Amine-reactive Neutron-encoded Labels for Highly Plexed Proteomic Quantitation

We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between ¹³C and ¹⁵N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference—relative to its nearest neighbor—so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration.Proteome quantification is an increasingly essential component of modern biology and translational medicine (1, 2). Whether targeted or global, stable isotope incorporation with mass spectrometry (MS) analysis is a core technique for protein abundance measurements. There are numerous approaches that can be used to introduce stable isotopes into peptides, the most frequently used being stable isotope labeling with amino acids in cell culture (SILAC)¹ and isobaric tagging (tandem mass tags or isobaric tags for relative and absolute quantitation) (3–7). Both of these methods incorporate heavy isotopes to increase mass by at least 1 Da. SILAC is the quantification gold standard for global proteomic analysis. However, the SILAC approach is not easily adapted for tissue sample analysis; SILAC mouse labeling, for example, requires feeding mice a specialized diet for multiple generations (8). Tissue samples can be analyzed if they are mixed with SILAC cell-culture-based labeled standards, but this strategy does not permit multiplexing (9). Isobaric labels, in contrast, are conjugated to the primary amines of peptides following proteolytic digestion and thus have the advantage of being completely compatible with samples from virtually any source (10, 11). That said, isobaric tagging suffers from dynamic range suppression caused by co-isolation of precursor peptides (12). Multiple studies have revealed that this problem greatly erodes quantitative accuracy—for example, 10-fold changes often are detected as much smaller ∼4-fold changes (13, 14). Here we propose a new approach to protein quantification, one that achieves tissue-compatible 4-plexed MS1-based quantification without increasing spectral complexity.Recently, we described the use of mass defects to expand SILAC quantification from 3-plex to 12-plex and beyond (i.e., NeuCode SILAC) (15). NeuCode SILAC exploits the subtle mass differences that exist in atoms as a result of the varying energies of nuclear binding in common stable isotopes (e.g., carbon, nitrogen, hydrogen, and oxygen) by using the extremely high resolving power of modern Fourier transform mass spectrometer systems (16, 17). For example, the multiplexing capability of tandem mass tagging was increased from 6 to 8 by incorporating a difference in mass of 6.3 mDa in specific reporter ions by swapping ¹⁴N for a ¹⁵N atom while concomitantly switching a ¹³C with a ¹²C atom (18, 19). This method requires only 30,000 resolving power to resolve the reporter ions, but it still suffers from the interference problem described above. Repetition of this process, within the context of an analyte molecule, can generate several chemically identical isotopologues that, when analyzed under normal MS analysis conditions (resolving power R) are indistinguishable (i.e., produce one m/z peak). Analysis of these NeuCode m/z peaks with high resolving power (480,000), however, often reveals distinct m/z peaks whose abundances can be extracted and used to determine analyte quantity across the sundry conditions. This strategy permits very high levels of MS1-based multiplexing (>10), which has several advantages. First, MS1 scans across the entire analyte elution profile can be averaged to increase quantitative accuracy and precision. Second, a tandem mass spectrum is not necessary for quantification. Mann and colleagues showed that once a peptide is identified via tandem MS analysis, it can be confidently identified in other runs with only the exact mass and elution profile matching (20). Third, MS1-based quantification does not suffer from the pervasive problem of precursor interference that cripples the quantitative accuracy of the isobaric tagging strategies (13).The NeuCode SILAC approach, however, relies on the use of amino acid isotopologues and still requires metabolic incorporation. Chemical labeling strategies for proteome quantification can be convenient and, for certain systems, are requisite. We reasoned that our NeuCode strategy could be extended to create novel chemical reagents for proteome quantification. We describe here the design, synthesis, and use of 12-plex NeuCode amine reactive labels for global proteome quantification.     

Nonparallelism in MHCIIβ diversity accompanies nonparallelism in pathogen infection of lake whitefish (Coregonus clupeaformis) species pairs as revealed by next‐generation sequencing

Major histocompatibility (MHC) immune system genes may evolve in response to pathogens in the environment. Because they also may affect mate choice, they are candidates for having great importance in ecological speciation. Here, we use next‐generation sequencing to test the general hypothesis of parallelism in patterns of MHCIIβ diversity and bacterial infections among five dwarf and normal whitefish sympatric pairs. A second objective was to assess the functional relationships between specific MHCIIβ alleles and pathogens in natural conditions. Each individual had between one and four alleles, indicating two paralogous loci. In Cliff Lake, the dwarf ecotype was monomorphic for the most common allele. In Webster Lake, the skew in the allelic distribution was towards the same allele but in the normal ecotype, underscoring the nonparallel divergence among lakes. Our signal of balancing selection matched putative peptide binding region residues in some cases, but not in others, supporting other recent findings of substantial functional differences in fish MHCIIβ compared with mammals. Individuals with fewer alleles were less likely to be infected; thus, we found no evidence for the heterozygote advantage hypothesis. MHCIIβ alleles and pathogenic bacteria formed distinct clusters in multivariate analyses, and clusters of certain alleles were associated with clusters of pathogens, or sometimes the absence of pathogens, indicating functional relationships at the individual level. Given that patterns of MHCIIβ and bacteria were nonparallel among dwarf and normal whitefish pairs, we conclude that pathogens driving MHCIIβ evolution did not play a direct role in their parallel phenotypic evolution.     

Epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thereby risk for arterial thrombosis. Activation of the renin-angiotensin system has been linked to the production of PAI-1 expression via the angiotensin II type 1 receptor (AT1R). In addition, bradykinin can induce the release of t-PA through a B2 receptor mechanism. In the present study, we aimed to investigate the epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels in a large population-based sample (n=2527). We demonstrated a strong significant interaction within genetic variations of the bradykinin B2 gene (P=0.002) and between ACE and bradykinin B2 (p=0.003) polymorphisms on t-PA levels in females. In males, polymorphisms in the bradykinin B2 and AT1R gene showed the most strong effect on t-PA levels (P=0.006). In both females and males, the bradykinin B2 gene interacted with AT1R gene on plasma PAI-1 levels (P=0.026 and P=0.039, respectively). In addition, the current study found a borderline significant interaction between PAI 4G5G and ACE I/D on plasma t-PA and PAI-1 levels. These results support the idea that the interplay between the renin-angiotensin, bradykinin, and fibrinolytic systems might play an important role in t-PA and PAI-1 biology.     

DNA Barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera)

Background

Many studies have shown the suitability of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group.

Methodology/Principal Findings

Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%.

Conclusions/Significance

This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage.     

Short Daily versus Conventional Hemodialysis for Hypertensive Patients: A Randomized Cross-Over Study

Background

Treatment of end stage renal disease patients with short daily hemodialysis has been associated with an improvement in blood pressure. It is unclear from these studies if anti-hypertensive management had been optimized prior to starting short daily hemodialysis. Also, the potential mechanism(s) of blood pressure improvement remain to be fully elucidated.

Study Design, Setting and Participants

We undertook a randomized cross-over trial in adult hypertensive patients with ESRD treated with conventional hemodialysis to determine: 1) if short-daily hemodialysis is associated with a reduction in systolic blood pressure after a 3-month blood pressure optimization period and; 2) the potential mechanism(s) of blood pressure reduction. Blood pressure was measured using Canadian Hypertension Education Program guidelines. Extracellular fluid volume (ECFV) was assessed with bioimpedance. Serum catecholamines were used to assess the sympathetic nervous system. Interleukin-6 (IL-6) and thiobarbituric acid reactive substances (T-BARS) were used as markers of inflammation and oxidative stress respectively.

Results

After a 3-month run-in phase in which systolic blood pressure improved, there was no significant difference in pre-dialysis systolic pressure between short-daily and conventional hemodialysis (p = 0.39). However, similar blood pressures were achieved on fewer anti-hypertensive medications with short daily hemodialysis compared to conventional hemodialysis (p = 0.01). Short daily hemodialysis, compared to conventional hemodialysis, was not associated with a difference in dry weight or ECFV (p = 0.77). Sympathetic nervous system activity as assessed by plasma epinephrine (p = 1.0) and norepinephrine (p = 0.52) was also not different. Markers of inflammation (p = 0.42) and oxidative stress (p = 0.83) were also similar between the two treatment arms.

Conclusions

Patients treated with short daily, compared to conventional hemodialysis, have similar blood pressure control on fewer anti-hypertensive medications. The mechanism(s) by which short daily hemodialysis allows for decreased anti-hypertensive medication use remains unclear but effects on sodium balance and changes in peripheral vascular resistance require further study.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00759967","term_id":"NCT00759967"}}NCT00759967     

A New Record of a Host-Parasitoid Interaction: Hymenoepimecis veranii Lofredo & Penteado-Dias, 2009 (Hymenoptera: Ichneumonidae) Parasitizing Araneus orgaos Levi, 1991 (Araneae: Araneidae)

The genus Hymenoepimecis only occurs in the Neotropics. In some species of this genus, the larval stage modifies the behavior of the spider hosts, inducing then to construct a modified web. The wasp Hymenoepimecis veranii has been previously described as an ectoparasitoid of the spider Araneus omnicolor. This study provides detailed information about the natural history of the host-parasitoid interaction involving this wasp with a new host, the spider Araneus orgaos (Araneae: Araneidae), which occurs sympatrically with A. omnicolor.     

Taste of a Pill: ORGANIC CATION TRANSPORTER-3 (OCT3) MEDIATES METFORMIN ACCUMULATION AND SECRETION IN SALIVARY GLANDS*

Drug-induced taste disturbance is a common adverse drug reaction often triggered by drug secretion into saliva. Very little is known regarding the molecular mechanisms underlying salivary gland transport of xenobiotics, and most drugs are assumed to enter saliva by passive diffusion. In this study, we demonstrate that salivary glands selectively and highly express OCT3 (organic cation transporter-3), a polyspecific drug transporter in the solute carrier 22 family. OCT3 protein is localized at both basolateral (blood-facing) and apical (saliva-facing) membranes of salivary gland acinar cells, suggesting a dual role of this transporter in mediating both epithelial uptake and efflux of organic cations in the secretory cells of salivary glands. Metformin, a widely used anti-diabetic drug known to induce taste disturbance, is transported by OCT3/Oct3 in vitro. In vivo, metformin was actively transported with a high level of accumulation in the salivary glands of wild-type mice. In contrast, active uptake and accumulation of metformin in salivary glands were abolished in Oct3^−/− mice. Oct3^−/− mice also showed altered metformin pharmacokinetics and reduced drug exposure in the heart. These results demonstrate that OCT3 is responsible for metformin accumulation and secretion in salivary glands. Our study uncovered a novel carrier-mediated pathway for drug entry into saliva and sheds new light on the molecular mechanisms underlying drug-induced taste disorders.