Title : A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum
Immunoglobulin M (
IgM )
Abstract :
- We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation
- It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for on-plate nanoliter reactions
- A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray
- In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots
- After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides , and one containing the intact glycopeptides
- Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace
- An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots
- Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment
- We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM
- By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-of-flight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain
- A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS
- We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy
- Besides complex type glycans on Asn 395, 332, 171 , and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171
- Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332