Simultaneous pre-concentration and separation on simple paper-based analytical device for protein analysis.

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.

Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells.

The peptide angiotensin IV (Ang IV) is a derivative of angiotensin II. While insulin regulated amino peptidase (IRAP) has been proposed as a potential receptor for Ang IV, the signalling pathways of Ang IV through IRAP remain elusive. We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A (N2A) cells treated with and without Ang IV using sta ble-isotope labeling by amino acids in cell culture (SILAC), and identified a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1 (PP1) upon Ang IV treatment. In addition, spinophilin (spn), a PP1 regulatory protein that plays important functions in the neural system, was expressed at higher levels. Immunoblotting revealed decreased phosphorylation of p70S6 kinase (p70(S6K)) and the major cell cycle modulator retinoblastoma protein (pRB). These changes are consistent with an observed decrease in cell proliferation. Taken together, our study suggests that Ang IV functions via regulating the activity of PP1.

Cupric ions induce the oxidation and trigger the aggregation of human superoxide dismutase 1.

BACKGROUND: Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1. METHODOLOGY/PRINCIPAL FINDINGS: As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn2-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu(2+) or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu(2+): a moderate-affinity site (10(6) M(-1)) and a high-affinity site (10(8) M(-1)). Furthermore, Cu(2+) binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form. CONCLUSIONS/SIGNIFICANCE: We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu(2+) plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.

[Differentially expressed urine protein of contrast induced acute kidney injury by two-dimensional differential in-gel electrophoresis and mass spectrometry].

OBJECTIVE: To explore the differentially expressed proteins of contrast-induced nephropathy through a comparison of urinary proteome so as to further elucidate the pathogenesis and discover the disease biomarker. METHODS: The urine samples of 12 patients were collected before and after coronary angiography. Two dimensional electrophoresis was performed after the urine samples were labeled by different dyes. The differences of urine proteome were analyzed by Decyder software and the differentially expressed spots identified by mass spectrometry. RESULTS: A total of 56 differentially expressed spots were detected. Among them, 39 spots were up-regulated and 17 spots down-regulated. And mannose binding lectin and mannose binding lectin associated serine protease 2, key proteins in complement body activation, were both significantly up-regulated. CONCLUSION: Urine proteomic study methods are constructed based on 2D-DIGE and mass spectrometry. The lectin pathway of complement body may be associated with contrast-induced acute kidney injury.

S-nitrosylation of AtSABP3 antagonizes the expression of plant immunity.

Changes in cellular redox status are a well established response across phyla following pathogen challenge. In this context, the synthesis of nitric oxide (NO) is a conspicuous feature of plants responding to attempted microbial infection and this redox-based regulator underpins the development of plant immunity. However, the associated molecular mechanism(s) have not been defined. Here we show that NO accretion during the nitrosative burst promotes increasing S-nitrosylation of the Arabidopsis thaliana salicylic acid-binding protein 3 (AtSABP3) at cysteine (Cys) 280, suppressing both binding of the immune activator, salicylic acid (SA), and the carbonic anhydrase (CA) activity of this protein. The CA function of AtSABP3 is required for the expression of resistance in the host against attempted pathogen infection. Therefore, inhibition of AtSBAP3 CA function by S-nitrosylation could contribute to a negative feedback loop that modulates the plant defense response. Thus, AtSABP3 is one of the first targets for S-nitrosylation in plants for which the biological function of this redox-based post-translational modification has been uncovered. These data provide a molecular connection between the changes in NO levels triggered by attempted pathogen infection and the expression of disease resistance.

C-terminal effect of Thermoanaerobacter tengcongensis ribosome recycling factor on its activity and conformation changes.

The in vivo activities and conformational changes of ribosome recycling factor from Thermoanaerobacter tengcongensis (TteRRF) with 12 successive C-terminal deletions were compared. The results showed that TteRRF mutants lacking one to four amino acid residues are inactive, those lacking five to nine are reactivated to a similar or a little higher level than wild-type TteRRF, and those lacking ten to twelve are inactivated again gradually. Conformational studies indicated that only the ANS binding fluorescence change is correlated well with the RRF in vivo activity change, while the secondary structure and local structure at the aromatic residues are not changed significantly. Trypsin cleavage site identification and protein stability measurement suggested that mutation only induced subtle conformation change and increased flexibility of the protein. Our results indicated that the ANS-detected local conformation changes of TteRRF and mutants are one verified direct reason of the in vivo inactivation and reactivation in Escherichia coli.