Structure and fragmentation chemistry of the peptide radical cations of glycylphenylalanylglycine (GFG).

Herein, we explore the generation and characterization of the radical cations of glycylphenylalanylglycine, or [GFG]•+, formed via dissociative electron-transfer reaction from the tripeptide to copper(II) within a ternary complex. A comprehensive investigation employing isotopic labeling, infrared multiple-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations elucidated the details and energetics in formation of the peptide radical cations as well as their dissociation products. Unlike conventional aromatic-containing peptide radical cations that primarily form canonical π-radicals, our findings reveal that 75% of the population of the experimentally produced [GFG]•+ precursors are [GFα•G]+, where the radical resides on the middle α-carbon of the phenylalanyl residue. This unexpected isomeric ion has an enthalpy of 6.8 kcal/mol above the global minimum, which has an N-terminal captodative structure, [Gα•FG]+, comprising 25% of the population. The [b2-H]•+ product ions are also present in a ratio of 75/25 from [GFα•G]+/ [Gα•FG]+, the results of which are obtained from matches between the IRMPD action spectrum and predicted IR absorption spectra of the [b2-H]•+ candidate structures, as well as from IRMPD isomer population analyses.

Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity.

Obesity is a global issue that warrants the identification of more effective therapeutic targets and a better understanding of the pivotal molecular pathogenesis. Annexin A1 (ANXA1) is known to inhibit phospholipase A2, exhibiting anti-inflammatory activity. However, the specific effects of ANXA1 in obesity and the underlying mechanisms of action remain unclear. Our study reveals that ANXA1 levels are elevated in the adipose tissue of individuals with obesity. Whole-body or adipocyte-specific ANXA1 deletion aggravates obesity and metabolic disorders. ANXA1 levels are higher in stromal vascular fractions (SVFs) than in mature adipocytes. Further investigation into the role of ANXA1 in SVFs reveals that ANXA1 overexpression induces lower numbers of mature adipocytes, while ANXA1-knockout SVFs exhibit the opposite effect. This suggests that ANXA1 plays an important role in adipogenesis. Mechanistically, ANXA1 competes with MYC binding protein 2 (MYCBP2) for interaction with PDZ and LIM domain 7 (PDLIM7). This exposes the MYCBP2-binding site, allowing it to bind more readily to the SMAD family member 4 (SMAD4) and promoting its ubiquitination and degradation. SMAD4 degradation downregulates peroxisome proliferator-activated receptor gamma (PPARγ) transcription and reduces adipogenesis. Treatment with Ac2-26, an active peptide derived from ANXA1, inhibits both adipogenesis and obesity through the mechanism. In conclusion, the molecular mechanism of ANXA1 inhibiting adipogenesis was first uncovered in our study, which is a potential target for obesity prevention and treatment.

Phosphate Migration versus the Loss of Phosphoric Acid in Protonated Phosphopeptides: A Computational Study.

