Optimization of Electron Transfer Dissociation via Informed Selection of Reagents and Operating Parameters

Electron transfer dissociation (ETD) has improved the mass spectrometric analysis of proteins and peptides with labile post-translational modifications and larger intact masses. Here, the parameters governing the reaction rate of ETD are examined experimentally. Currently, due to reagent injection and isolation events as well as longer reaction times, ETD spectra require significantly more time to acquire than collision-induced dissociation (CID) spectra (>100 ms), resulting in a trade-off in the dynamic range of tandem MS analyses when ETD-based methods are compared to CID-based methods. Through fine adjustment of reaction parameters and the selection of reagents with optimal characteristics, we demonstrate a drastic reduction in the time taken per ETD event. In fact, ETD can be performed with optimal efficiency in nearly the same time as CID at low precursor charge state (<i>z</i> = +3) and becomes faster at higher charge state (<i>z</i> > +3)

Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma

The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion

Substrate Specificity of Mammalian N-Terminal α-Amino Methyltransferase NRMT

N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products

Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/ M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases

The CDK1-9 sites of Rad9 specifically function in the Chk1 branch of the G2/M checkpoint.

<p><i>rad9^CDK1-9A</i> mutant cells arrested in G2/M by nocodazole treatment are neither defective for Rad9 (A) nor for Rad53 (B) phosphorylations induced by bleocin treatment started at time 0. (C) The CDK1-9 sites of Rad9 function specifically in the Chk1 branch and not the Rad53 branch of the G2/M checkpoint. The indicated strains were examined for epistatic relationships using the G2/M checkpoint assay, in which cells synchronized in G2/M using nocodazole are released after irradiation into medium without nocodazole, but containing α-factor, preventing cells that have successfully completed mitosis from cycling further by arresting them in G1. This assay measures the delay in completing mitosis under DNA damaging conditions by comparing the behavior of cells that have been irradiated with IR (+IR) or not (−IR). All strains contained the <i>sml1Δ</i> mutation necessary for the viability of <i>rad53Δ</i> cells. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s003" target="_blank">Figure S3</a>.</p

CDK sites 6 (T125) and 7 (T143) regulate the Rad9/Chk1 interaction.

<p>(A) The Y2H interaction between Rad9 and Chk1 mostly requires the 6^th and 7^th CDK sites, T125 and T143, of the CAD region (B) Peptides pull down experiments identified phosphorylated T143 (CDK site 7) and unphosphorylated T125 (CDK site 6) as the best combination of these sites required for maximum Chk1/Rad9 interaction. Chk1-3FLAG was immunopurified from <i>rad9Δ</i> cell clarified crude extracts (CCE) and incubated with four different biotinylated 35 amino-acid peptides. As represented in red, the T125 T143, T125p, T143p and T125p143p peptides were un-, mono- or di-phosphorylated on residues T125 and T143. Magnetic streptavidin beads were boiled to analyse both the presence of Chk1-3FLAG (anti-FLAG western blot) and that equal amount of peptides were used in each pull down (see Ponceau S stain). The dependency on the phosphorylation of T143 was confirmed by lambda phosphatase treatment in the absence (λ) or in the presence of phosphatase inhibitors (λ+Inh). Empty lanes are indicated by ‘x’ above the relevant lanes. (C) The Rad9 CAD peptide array phosphorylation profile shows that peptides containing consensus CDK sites 6 (T125), 7 (T143), 8 (T155) and 9 (T218) are phosphorylated by Cdc28/Clb2 <i>in vitro</i>. Peptides arrays of immobilized overlapping 19-mer peptides, each shifted to the right by 3 amino acids encompassing the first 260 amino acids of Rad9 sequence, were generated and are schematically represented at the top of the panel. The arrays were used in a kinase assay with (+) or without (−) the purified Cdc28/Clb2 complex. Target peptides of Cdc28, CK2 and Cdc7 were also spotted as controls as indicated. (D) Phosphorylation levels for CDK site-containing peptides within Rad9 isolated from asynchronous, G1- and G2/M arrested cells. Relative abundances were determined by mass spectrometry (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#s4" target="_blank">Material and Methods</a> section). Residues T125 and T143 (CDK sites 6 and 7 respectively) are differentially phosphorylated <i>in vivo</i>. ○ indicates phosphorylated peptide containing this CDK site is assigned by HPLC retention time and accurate mass only. ND = Not detected. Non-phosphorylated and phosphorylated peptides were not detected. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s007" target="_blank">Figure S7</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s008" target="_blank">Table S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s012" target="_blank">Table S5</a>.</p

CDK is required for the activation of Chk1-dependent signaling.

