( rate constituents of the cleav...)Structural Analysis of Glycosylated Peptides in Complex Mixtures with ,,, Ion Trap MSn

rate constituents of the cleaved glycoform. Generally, neither the oligosaccharide structure nor the exact site of attachment to the peptide can be determined. This technique is labor-intensive, time-consuming, and it requires a large amount of sample all of which severely limit its general utility. Goal In this report, we describe a novel, highthroughput technique that will allow researchers to determine:

1. The amino acid sequence of glycopeptides
2. The exact site of attachment of the oligosaccharide
3. Accurate, detailed structures of the attached oligosaccharide

Starting materials for this technique are tryptic fragments of the protein of interest. Treatment with glycosidase is not required, and very little sample is needed. Analysis can be accomplished in a single, automated process using any LCQ ion trap mass spectrometer with Data Dependent^TM MSⁿ in this case up to MS⁴ and Dynamic Exclusion^TM to successively isolate, fragment, and analyze the peptide and oligosaccharide structures. In this case, as is the case for most glycoprotein analyses, MS/MS is simply not enough. Experimental Conditions Thermo Finnigan LCQ Deca XP mass spectrometer fitted with orthogonal ESI probe (positive ion mode)
Ion capillary tube temp: 140 C
Needle voltage: +3.8 kV
Sheath gas: 9 units
Normalized Collision Energy: 65%
Dynamic Exclusion duration: 3 min
Surveyor Autosampler with Surveyor MS Pump: 120 L/min before splitting, 1 L/minute after splitting
HPLC column: Microte
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