Residue-specific phosphorylation is a protein post-translational modification that regulates cellular functions. Experimental determination of the exact sites of protein phosphorylation provides an understanding of the signaling and processes at work for a given cellular state. Any experimental artifact that involves migration of the phosphate group during measurement is a concern, as the outcome can lead to erroneous conclusions that may confound studies on cellular signal transduction. Herein, we examine computationally the mechanism by which a phosphate group migrates from one serine residue to another serine in monoprotonated pentapeptides [BA-pSer-Gly-Ser-BB + H]+ → [BA-Ser-Gly-pSer-BB + H]+ (where BA and BB are different combinations of the three basic amino acids, histidine, lysine, and arginine). In addition to moving the phosphate group, the overall mechanism involves transferring a proton from the N-terminal amino acid, BA, to the C-terminal amino acid, BB. This is not a synchronous process, and there is a key high-energy intermediate, structure C, that is zwitterionic with both the basic amino acids protonated and the phosphate group attached to both serine residues and carrying a negative charge. The barriers to moving the phosphate group are calculated to be in the range of 219-274 kJ mol-1 at the B3LYP/6-31G(d) level. These barriers are systematically slightly lower and in good agreement with single-point energy calculations at both M06-2X/6-311++G(d,p) and MP2/6-31++G(d,p) levels. The competitive reaction, loss of phosphoric acid from the protonated pentapeptides, has a barrier in the range of 176-202 kJ mol-1 at the B3LYP/6-31G(d) level. Extension of the theory to M06-2X/6-311++G(d,p)//B3LYP/6-31G(d) and MP2/6-31++G(d,p)// B3LYP/6-31G(d) gives higher values for the loss of phosphoric acid, falling in the range of 196-226 kJ mol-1; these are comparable to the barriers against phosphate migration at the same levels of theory. For larger peptides His-pSer-(Gly)n-Ser-His, where n has values from 2 to 5, the barriers against the loss of phosphoric acid are higher than those against the phosphate group migration. This difference is most pronounced and significant when n = 4 and 5 (the differences are approximately 80 kJ mol-1 under the single-point energy calculations at the M06-2X and MP2 levels). Energy differences using two more recent functionals, M08-HX and MN15, on His-pSer-(Gly)n-Ser-His, where n = 1 and 5, are in good agreement with the M06-2X and MP2 calculations. These results provide the mechanistic rationale for phosphate migration versus other competing reactions in the gas phase under tandem mass spectrometry conditions.

Averting an Outbreak of SARS-CoV-2 in a University Residence Hall through Wastewater Surveillance.

A wastewater surveillance program targeting a university residence hall was implemented during the spring semester 2021 as a proactive measure to avoid an outbreak of COVID-19 on campus. Over a period of 7 weeks from early February through late March 2021, wastewater originating from the residence hall was collected as grab samples 3 times per week. During this time, there was no detection of SARS-CoV-2 by reverse transcriptase quantitative PCR (RT-qPCR) in the residence hall wastewater stream. Aiming to obtain a sample more representative of the residence hall community, a decision was made to use passive samplers beginning in late March onwards. Adopting a Moore swab approach, SARS-CoV-2 was detected in wastewater samples just 2 days after passive samplers were deployed. These samples also tested positive for the B.1.1.7 (Alpha) variant of concern (VOC) using RT-qPCR. The positive result triggered a public health case-finding response, including a mobile testing unit deployed to the residence hall the following day, with testing of nearly 200 students and staff, which identified two laboratory-confirmed cases of Alpha variant COVID-19. These individuals were relocated to a separate quarantine facility, averting an outbreak on campus. Aggregating wastewater and clinical data, the campus wastewater surveillance program has yielded the first estimates of fecal shedding rates of the Alpha VOC of SARS-CoV-2 in individuals from a nonclinical setting. IMPORTANCE Among early adopters of wastewater monitoring for SARS-CoV-2 have been colleges and universities throughout North America, many of whom are using this approach to monitor congregate living facilities for early evidence of COVID-19 infection as an integral component of campus screening programs. Yet, while there have been numerous examples where wastewater monitoring on a university campus has detected evidence for infection among community members, there are few examples where this monitoring triggered a public health response that may have averted an actual outbreak. This report details a wastewater-testing program targeting a residence hall on a university campus during spring 2021, when there was mounting concern globally over the emergence of SARS-CoV-2 variants of concern, reported to be more transmissible than the wild-type Wuhan strain. In this communication, we present a clear example of how wastewater monitoring resulted in actionable responses by university administration and public health, which averted an outbreak of COVID-19 on a university campus.

Generation, Characterization, and Dissociation of Radical Cations Derived from Prolyl-glycyl-glycine.

