Tandem mass spectrometry for the structural determination of backbone-modified peptides

AbstractA variety of backbone-modified peptides were desorbed by fast atom bombardment and collisionally activated. These peptide modifications involve the replacement of a normal [CONH] peptide linkage with such groups as thiomethylene ether (CH2S), thioamide (CSNH), methyleneamine (CH2NH), and thiomethylene sulfoxide (CH2SO) moieties. Modified linear peptides decompose to give fragmentations characteristic of the modifications as well as typical peptide bond fragments. The presence of a replacement group in cyclic peptides can induce new fragmentations. The presence of other functional groups, such as exocyclic N-terminal residue, however, can dominate the observed fragmentations. Upon collisional activation, unmodified linear peptides fragment to give N-terminal ions as the most abundant daughter ions. In comparison, ψ[CH2NH] and ψ[CH2S] modified linear peptides decompose to give prominent C-terminal sequence ions. The ψ[CH2SO] modified linear peptides, however, fragment in both N- and C-terminal ions of high relative abudance. Depending on the modification, daughter ions or internal fragment ions are observed that are characteristic of the amide bond replacement. Useful structural information can therefore be obtained

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium ho-meostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Sur-prisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.Peer reviewe

Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics

Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry.

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83-103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP

Simplified Synthesis of Isotopically Labeled 5,5-Dimethyl-pyrroline N-Oxide

5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment

Expression and localization of the wild-type and mutant hTTP in transfected human cells.

<p>(A) Immunoblotting. HEK293 cells were transfected with pHis-hTTP plasmids. Proteins in the soluble extracts (10,000<i>g</i>, 10 µg/lane) were separated by SDS-PAGE (10% Tris-glycine gel) and transferred onto nitrocellulose membrane. The membrane was incubated in anti-MBP-hTTP serum (1∶10,000 dilution, 2 h) followed by secondary antibodies (1∶10,000 dilution, 1 h). The blot was incubated in Super Signal for 5 min and exposed to X-ray film for 5 sec. The underlined numbers in the plasmids 1–9 below the gel represent the sites of serine/threonine residues mutated to alanine residues in addition to the mutations of hTTP in the preceding plasmid. (B) Immunostaining. HEK293 cells were transfected with pBS+ control plasmid and pHis-hTTP plasmids encoding wild-type His-hTTP and mutant His-hTTP with S(214,218,228)A and S(88, 90, 93, 197, 214, 218, 228, 296)A mutations. The cells were stained with anti-MBP-hTTP antibodies (1∶5,000 dilution, overnight) and labeled with goat anti-rabbit Alexa Fluor 488 (1∶1,000 dilution, 1 h). Immunofluorescence was recorded by confocal microscopy.</p

MAIDI-MS analysis of HPLC fractions with major radioactivity.

1<p>The wild-type hTTP protein was purified from transfected human cells after <i>in vivo</i> radiolabeling with [³²P]-orthophosphate. The protein was purified and digested by trypsin to completion. The phosphopeptides were identified by radioactivity peak on HPLC chromatogram (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100977#pone-0100977-g006" target="_blank">Figure 6A</a>).</p>2<p>The observed peptide mass of [M+H] ion was obtained after phosphopeptides were sequenced by MAIDI-MS.</p>3<p>The unmodified peptide mass of [M+H] ion was obtained after theoretical digestion of His-hTTP with trypsin.</p>4<p>The differential mass was obtained by subtraction the unmodified ion mass from the observed ion pass. Phosphorylation results in a peptide ion with a +80 Da mass increase compared to the unmodified peptide for each phosphorylated Ser, Thr or Tyr residue (HPO₃⁻ = 79.97 Da).</p

Multiple distinct bands of the wild-type and mutant hTTP purified from transfected human cells.

<p>HEK293 cells were transfected with the selected plasmids. Proteins in the soluble extracts were bound to Ni-NTA beads and eluted with 100 mM imidazole solution. Proteins were separated by SDS-PAGE (10% Tris-glycine gel). (A) Coomassie brilliant blue staining (20 µL of protein). The gel was fixed with 10% acetic acid/25% isopropanol, stained with 0.006% Coomassie brilliant blue in 10% acetic acid overnight and destained with 10% acetic acid. (B) Silver staining (2 µL of protein). The identical proteins used in both panels are linked with a line.</p

Phosphopeptide mapping of mutant hTTP proteins from transfected human cells.

<p>The methods for generating the phosphopeptide mapping profiles were identical to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100977#pone-0100977-g006" target="_blank">Figure 6</a> legend. The phosphopeptide profiles in each pair of hTTP proteins are (A) S(197,214,218,228)A vs. S(197,214,218,228,296)A, (B) S(197,214,218,228,296)A vs. S(88,197,214,218,228,296)A, (C) S(88,197,214,218,228,296)A vs. S(88,186,197,214,218,228,296)A, and (D) S(88,197,214,218,228,296)A vs. S(88,197,214,218,228,296)T271A.</p

Trypsin digestion of the wild-type and mutant hTTP proteins from transfected human cells.

<p>HEK293 cells were transfected with the wild-type and 9 mutant plasmids followed by <i>in vivo</i> radiolabeling with [³²P]-orthophosphate. Proteins in the soluble extracts were bound to Ni-NTA beads and eluted with imidazole solution. Proteins were digested with trypsin. The undigested protein and digested peptides were separated by SDS-PAGE (8–16% Tris-glycine gel). The gel was dried and exposed to X-ray film. The underlined numbers in the plasmids 1–10 below the gel represent the sites of serine/threonine residues mutated to alanine residues in addition to the mutations of hTTP in the preceding plasmid.</p