W. Andy Tao

Aptamer in bioanalytical applications.

With over 2,000 publications, including about 250 reviews, resulting from a SciFinder search in just a two year period (2009–2010), the field of aptamer research has continuously generated lots of interest in the scientific community. Aptamers, first reported by three groups independently in 1990,1–3 are the artificial single-stranded DNA or RNA sequences (more recently, peptides) that fold into secondary and tertiary structures making them bind to certain targets with extremely high specificity. Owing to the high specific affinity of an aptamer to its target molecule (small molecules, proteins and even entire cells), it is thought to resemble chemical antibodies, with the dissociation constants ranging from nanomolar to picomolar level. Aptamers have a number of unique features which make them a more effective choice than antibodies. First, aptamers can be screened via in vitro process against a synthetic library, making it possible to target any molecules (from small inorganic ions to intact cells), overcoming the limit of having to use cell lines or animals, as is necessary for antibodies. Second, aptamers, once selected, can undergo subsequent amplification through polymerase chain reaction to produce a large quantity with high purity. Third, the simple chemical structure of aptamer makes it easily amendable to further modifications with functional groups according to different purposes. Finally, aptamers are much more stable than antibodies, making them suitable in applications requiring harsh conditions (e.g., high temperature or extreme pH). The applications of aptamers remain very dynamic, with increasing explorations in the fields of biosensing, diagnostics and therapeutics (some aptamer-based applications are illustrated in Figure 1). There have been a numbers of excellent reviews in recent years with different emphases.4–8 Herein, as the first review of aptamers on Analytical Chemistry, we attempt to cover major progresses in bioanalytical applications of aptamers in the past 2 years. Figure 1 The widespread use of aptamers for numerous analytical and biological applications. Bioanalytical applications of aptamers are highlighted in red.

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1

Significance Drought stress induces the accumulation of the plant stress hormone abscisic acid (ABA). ABA then quickly activates the protein kinase OST1/SnRK2.6 to phosphorylate a number of proteins in guard cells, resulting in stomatal closure to reduce transpirational water loss. How SnRK2.6 is deactivated and how ABA signaling may be desensitized are unclear. This study found that nitric oxide (NO) resulting from ABA signaling causes S-nitrosylation of SnRK2.6 at a cysteine residue close to the kinase catalytic site, which blocks the kinase activity. Dysfunction of S-nitrosoglutathione (GSNO) reductase causes GSNO overaccumulation in guard cells and ABA insensitivity in stomatal regulation. This work thus reveals how ABA-induced NO functions in guard cells to inactivate SnRK2.6 to negatively feedback regulate ABA signaling. The phytohormone abscisic acid (ABA) plays important roles in plant development and adaptation to environmental stress. ABA induces the production of nitric oxide (NO) in guard cells, but how NO regulates ABA signaling is not understood. Here, we show that NO negatively regulates ABA signaling in guard cells by inhibiting open stomata 1 (OST1)/sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6) through S-nitrosylation. We found that SnRK2.6 is S-nitrosylated at cysteine 137, a residue adjacent to the kinase catalytic site. Dysfunction in the S-nitrosoglutathione (GSNO) reductase (GSNOR) gene in the gsnor1-3 mutant causes NO overaccumulation in guard cells, constitutive S-nitrosylation of SnRK2.6, and impairment of ABA-induced stomatal closure. Introduction of the Cys137 to Ser mutated SnRK2.6 into the gsnor1-3/ost1-3 double-mutant partially suppressed the effect of gsnor1-3 on ABA-induced stomatal closure. A cysteine residue corresponding to Cys137 of SnRK2.6 is present in several yeast and human protein kinases and can be S-nitrosylated, suggesting that the S-nitrosylation may be an evolutionarily conserved mechanism for protein kinase regulation.

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.

Rapid ambient mass spectrometric profiling of intact, untreated bacteria using desorption electrospray ionization

Desorption electrospray ionization (DESI) allows the rapid acquisition of highly reproducible mass spectra from intact microorganisms under ambient conditions; application of principal component analysis to the data allows sub-species differentiation.

Differentiation and quantitation of isomeric dipeptides by low-energy dissociation of copper(II)-bound complexes.

Application of the kinetic method based on the dissociation of transition metal centered cluster ions is extended from chiral analysis (Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc.2000, 122, 10598) to quantitative analysis of isomeric mixtures, including those with Leu/Ile substitutions. Copper(II)-bound complexes of pairs of peptide isomers are generated by electrospray ionization mass spectrometry and the trimeric complex [CuII(ref)2(A) − H]+ (analyte A, a mixture of isomeric peptides; reference compound ref, usually a peptide) is caused to undergo collisional dissociation. Competitive loss of the neutral reference compound or the neutral analyte yields two ionic products and the ratio of rates of the two competitive dissociations, viz. the product ion branching ratio R is shown to depend strongly on the regiochemistry of the analyte in the precursor [CuII(A)(ref)2 − H]+ complex ion. Calibration curves are constructed by relating the branching ratio measured by the kinetic method, to the isomeric composition of the mixture to allow rapid quantitative isomer analysis.

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

Background: Glycolytic enzymes (GEs) are membrane-bound in oxygenated erythrocytes, but some GEs do not bind to the NH2 terminus of band 3. Results: Additional GE binding sites are identified on erythrocyte membrane proteins that associate with band 3. Conclusion: Complexes of GEs exist on the membrane in areas where ATP is consumed. Significance: The architecture of the GE complex is defined in greater detail. Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

Playing tag with quantitative proteomics

AbstractThere is steady need for new proteomic strategies on quantitative measurements that provide essential components for detailing dynamic changes in many cellular functions and processes. Stable isotope labeling is a rapidly evolving field, which can be used either after protein extraction with chemical labeling, or in cell culture with metabolic incorporation. In this review, we explore the most frequently utilized quantitation techniques with particular attention paid to chemical labeling using different isotopic tags, including a recent labeling strategy—soluble polymer-based isotopic labeling (SoPIL)—that achieves efficient labeling in homogeneous conditions. Special care should be devoted to the selection of appropriate quantitation approaches according to the needs of the sample and overall experimental design. We evaluate recent advances in quantitative proteomics using stable isotope labeling and their applications to current insightful biological inquiries. FigureChemical modules of isotopic tags for quantitative proteomics.

Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.

Facile Determination of Double Bond Position in Unsaturated Fatty Acids and Esters by Low Temperature Plasma Ionization Mass Spectrometry

Unsaturated fatty acids and esters can be oxidized in situ during ionization using a low temperature plasma (LTP) probe. The discharge generates ozone from air that reacts with and cleaves olefins. The molecular ions of the resulting acid/ester oxidation products are present in the full scan mass spectra and are confirmed by exact mass measurements. The fragmentation information can be used to assign double bond positions. We have successfully applied this strategy to a range of mono-/polyunsaturated fatty acids and fatty acid methyl/ethyl esters to assign their double bond locations. The procedure allows rapid and direct identification of double bond positions in situ at atmospheric pressure without sample preparation prior to mass spectrometric analysis. Microbial fatty acid ethyl ester (FAEE) mixtures from complex bacterial samples were directly analyzed by this method. Structural confirmation of their diagnostic ions by using exact mass measurements and tandem mass spectrometry confirms double bond positions in unsaturated bacterial FAEEs.