Radical cations of an aliphatic tripeptide prolyl-glycyl-glycine (PGG•+) and its sequence ions [a3 + H]•+ and [b2 - H]•+ have been generated by collision-induced dissociation of the [Cu(Phen)(PGG)]•2+ complex, where Phen = 1,10-phenanthroline. Infrared multiple photon dissociation spectroscopy, ion-molecule reaction experiments, and theoretical calculations have been used to investigate the structures of these ions. The unpaired electron in these three radical cations is located at different α-carbons. The PGG•+ radical cation has a captodative structure with the radical at the α-carbon of the proline residue and the proton on the oxygen of the first amide group. This structure is at the global minimum on the potential energy surface (PES). By contrast, the [a3 + H]•+ and [b2 - H]•+ ions are not the lowest-energy structures on their respective PESs, and their radicals are formally located at the C-terminal and second α-carbons, respectively. Density functional theory calculations on the structures of the ternary copper(II) complex ion suggest that the charge-solvated isomer of the metal complex is the precursor ion that dissociates to give the PGG•+ radical cation. The isomer of the complex in which PGG is bound as a zwitterion dissociates to give the [a3 + H]•+ and [b2 - H]•+ ions.

Thermo-mechanical processing of brass components for potable-water usage increases risks of Pb leaching.

The problem of lead contamination in potable water has been a serious concern in different countries. Although the use of leaded welding solder has been banned and brass components used in potable water pipework have to be of the nominally "lead-free" grade in most jurisdictions, incidents of excessive lead leaching are still reported. The widely advocated explanation of lead leaching from brass components in terms of corrosion and the formation of electrochemical cells is inadequate since mechanical cutting is also known to cause lead segregation on brass surfaces. In this study, the effects of lead segregation on brass surfaces and subsequent leaching to contacting water resulting from thermo-mechanical processing of the brass are studied. The results indicate that mechanical milling and polishing that replicate the common processing involved in pipeline installation yield a significant increase in surface lead, and a strong correlation exists between lead leaching and the plastic deformation of the brass surface. Furthermore, flame-torch treatment that replicates the common brazing of brass also results in a significant increase in surface lead. These results indicate that the common thermo-mechanical processing of brass piping components poses a real risk of lead contamination in potable water, and revision in the common protocols for handling lead components may be necessary.

rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond.

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.

Dissociative electron transfer of copper(ii) complexes of glycyl(glycyl/alanyl)tryptophan in vacuo: IRMPD action spectroscopy provides evidence of transition from zwitterionic to non-zwitterionic peptide structures.

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]˙2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]˙2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]˙2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.

Doubly Charged Small Organic Fragments Derived from [Ce(tripeptide)(CH3CN)m]3+ Complexes: Observation of the Elusive [bn + H]2+ Ions.

[a3 + H]2+ ions generated from Ln3+/tripeptide complexes, where Ln = La or Ce, have similar structures to the linear [an]+ ions but with protonation at both the terminal NH2 and N═CH2 groups. Ion stability is favored by having the basic secondary amine of the proline residue at the N-terminus and by an amino acid residue accommodating one of the protons on the side chain. Dissociation of [a3 + H]2+ ions derived from peptides containing only aliphatic residues is by cleavage of the second amide bond to give [b2]+ or [a2]+ ions along with internal [a1]+ ions. For [a3 + H]2+ ions containing a tryptophan residue in the central location, in addition to cleavage of the amide bond, losses of neutrals NH3, HN═CHR, (NH3 + CO), and HNCO were observed. Dissociations of some unsolvated Ln3+/tripeptide complexes gave [b3 + H]2+ ions in low abundance; formation of these [b3 + H]2+ ions was favored by the presence of a proline residue at the N-terminus and by either a histidine or tryptophan residue in the central position. Dissociation of these [b3 + H]2+ ions was by the loss of (H2O + CO) and not only CO, indicating that these ions did not have the same type of oxazolone structure as found for [bn]+ ions. Density functional theory calculations suggest that the observed [b3 + H]2+ ions of ProGlyGly were formed from [Ce(ProGlyGly)]3+ complexes in which the peptide was bound to the metal ion as an enolate. Dissociation of the slightly lower-energy complex, where the peptide is bound in the keto form, would produce an oxazolone but the high barrier required to create this isomer of the [b3 + H]2+ ion would be sufficient to result in further dissociation. Two isomers of the [b3 + H]2+ ion of ProHisGly have been created, one from the [Ce(ProHisGly)]3+ complex that characteristically dissociates by the combined loss of (H2O + CO) and the other by the loss of glycine from [ProHisGlyGly + 2H]2+. The [b3 + H]2+ ion derived from [ProHisGlyGly + 2H]2+ dissociated by the loss of only CO.