<p>Cdc28 activity was regulated using the 1-NMPP1 inhibitor in G2/M arrested <i>cdc28-as1</i> cells treated with bleocin or 4-NQO to examine the activation of Chk1 signaling. Rad9 and Rad53 were followed as markers of checkpoint activation, while Orc6 phosphorylation serves as a marker for Cdc28 inactivation. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s004" target="_blank">Figure S4</a>.</p

N-terminal CDK consensus sites are phosphorylated by Cdc28/Clb2 and are required for activation of Chk1 <i>in vivo</i>.

<p>(A) Schematic representation of the Rad9 N-terminus showing the 9 consensus sites for phosphorylation by Cdc28 and those mutated to alanine in Rad9^CDK1-9A mutant protein. (B) <i>In vitro</i> phosphorylation of Rad9 CAD^WT, but not CAD^CDK1-9A, requires Cdc28/Clb2. <i>In vitro</i> kinase assays were performed on the indicated substrates with four specific purified Cdc28 complexes. (C) DNA damage-dependent Chk1 phosphorylation is defective in <i>rad9^CDK1-9A, rad9^CADΔ</i> and <i>rad9 Δ</i> cells. Asynchronously growing cells were treated with bleocin for the indicated times and Chk1 phosphorylation analysed by western blotting. (D&E) Chk1 activation is mostly dependent on the 9 CAD CDK consensus sites in G2/M cells. Cells were grown and arrested in the cell cycle as indicated with either α-factor (G1 cells in D) or nocodazole (G2/M cells in E), treated with bleocin for the indicated times and analysed by western blotting of Chk1-3HA (D & E). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s002" target="_blank">Figure S2</a> for related experiments.</p

CDK-dependent phosphorylation of the nine N-terminal CDK sites in Rad9 regulates a physical interaction between Rad9 and Chk1.

<p>(A) Rad9/Chk1 interaction measured <i>in vivo</i> using a yeast two-hybrid (Y2H) assay is dependent on the CDK1-9 sites. Y2H interaction of specific bait and prey plasmids shown on the left is indicated by the white colour of the otherwise red cells, their resistance to Aureobasidin A and their blue colour on media containing the X-α-gal substrate, as for the p53/T antigen interaction control. Six independent clones are presented for each vector combination. (B) The Y2H interaction between Rad9 and Chk1 is dependent on CDK activity in G2/M cells. The indicated bait and prey plasmids were introduced into <i>cdc28-as1</i> cells mock treated or treated with 1-NMPP1 1 h after synchronization of cells with either nocodazole or alpha factor and prior to induction of expression of each bait protein. The recently reported CDK-dependent Rad9/Dpb11 interaction was used as a positive control. (C) The Rad9/Chk1 interaction measured using co-immunoprecipitation (co-IP) occurs both in the absence and presence of DNA damage. Chk1 (anti-FLAG) and Dpb11 (anti-MYC) immunoprecipitations (IPs) were performed as indicated on extracts prepared from nocodazole-arrested cells, expressing both Chk1-3FLAG and Dpb11-13MYC, and either mock treated or treated with 20 µg/ml of bleocin for 45 min. Mock (IgG) or Dpb11 (MYC) IPs were performed as controls. Rad9, Chk1-3FLAG and Dpb11-13MYC specific bands were detected in western blots. Lower exposures, to facilitate their visualisation, of the western blots of the starting extracts are shown to the left. (D) Chk1 interaction with Rad9 is dependent on the CDK1-9 sites. As in panel C, except <i>rad9^CDK1-9A</i> cells were used. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s005" target="_blank">Figure S5</a> for related experiments.</p

PBMC from a healthy HLA-B*57:01 positive donor (Donor 1) where primed with abacavir at day 0, cultured for 14 days and then restimulated 1:10 with (A) HLA-B*57:01 single antigen line (C1R.B57), (B) with O/N abacavir treated C1R.B57 (C1R.B57.ABC) or (C) with O/N acyclovir treated C1R.B57 (C1R.B57.ACY).

<p>Antigen activated cells were detected by ICS for IFN-γ production and CD8+/ IFN-γ T-cells quantitated using flow cytometry. (D) PBMC from two healthy HLA-B*57:01 positive donors were either primed with abacavir (ABC primed), primed with acyclovir (ACY primed) or had no treatment (Control. PBMC were cultured for 14 days and then stimulated 1:10 with treated and untreated single antigen line, C1R.B57, as indicated.</p