Streamlined microfluidic analysis of phosphopeptides using stable isotope-labeled synthetic peptides and MRM-MS detection.

Modern high-throughput and high-content biological research is performed with advanced instrumentation and complex and time-consuming protocols, which, as a whole, pose a challenge for routine implementation in a research laboratory. In support of a "bioanalytical toolbox" with potential utility for exploring cellular functions mediated via protein phosphorylation-a post-translational modification (PTM) with essential regulatory roles in a variety of cellular processes-in this work, we describe the development of a simple, integrated microfluidic chip that can perform targeted, quantitative analysis of phosphopeptides involved in cancer-relevant signaling pathways. The microfluidic device comprises microreactors packed with C18 and TiO2 particles for on-chip solid phase extraction (SPE) and phosphopeptide enrichment, and an ESI interface for facilitating multiple reaction monitoring (MRM)-mass spectrometry (MS) detection. The chips are demonstrated for the detection of three phosphopeptides involved in ERBB2/MAPK signaling pathways, selected from the outcome of a proteomic study involving EGF stimulation of SKBR3/HER2+ breast cancer cells. The data demonstrate that the proposed microfluidic strategy can be used for the MS quantification of phosphopeptides in the low nM range from cell lysates without any prior sample pretreatment, fractionation or bioaffinity enrichment, and is generally applicable to the analysis of any phosphopeptide targets.

Exploring the glycoproteomics landscape with advanced MS technologies.

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.

N(2)O emission from degraded soybean nodules depends on denitrification by Bradyrhizobium japonicum and other microbes in the rhizosphere.

A model system developed to produce N(2)O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N(2)O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N(2)O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N(2)O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N(2)O from the soybean rhizosphere. The N(2)O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N(2)O emission. A (15)N tracer experiment indicated that N(2)O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N(2)O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N(2)O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N(2)O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N(2)O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N(2)O from nodule N via their denitrification processes (N(2)O source), whereas nosZ-competent B. japonicum exclusively takes up N(2)O (N(2)O sink). Net N(2)O flux from soybean rhizosphere is likely determined by the balance of N(2)O source and sink.