Structures and Dissociation Products of Ce/Peptide Complexes: Competition between Coordination and Charge Delocalization.

Structures of [Ce(GGG)]3+ and [Ce(GGG ? H)]2+ have been investigated by DFT calculations. The two lowest-energy structures of the triply charged metal complex have the peptide in either the iminol or conventional zwitterionic form, and these ions have almost identical energies. In the doubly charged complex, the iminol and charge-solvated structures are the best structures on the potential energy surface, but the latter is favored. In both iminol structures, the metal ion coordinates to the iminol oxygen rather than to the nitrogen, unlike in previously reported iminol-containing complexes. Triply charged [Ce(peptide)]3+ complexes are fragile and not easily isolated in a mass spectrometer, whereas the doubly charged [Ce(peptide ? H)]2+ complexes are more robust. Here, we studied the fragmentations of 37 [Ce(peptide ? H)]2+ and 30 [Ce(peptide)(peptide ? H)]2+ complexes and the results are systematically summarized. Losses of CO and/or H2O are the most commonly observed fragmentation channels for [Ce(peptide ? H)]2+ complexes and these dissociation pathways are modeled by DFT calculations. For [Ce(peptide)(peptide ? H)]2+ complexes the neutral peptide plays the role of a solvent molecule but, unlike in the dissociations of [Ce(CH3CN)(peptide ? H)]2+ complexes, the loss of the solvent molecule is not observed. Instead, fragmentation occurs by cleavage of the second amide bond of the solvating peptide molecule.

The plasma peptides of breast versus ovarian cancer.

BACKGROUND: There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC-ESI-MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. CONCLUSION: There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC-ESI-MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti-cancer, -viral, and -parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti-inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2-hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein-protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero- and homo-oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.-Grañe-Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor-Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

The plasma peptidome.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC-ESI-MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. METHODS: A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC-ESI-MS/MS experiments analyzed in SQL Server R. RESULTS: Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 µL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey-Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes. CONCLUSIONS: The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC-ESI-MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

Loss of water from protonated polyglycines: interconversion and dissociation of the product imidazolone ions.

Collision-induced dissociation of isotopically labelled protonated pentaglycine produced two abundant [b5]+ ions, the products of the loss of water from the first and second amide groups, labelled [b5]+I and [b5]+II. IRMPD spectroscopy and DFT calculations show that these two [b5]+ ions feature N1-protonated 3,5-dihydro-4H-imidazol-4-one structures. 15N-Labelling established that some interconversion occurs between these two ions but dissociations are preferred. For both ions, DFT calculations show that the barrier to interconversion is slightly higher than those to dissociation. Dehydration of protonated hexaglycine produced three imidazolone ions. Ions [b6]+I and [b6]+II exhibit analogous CID spectra to those from [b5]+I and [b5]+II; however, the spectrum of the [b6]+III ion was dramatically different, showing losses predominantly of a further water molecule or cleavage of the second amide bond to give the glycyloxazolone (a deprotonated [b2]+ ion, labelled GlyGlyox (114 Da)) from the N-terminus. Protonated polyglycines [Glyn + H]+, where n = 7-9, all readily lose at least one water molecule. The corresponding [bn]+ ions lose either a further water molecule, an oxazolone from the N-terminus or a truncated peptide from the C-terminus. The number of amino acid residues in the latter two eliminated neutral molecules provides insight into the location of the imidazolone in the peptide chain and which oxygen was lost in the initial dehydration reaction. From this analysis, it appears that water loss from the longer protonated polyglycines is predominantly from the central residues.

