NCGG Workshop on Glycomics, Glycoproteomics & Proteomics
Hands-on Lab Experiences - Details
The hands-on experience of the glycomics workshop focuses on techniques related to glycomic analysis. N-Glycans of human blood serum samples will be enzymatically released using PNGase F prior to clean-up. The released N-glycans will be profiled using C-GlycoMAP which involves solid-phase isotopic permethylation using a spin-column method. Permethylated glycans and deuteromethylated glycans will be then analyzed by MALDI-TOF mass spectrometer where glycomic maps of the samples will be generated. The use of isotopically labeled glycans for direct comparison of samples and tandem MS analysis of permethylated and deuteromethylated glycans will be investigated. The ultimate goal of these activities is to train you to be able to perform glycomic analysis and C-GlycoMAPs on your own.
Glycoproteomics / LC-MS / MALDI-PMF
In this part of the hands-on lab experience, you will perform LC-MS on a trypsin digest of a protein and a simple mixture of proteins (time permitting). The LC-MS experiment will be conducted in a manner allowing the simultaneous generation of CID and ETD tandem mass spectra for both peptides and glycopeptides. The two techniques will then be compared, thus highlighting the strengths and weaknesses of each. Resulting data will then be processed and database searched. You will also explore MALDI-MS, including basic instrumentation and common matrices used for biological studies. Peptide-mass fingerprinting and tandem MS approaches will also be covered. Time permitting, LC-MALDI and automation of data collection will also be discussed.
Lectin enrichment / Top-down glycoproteomics
The usefulness of lectin enrichment to facilitate the top-down analysis of various glycoproteins will be demonstrated in this part of the hands-on lab experience. A mixture of standard proteins and glycoproteins bearing different types of N- and O-glycans will be subjected to Con A-agarose based lectin affinity chromatography. After the elution of bound glycoproteins with an appropriate buffer and buffer exchange, these proteins will be separated and analyzed by LC-MS, using a Dionex Ultimate 3000 LC system interfaced to an LTQ-FT Ultra mass spectrometer. Samples will be on-line desalted and pre-concentrated on a C4 trapping cartridge, while the separation of intact proteins and glycoproteins will be attained using a C4 or C8 capillary column. Data acquired from high-resolution MS scanning will be evaluated for each of the separated glycoproteins with focus on the evaluation of post-translational modification. The training will also include a thorough explanation of the system setup and the utility of FT-MS for the analysis of peptides and intact proteins.