Chromatographic separation is often an important part of mass-spectrometry-based proteomic analysis. It reduces the complexity of the initial samples before they are introduced to mass-spectrometric ...detection and chromatographic characteristics (such as retention time) add analytical features to the analyte. The acquisition and analysis of chromatographic data are thus of great importance, and specialized software is used for the extraction of quantitative information in an efficient and optimized manner. However, occasionally, automatic peak picking and correct peak boundary setting is challenged by, for instance, aberration of peak shape, peak truncation, and peak tailing, and a manual review of a large number of peaks is frequently required. To support this part of the analysis, we present here a software tool, Peakfit, that fits acquired chromatographic data to the log-normal peak equation and reports the calculated peak parameters. The program is written in R and can easily be integrated into Skyline, a popular software packages that is frequently used for proteomic parallel reaction monitoring applications. The program is capable of processing large data sets (>10 000 peaks) and detecting sporadic outliers in peak boundary selection performed, for instance, in Skyline. In an example data set, available via ProteomeXchange with identifier PXD026875, we demonstrated the capability of the program to characterize chromatographic peaks and showed an example of its ability to objectively and reproducibly detect and solve problematic peak-picking situations.
Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while ...glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics.
The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared.
Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified.
Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.
Carotid atherosclerosis is a risk factor for ischemic stroke, one of the main causes of mortality and disability worldwide. The disease is characterized by plaques, heterogeneous deposits of lipids, ...and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis, but the nature of their involvement is not fully understood. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 different lipid signals, originating of >90 uniquely assigned species, in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-directed multivariate analysis. These data allowed us to extract the spatial lipid patterns associated with morphological plaque features in advanced plaques from a symptomatic population, revealing spatial lipid patterns in atherosclerosis and their relation to histological tissue type. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, whereas diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. These results demonstrate a clear colocalization between plaque features and specific lipid classes, as well as individual lipid species in high-risk atherosclerotic plaques.
Abstract
Background
While thyroxine (T4), 3,3’,5-triiodothyronine (T3), and 3,3’,5’-triiodothyronine (rT3) have routine methods available for evaluating patients with suspected thyroid disease, ...appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. The effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed, including associated reference intervals.
Methods
Clinical and Laboratory Standards Institute guidelines, European Medicines Agency guidelines, and the National Institute of Standards and Technology protocol were used for the method validation and reference intervals. Reference intervals were determined in 132 healthy males and 121 healthy females. Serum samples were deproteinized with acetonitrile, followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight 13C6-internal standards
Results
The analytical method validation was performed for all nine THMs. Reference intervals (2.5th to 97.5th percentile) were determined for L-thyronine (4.9–11.3 ng/dL), 3-monoiodothyronine (0.06 --0.41 ng/dL), 3,5-diiodothyronine (<0.13 ng/dL), 3,3’-diiodothyronine (0.25--0.77 ng/dL), T3 (66.4--129.9 ng/dL), rT3 (15.0--64.1 ng/dL), T4 (4.3--10.0 µg/dL), triac/3,3’,5-triiodothyroacetic acid (not detected), and tetrac/3,3’,5,5’-tetraiodothyroacetic acid (2.2--27.2 ng/dL).
Conclusions
A broad dynamic concentration range exists among the nine THMs. This method should help to develop a better understanding of the clinical relevance of other THMs, as well as an understanding of thyroid hormone metabolism in health and disease.
The recent accurate and precise determination of the electron affinity (EA) of the astatine atom At0 warrants a re‐investigation of the estimated thermodynamic properties of At0 and astatine ...containing molecules as this EA was found to be much lower (by 0.4 eV) than previous estimated values. In this contribution we estimate, from available data sources, the following thermodynamic and physicochemical properties of the alkali astatides (MAt, M = Li, Na, K, Rb, Cs): their solid and gaseous heats of formation, lattice and gas‐phase binding enthalpies, sublimation energies and melting temperatures. Gas‐phase charge‐transfer dissociation energies for the alkali astatides (the energy requirement for M+At− ➔ M0 + At0) have been obtained and are compared with those for the other alkali halides. Use of Born‐Haber cycles together with the new AE (At0) value allows the re‐evaluation of ΔHf (At0)g (=56 ± 5 kJ/mol); it is concluded that (At2)g is a weakly bonded species (bond strength <50 kJ/mol), significantly weaker bonded than previously estimated (116 kJ/mol) and much weaker bonded than I2 (148 kJ/mol), but in agreement with the finding from theory that spin‐orbit coupling considerably reduces the bond strength in At2. The hydration enthalpy (ΔHaq) of At− is estimated to be −230 ± 2 kJ/mol (using ΔHaqH+ = −1150.1 kJ/mol), in good agreement with molecular dynamics calculations. Arguments are presented that the largest alkali halide, CsAt, like the smallest, LiF, will be only sparingly soluble in water, following the generalization from hard/soft acid/base principles that “small likes small” and “large likes large.”
DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the ...NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not$DNA-PK_{cs}$. We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.
The discovery of the IDH1 R132H (IDH1 mut) mutation in low‐grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, ...we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α‐ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA‐cycle activity in IDH1 mut gliomas.
Abstract
Background
Minimal residual disease (MRD) status assessed on bone marrow aspirates is a major prognostic biomarker in multiple myeloma (MM). In this study we evaluated blood-based targeted ...mass spectrometry (MS-MRD) as a sensitive, minimally invasive alternative to measure MM disease activity.
