Amidated peptides identified in adult male mouse pituitary extracts.

<p>All masses listed here are monoisotopic. Abbreviations: MSH, melanocyte stimulating hormone; JP, joining peptide.</p

Conversion of ACTH(1-13)-Gly-OH to its amidated form ACTH(1-13)-NH₂ during the measurement process when analyzed by MALDI-TOF MS and ESI-IT MS².

<p>(<b>A</b>) The table shows the assessment of the amidated and hydroxyglycine-extended ACTH(1-13) peaks in MALDI-TOF MS. (<b>B</b>) With manual ESI-IT MS², ACTH(1-13)-NH₂ and ACTH(1-13)-Gly-OH were respectively isolated, and then fragmented. The MS² of ACTH(1-13)-NH₂ matches the MS³ of ACTH(1-13)-Gly-OH.</p

Effects of PAM haploinsufficiency and dietary copper (Cu) on the amidation of joining peptide (JP) and arginine-vasopressin (AVP).

<p>Pituitary samples were taken from WT and PAM^+/− mice maintained on normal, copper-deficient or copper-supplemented diets. In order to take into account differences in the sample preparation and mass spectrometry, for each peptide in each sample, the ratio of glycine-extended to amidated peptide was calculated, with higher numbers indicating an accumulation of glycine-extended peptide. The effects of (<b>A</b>) haploinsufficiency, (<b>B</b>) copper deficiency and (<b>C</b>) copper supplementation were then assessed by comparing the ratios between the pairs of samples to indicate the extent of the glycine-extended peptide accumulation. For example, (A) reports the WT normal diet value divided by the PAM^+/− normal diet value. N is the number of biological replicate pairs. Error bar, SEM. Student's <i>t</i>-test: ***, <i>p</i><0.0005; **, <i>p</i><0.05; *, <i>p</i><0.1.</p

Analysis of mouse pituitary extracts using capillary LC coupled to ESI-IT MS².

<p>Mass spectra of amidated (top) and glycine-extended (bottom) forms of (<b>A</b>) synthetic adrenocorticotropic hormone (ACTH) (1-13), and (<b>B</b>) endogenous joining peptide (JP). The assignment of b- and y-ions matches expected fragments within 0.2 Da.</p

Proteins uniquely associated with ARC-purified hmCD40 as determined by mass spectrometry analysis.

<p>*All protein assignments listed met or exceeded the Scaffold 95% confidence filter.</p><p>**TRAF1 was detected in gel slices containing material at or above the known molecular weight for TRAF1 in hmCD40 ARC samples, but was also detected in both hmCD40Δ67 and hmCD40 gel slices containing material of approximately 35 kD.</p

Western blot verification of mass spectrometry results.

<p>hmCD40 and hmCD40Δ67 ARC samples (3.3×10⁶ cell equivalents per lane) were fractionated by SDS-PAGE and transferred to PVDF membrane for Western blotting with the indicated antibodies. Lanes containing whole cell lysates (1.0×10⁵ cell equivalents per lane) were run in parallel. Each blot is representative of three or more experiments.</p

TRAF-dependent recruitment of proteins to CD40.

<p>Endogenous CD40 from CH12.LX cells (WT), TRAF2-deficient CH12.LX (T2-), or TRAF3 deficient CH12.LX cells (T3-) was isolated using ARC (lane loading as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011380#pone-0011380-g002" target="_blank">Fig. 2</a>). Western blotting was performed with the indicated antibodies. Each blot is representative of three or more experiments.</p

HOIPΔRBR inhibits IκBα phosphorylation and degradation.

<p>TRAF6-deficient A20.2J cells were stably transfected with FLAG2X-tagged HOIP or HOIPΔRBR in an IPTG-inducible expression vector. A, Expression of FLAG2X-tagged HOIP or HOIPΔRBR in stably transfected cell lines. Western blots of cell lysates from uninduced and IPTG-induced cell lines were probed with anti-FLAG (left) and anti-HOIP (right). Blots were reprobed for actin to verify equivalent lane loading. B, The two cell lines were stimulated with CD154 (CD40 ligand) for the times indicated and then processed for Western blotting. Unstimulated cells (first lane) and cells incubated for 5 minutes with insect cells lacking CD154 served as negative controls. Western blotting for phosphorylated IκBα was performed; blots were then stripped and reprobed for total IκBα and actin. C, Quantification of IκBα degradation (panel B) in TRAF6-deficient A20.2J cells stably transfected with Lac repressor only (◊;♦), Lac repressor plus full-length HOIP (□;▪), or Lac repressor plus HOIPΔRBR (Δ; ▴). Filled symbols indicate cultures pre-treated with IPTG. IκBα bands in each lane were normalized to the actin signal. The degradation index is the fraction of IκBα remaining at each time point relative to the amount of IκBα present in cells incubated for 5 minutes with insect cells lacking CD154. The results presented are the mean of four experiments. Error bars indicate standard error of the mean.*, p<0.05 (one-sided Student's t test).</p

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long-term potentiation and in the effects of cocaine on behavior and spine morphology. The <i>KALRN</i> gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multisite phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity, and reduced GEF activity