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.

Phantom assessment of three-dimensional geometric distortion of a dedicated wide-bore MR-simulator for radiotherapy.

This study evaluated the machine-dependent three-dimensional geometric distortion images acquired from a 1.5T 700 mm-wide bore MR-simulator based on a large geometric accuracy phantom. With the consideration of radiation therapy (RT) application requirements, every sequence was examined in various combinations of acquisition-orientations and receiver-bandwidths with console-integrated distortion correction enabled. Distortion was repeatedly measured over a six-month period. The distortion measured from the images acquired at the beginning of this period was employed to retrospectively correct the distortion in the subsequent acquisitions. Geometric distortion was analyzed within the largest field-of-view allowed. Six sequences were examined for comprehensive distortion analysis-VIBE, SPACE, TSE, FLASH, BLADE and PETRA. Based on optimal acquisition parameters, their diameter-sphere-volumes (DSVs) of CT-comparable geometric fidelity (where 1 mm distortion was allowed) were 333.6 mm, 315.1 mm, 316.0 mm, 318.9 mm, 306.2 mm and 314.5 mm respectively. This was a significant increase from 254.0 mm, 245.5 mm, 228.9 mm, 256.6 mm, 230.8 mm and 254.2 mm DSVs respectively, when images were acquired using un-optimized parameters. The longitudinal stability of geometric distortion and the efficacy of retrospective correction of console-corrected images, based on prior distortion measurements, were inspected using VIBE and SPACE. The retrospectively corrected images achieved over 500 mm DSVs with 1 mm distortion allowed. The median distortion was below 1 mm after retrospective correction, proving that obtaining prior distortion map for subsequent retrospective distortion correction is beneficial. The systematic evaluation of distortion using various combinations of sequence-type, acquisition-orientation and receiver-bandwidth in a six-month time span would be a valuable guideline for optimizing sequence for various RT applications.

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.

Validation of the Chinese Version of Dementia Quality of Life Measure - Proxy in Care Home Residents with Dementia.

OBJECTIVE: To evaluate psychometric properties of the Chinese version of Dementia Quality of Life Measure - Proxy (C-DEMQoL-Proxy). METHODS: Care home residents aged ≥60 years who were diagnosed with dementia or demonstrated impairment in cognition were recruited from four care facilities in Hong Kong. Caregivers of these participants were also invited to participate. The original DEMQoL-Proxy was translated into Chinese (Cantonese) by a trained translator. The forward-translated version was reviewed by an expert panel of six experienced healthcare professionals. Revisions were made based on comments. The instrument was back-translated to English to check whether further changes were necessary. Demographic data (age, sex, type and severity of dementia, and Mini-Mental State Examination [MMSE] score) were collected from medical records of participants with dementia. Caregivers were interviewed by an occupational therapist or personnel supervised by the occupational therapist using the C-DEMQoL-Proxy and the Chinese version of Quality of Life-Alzheimer's Disease-Proxy (C-QoL-AD-Proxy). Acceptability, reliability, and validity of the C-DEMQoL-Proxy were evaluated using standard psychometric methods. RESULTS: 90 individuals (82.2% women) with dementia aged 72 to 102 years were included. Their diagnosis included Alzheimer's disease (23.3%), vascular dementia (15.6%), mixed and other types of dementias (51.1%), and missing (10%). Severity was mild in 12.2%, moderate in 62.2%, and severe in 25.6%. The mean MMSE score was 12.0 ± 4.9. 20% of the caregivers were family members and the rest were professional carers. The C-DEMQoL-Proxy had good acceptability, with no floor or ceiling effects or missing data. It had good internal consistency (Cronbach alpha = 0.91) and test-retest reliability (intraclass correlation coefficients = 0.83). It was mildly correlated with C-QoL-AD-Proxy (r = 0.29, p < 0.01). Age and sex were not correlated with C-DEMQoL-Proxy scores. C-DEMQoL-Proxy scores were not significantly different between dementia types, severity levels, or between those with higher or lower MMSE scores. CONCLUSION: The C-DEMQoL-Proxy is a valid and reliable instrument to assess health-related quality of life in individuals with dementia.

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.

Orthodontic plate for management of obstructive sleep apnoea in infants with Pierre Robin sequence: experience and protocol in Hong Kong.