The plasma peptides of ovarian cancer.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation. METHODS: The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 µl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC-ESI-MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey-Kramer HSD test. CONCLUSION: Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC-ESI-MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research.

Interconversion between 4-Imidazolone Ions; Isomers of [b4]+ Derived from Protonated Tetraglycine.

Collision-induced dissociations of isotopically labeled protonated tetraglycines establish that the [b4]+ ion formed by loss of water from the second amide bond (structure II) rearranges to form N1-protonated 3,5-dihydro-4H-imidazol-4-one (structure I), the product of water loss from the first amide bond. Structure II is slightly higher in energy than I (ΔH at 0 K is 5.1 kJ mol-1, as calculated at M06-2X/6-311++G-(d,p)), and the barrier to interconversion is 139.8 kJ mol-1 above I. The dominant dissociation pathway is the loss of methanimine (HN=CH2) from ion I with a barrier of 167.1 kJ mol-1, giving [GlyGlyGlyGly + H - H2O - HN=CH2]+, ion III; a minor channel, loss of NH3, has a slightly higher barrier (181.5 kJ mol-1). Using labeled glycine (13Cα) it was determined that loss of the imine is from the same residue as that from which water was initially lost. The collision-induced dissociation spectra of ion III derived from both I and II were identical, and their energy-resolved curves were also very similar. Ion III fragments by losses of a glycine molecule (the dominant channel), a water molecule, and a glycine residue (57 Da), giving ions IV, V, and VII, respectively. Isotopic labeling established the origins of each of the neutral molecules that are lost. Using glycine (2,2 D2), rapid deuterium exchange was observed for both ions I and II for the α-hydrogens that are from the same residue as that from which the water had been eliminated.

Isomerization versus dissociation of phenylalanylglycyltryptophan radical cations.

Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ˙]+, α-carbon- [FGα˙W]+, N-centered [FGWN˙]+ and ζ-carbon- [Fζ˙GW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal-ligand-peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen-nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα˙W]+, and ζ-phenyl radical [Fζ˙GW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ˙]+ and [FGWN˙]+ is only 31.1 kcal mol-1, much lower than the barriers to their dissociation (40.3 kcal mol-1). For the [FGWπ˙]+, [FGα˙W]+, and [Fζ˙GW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 - H]˙+ ions isolated from [FGα˙W]+ and [Fζ˙GW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.

Characterization of the Molecular Mechanisms Underlying the Chronic Phase of Stroke in a Cynomolgus Monkey Model of Induced Cerebral Ischemia.

Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.

Radical-induced dissociation leading to the loss of CO2 from the oxazolone ring of [b5- H]˙(+) ions.

Macrocyclization is commonly observed in large bn(+) (n≥ 4) ions and as a consequence can lead to incorrect protein identification due to sequence scrambling. In this work, the analogous [b5- H]˙(+) radical cations derived from aliphatic hexapeptides (GA5˙(+)) also showed evidence of macrocyclization under CID conditions. However, the major fragmentation for [b5- H]˙(+) ions is the loss of CO2 and not CO loss, which is commonly observed in closed-shell bn(+) ions. Isotopic labeling using CD3 and (18)O revealed that more than one common structure underwent dissociations. Theoretical studies found that the loss of CO2 is radical-driven and is facilitated by the radical being located at the Cα atom immediately adjacent to the oxazolone ring. Comparable energy barriers against macrocyclization, hydrogen-atom transfer, and fragmentations are found by DFT calculations and the results are consistent with the experimental observations that a variety of dissociation products are observed in the CID spectra.