Research - Proteomics

Electrospray–assisted Laser Desorption Ionization (ELDI)

Summary

ELDI is a new, soft ionization method which combines features of both ESI and MALDI. In ELDI, neutral protein molecules are desorbed from solid samples by a nitrogen laser pulse and post-ionized through fusion with charged solvent droplets generated by ESI. The advantages of ELDI include: (1) it works under ambient conditions, (2) laser desorption has a higher power for desorption compared to DESI (Desorption ESI), (3) matrices are unnecessary in ELDI, (4) ESI-like ions give more accurate molecular weight and reduce the mass requirement of the mass analyzer for large protein analysis.

Electrowetting–on–dielectric (EWOD)

Summary

Microfluidics is a technology which can integrate multiple functions onto a single device. Applications of microfluidics include separations, reactions, immunoassays, enzymatic digests, and cell transport and analysis. The capacity for integrating multiple laboratory processes makes microfluidics an attractive technology for proteomics applications.

EWOD is a new microfluidic technique which transports fluid as discrete droplets on a surface. The movement of droplets on the EWOD surface is based on the buildup of charge on the dielectric material, which makes the surface more hydrophilic and decreases the contact angle of the droplet, θ. EWOD chip can serve as a media for protein purification and is comparable to MALDI-TOF. In collaboration with Professor Robin Garrell, we hope to develop EWOD into a useful technique for high-throughput proteomic studies.

Publications

Human Salivary Proteome

Summary

The Human Salivary Proteome Project is an effort to generate a catalog of proteins and their posttranslational modifications in the secretions from the three major salivary glands namely parotid, submandibular and sublingual. The tools currently being employed to map the salivary proteome include:

1. Shotgun Proteomics. In the shotgun approach salivary proteins are first prefractionated either by their molecular weight using a MW cutoff filter or by their pI by in-solution Zoom IEF fractionation. The proteins in each fraction are then digested with trypsin. The digests are further analyzed and proteins are identified by 1D and 2D-LC mass spectrometry.
2. Two-dimensional gel electrophoresis-mass spectrometry (2-DE-MS). Proteins from the saliva are separated on a 2D-gel. The gel is stained with total protein stain like Sypro Ruby. The gel spots are then excised, proteins therein are digested in-gel and identified by mass spectrometry.

We also aim to identify posttranslational modifications on salivary proteins like glycosylation and phosphorylation. The technique currently being employed to identify N-glycosylated proteins is the hydrazide coupling and release method. In this approach, glycoproteins are coupled onto a hydrazide resin, the proteins are then digested and formerly N-glycosylated peptides are selectively released with the enzyme PNGase F and analyzed by LC-MS/MS. Employing this method we have identified more than 40 N-linked salivary glycoproteins.

Publications

Virtual 2D Gel Electrophoresis

Summary

In the virtual 2D gel approach, mass spectrometry is substituted for SDS-PAGE size-based separation. Use of mass spectrometry for the second dimension provides advantages in mass resolution, mass accuracy, and sensitivity over classical 2D gels. Post-translational modifications can be readily detected by the mass-resolved heterogeneity of component hydrocarbon chains. Direct comparison to classical 2D gels provides a link between 2D gel spots and accurate, higher resolution molecular weight measurements.

Publications