OBJECTIVE: To present the application of the pre-epiglottic baton plate (PEBP) in infants with Pierre Robin sequence (PRS) in the Southern Chinese population (Hong Kong) and to present the diagnosis and management protocol of these infants in our centre. DESIGN: Retrospective case series of three patients with PRS. SETTING: Neonatal Intensive Care Unit in Kwong Wah Hospital and Craniofacial Orthodontic Centre in United Christian Hospital, Hong Kong. PARTICIPANTS: Three new-born infants (two girls, one boy) with PRS and upper airway obstruction due to glossoptosis. METHODS: A protocol for the diagnosis and management of these infants in the Southern Chinese population (Hong Kong) was presented. The three patients received nasal high-flow oxygen and/or continuous positive airway pressure (CPAP) as first-line respiratory support, followed by PEBP for 3-5 months. A two-stage approach was undertaken to ensure accurate positioning of the PEBP. RESULTS: All three infants had improvement in clinical signs, symptoms and polysomnography upon discharge. PEBP and other respiratory aids were weaned off at 3-6 months. CONCLUSIONS: The PEBP, combined with other respiratory support, is a useful modality in the treatment of obstructive sleep apnoea in infants with PRS.

Abundant neuroprotective chaperone Lipocalin-type prostaglandin D synthase (L-PGDS) disassembles the Amyloid-ß fibrils.

Misfolding of Amyloid ß (Aß) peptides leads to the formation of extracellular amyloid plaques. Molecular chaperones can facilitate the refolding or degradation of such misfolded proteins. Here, for the first time, we report the unique ability of Lipocalin-type Prostaglandin D synthase (L-PGDS) protein to act as a disaggregase on the pre-formed fibrils of Aß(1-40), abbreviated as Aß40, and Aß(25-35) peptides, in addition to inhibiting the aggregation of Aß monomers. Furthermore, our proteomics results indicate that L-PGDS can facilitate extraction of several other proteins from the insoluble aggregates extracted from the brain of an Alzheimer's disease patient. In this study, we have established the mode of binding of L-PGDS with monomeric and fibrillar Aß using Nuclear Magnetic Resonance (NMR) Spectroscopy, Small Angle X-ray Scattering (SAXS), and Transmission Electron Microscopy (TEM). Our results confirm a direct interaction between L-PGDS and monomeric Aß40 and Aß(25-35), thereby inhibiting their spontaneous aggregation. The monomeric unstructured Aß40 binds to L-PGDS via its C-terminus, while the N-terminus remains free which is observed as a new domain in the L-PGDS-Aß40 complex model.

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.

Bacteriology and risk factors associated with periprosthetic joint infection after primary total knee arthroplasty: retrospective study of 2543 cases.

INTRODUCTION: Periprosthetic joint infection after total knee arthroplasty is a serious complication. This study aimed to identify risk factors and bacteriological features associated with periprosthetic joint infection after primary total knee arthroplasty performed at a teaching hospital. METHODS: We reviewed 2543 elective primary total knee arthroplasties performed at our institution from 1993 to 2013. Data were collected from the Hong Kong Hospital Authority's Clinical Data Analysis and Reporting System, the Infection Control Team, and the joint replacement division registry. The association between potential risk factors and periprosthetic joint infection was examined by univariable analysis and multivariable logistic regression. Univariable analyses were also performed to examine the association between potential risk factors and bacteriology and between potential risk factors, including bacteriology, and early-onset infection. RESULTS: The incidence of periprosthetic joint infection in our series was 1.34% (n=34). The incidence of early-onset infection was 0.39% (n=24). Of the periprosthetic joint infections, 29.4% were early-onset infections. In both univariable and multivariable analyses, only rheumatoid arthritis was a significant predictor of periprosthetic joint infection. Methicillin-sensitive Staphylococcus aureus was the most common causative organism. We did not identify any significant association between potential risk factors and bacteriology. Periprosthetic joint infection caused by skin flora was positively associated with early-onset infection but the association was not statistically significant. CONCLUSION: The incidence of periprosthetic joint infection after elective primary total knee arthroplasty performed at our institution from 1993 to 2013 was 1.34%. Rheumatoid arthritis was a significant risk factor for periprosthetic joint infection.

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.

Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.