Methods
Therapy response of 41 MM patients in the IFM-2009 clinical trial (NCT01191060) was assessed with MS-MRD on frozen sera and compared to routine state-of-the-art monoclonal protein (M-protein) diagnostics and next-generation sequencing (NGS-MRD) at 2 time points.
Results
In all 41 patients we were able to identify clonotypic M-protein-specific peptides and perform serum-based MS-MRD measurements. MS-MRD is significantly more sensitive to detect M-protein compared to either electrophoretic M-protein diagnostics or serum free light chain analysis. The concordance between NGS-MRD and MS-MRD status in 81 paired bone marrow/sera samples was 79%. The 50% progression-free survival (PFS) was identical (49 months) for patients who were either NGS-positive or MS-positive directly after maintenance treatment. The 50% PFS was 69 and 89 months for NGS-negative and MS-negative patients, respectively. The longest 50% PFS (96 months) was observed in patients who were MRD-negative for both methods. MS-MRD relapse during maintenance treatment was significantly correlated to poor PFS (P < 0.0001).
Conclusions
Our data indicate proof-of-principle that MS-MRD evaluation in blood is a feasible, patient friendly alternative to NGS-MRD assessed on bone marrow. Clinical validation of the prognostic value of MS-MRD and its complementary value in MRD-evaluation of patients with MM is warranted in an independent larger cohort.
Changes to extracellular matrix (ECM) structures are linked to tumor cell proliferation and metastasis. We previously reported that naturally occurring peptides of collagen type I are elevated in ...urine of patients with colorectal liver metastasis (CRLM). In the present study, we took an MS-based proteomic approach to identify specific collagen types that are up-regulated in CRLM tissues compared with healthy, adjacent liver tissues from the same patients. We found that 19 of 22 collagen-α chains are significantly up-regulated (p < 0.05) in CRLM tissues compared with the healthy tissues. At least four collagen-α chains were absent or had low expression in healthy colon and adjacent tissues, but were highly abundant in both colorectal cancer (CRC) and CRLM tissues. This expression pattern was also observed for six noncollagen colon-specific proteins, two of which (CDH17 and PPP1R1B/DARP-32) had not previously been linked to CRLM. Furthermore, we observed CRLM-associated up-regulation of 16 proteins (of 20 associated proteins identified) known to be required for collagen synthesis, indicating increased collagen production in CRLM. Immunohistochemistry validated that collagen type XII is significantly up-regulated in CRLM. The results of this study indicate that most collagen isoforms are up-regulated in CRLM compared with healthy tissues, most likely as a result of an increased collagen production in the metastatic cells. Our findings provide further insight into morphological changes in the ECM in CRLM and help explain the finding of tumor metastasis–associated proteins and peptides in urine, suggesting their utility as metastasis biomarkers.
Seasonal influenza vaccination takes into account primarily hemagglutinin (HA)-specific neutralizing antibody responses. However, the accumulation of substitutions in the antigenic regions of HA ...(i.e., antigenic drift) occasionally results in a mismatch between the vaccine and circulating strains. To prevent poor vaccine performance, we investigated whether an antigenically matched neuraminidase (NA) may compensate for reduced vaccine efficacy due to a mismatched HA. Ferrets were vaccinated twice with adjuvanted split inactivated influenza vaccines containing homologous HA and NA (vacH3N2), only homologous HA (vacH3N1), only homologous NA (vacH1N2), heterologous HA and NA (vacH1N1), or phosphate-buffered saline (vacPBS), followed by challenge with H3N2 virus (A/Netherlands/16190/1968). Ferrets vaccinated with homologous HA (vacH3N2 and vacH3N1) displayed minimum fever and weight loss compared to vacH1N1 and vacPBS ferrets, while ferrets vaccinated with NA-matched vacH1N2 displayed intermediate fever and weight loss. Vaccination with vacH1N2 further led to a reduction in virus shedding from the nose and undetectable virus titers in the lower respiratory tract, similarly to when the homologous vacH3N2 was used. Some protection was observed upon vacH1N1 vaccination, but this was not comparable to that observed for vacH1N2, again highlighting the important role of NA in vaccine-induced protection. These results illustrate that NA antibodies can prevent severe disease caused by influenza virus infection and that an antigenically matched NA in seasonal vaccines might prevent lower respiratory tract complications. This underlines the importance of considering NA during the yearly vaccine strain selection process, which may be particularly beneficial in seasons when the HA component of the vaccine is mismatched.
Despite the availability of vaccines, influenza virus infections continue to cause substantial morbidity and mortality in humans. Currently available influenza vaccines take primarily the hemagglutinin (HA) into account, but the highly variable nature of this protein as a result of antigenic drift has led to a recurrent decline in vaccine effectiveness. While the protective effect of neuraminidase (NA) antibodies has been highlighted by several studies, there are no requirements with regard to quantity or quality of NA in licensed vaccines, and NA immunity remains largely unexploited. Since antigenic changes in HA and NA are thought to occur asynchronously, NA immunity could compensate for reduced vaccine efficacy when drift in HA occurs. By matching and mismatching the HA and NA components of monovalent split inactivated vaccines, we demonstrated the potential of NA immunity to protect against disease, virus replication in the lower respiratory tract, and virus shedding in the ferret model.
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