localization and characterization of the respective carbohydrate substituents. Copyright © 2004 John Wiley & Sons, Ltd.