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.

The response of cells to physical or chemical stimuli is complex, unfolding on time-scales from seconds to days, with or without de novo protein synthesis, and involving signaling processes that are transient or sustained. By combining the technology of microfluidics that supports fast and precise execution of a variety of cell handling operations, with that of mass spectrometry detection that facilitates an accurate and complex characterization of the protein complement of cells, in this work, we developed a platform that supports (near) real-time sampling and proteome-level capturing of cellular responses to a perturbation such as treatment with mitogens. The geometric design of the chip supports three critical features: (a) capture of a sufficient number of cells to meet the detection limit requirements of mass spectrometry instrumentation, (b) fluid delivery for uniform stimulation of the resident cells, and (c) fast cell recovery, lysis and processing for accurate sampling of time-sensitive cellular responses to a stimulus. COMSOL simulations and microscopy were used to predict and evaluate the flow behavior inside the microfluidic device. Proteomic analysis of the cellular extracts generated by the chip experiments revealed that the identified proteins were representative of all cellular locations, exosomes, and major biological processes related to proliferation and signaling, demonstrating that the device holds promising potential for integration into complex lab-on-chip work-flows that address systems biology questions. The applicability of the chips to study time-sensitive cellular responses is discussed in terms of technological challenges and biological relevance.

Phafin2 is a phosphatidylinositol 3‐phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ∼28 kDa with an intensity‐average hydrodynamic diameter of ∼7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/β structure and predicts ∼40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding‐unfolding curves, obtained using urea‐ and guanidine hydrochloride‐mediated denaturation, indicate that Phafin2 undergoes a two‐state native‐to‐denatured transition. Analysis of these transitions shows that the free energy change for urea‐ and guanidine hydrochloride‐induced Phafin2 denaturation in water is ∼4 kcal mol−1. PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P‐binding domains of the protein in endomembrane compartments.

We demonstrate that partial enzymatic reactions conducted on short time-scales represent a valuable asset to proteomic studies, and propose their implementation either as simple, cost-effective, stand-alone protocols for substantially streamlining the analysis of biological samples, or as complementary protocols, for improving protein sequence and proteome coverage.

Abstract. 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 TiO 2 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 TiO 2 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.