fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Virginia Tech

The advance of glycoproteomic technologies has offered unique insights into the importance of glycosylation in determining the functional roles of a protein within a cell. Biologically active glycoproteins include the categories of enzymes, hormones, proteins involved in cell proliferation, cell membrane proteins involved in cell-cell recognition, and communication events or secreted proteins, just to name a few. The recent progress in analytical instrumentation, methodologies, and computational approaches has enabled a detailed exploration of glycan structure, connectivity, and heterogeneity, underscoring the staggering complexity of the glycome repertoire in a cell. A variety of approaches involving the use of spectroscopy, MS, separation, microfluidic, and microarray technologies have been used alone or in combination to tackle the glycoproteome challenge, the research results of these efforts being captured in an overwhelming number of annual publications. This work is aimed at reviewing the major developments and accomplishments in the field of glycoproteomics, with focus on the most recent advancements (2012-2014) that involve the use of capillary separations and MS detection.

Exploring the glycoproteomics landscape with advanced MS technologies.

The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples. Graphical Abstract ᅟ.

Article	Year
Exploring the glycoproteomics landscape with advanced MS technologies. Electrophoresis\| PubMed ID: 25311661	2015
Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins. Journal of the American Society for Mass Spectrometry\| PubMed ID: 26883530	2016

Jingren Deng

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