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Tara Pukala 《Rapid communications in mass spectrometry : RCM》2019,33(Z3):72-82
The field of ion mobility mass spectrometry (IM‐MS) has developed rapidly in recent decades, with new fundamental advances underpinning innovative applications. This has been particularly noticeable in the field of biomacromolecular structure determination and structural biology, with pioneering studies revealing new structural insight for complex protein assemblies which control biological function. This perspective offers a review of recent developments in IM‐MS which have enabled expanding applications in protein structural biology, principally focusing on the quantitative measurement of collision cross sections and their interpretation to describe higher order protein structures. 相似文献
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Sánchez L Madurga S Pukala T Vilaseca M López-Iglesias C Robinson CV Giralt E Carulla N 《Journal of the American Chemical Society》2011,133(17):6505-6508
A critical aspect to understanding the molecular basis of Alzheimer's disease (AD) is the characterization of the kinetics of interconversion between the different species present during amyloid-β protein (Aβ) aggregation. By monitoring hydrogen/deuterium exchange in Aβ fibrils using electrospray ionization mass spectrometry, we demonstrate that the Aβ molecules comprising the fibril continuously dissociate and reassociate, resulting in molecular recycling within the fibril population. Investigations on Aβ40 and Aβ42 amyloid fibrils reveal that molecules making up Aβ40 fibrils recycle to a much greater extent than those of Aβ42. By examining factors that could influence molecular recycling and by running simulations, we show that the rate constant for dissociation of molecules from the fibril (k(off)) is much greater for Aβ40 than that for Aβ42. Importantly, the k(off) values obtained for Aβ40 and Aβ42 reveal that recycling occurs on biologically relevant time scales. These results have implications for understanding the role of Aβ fibrils in neurotoxicity and for designing therapeutic strategies against AD. 相似文献
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Pukala TL Urathamakul T Watt SJ Beck JL Jackway RJ Bowie JH 《Rapid communications in mass spectrometry : RCM》2008,22(22):3501-3509
Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure. 相似文献
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Calabrese AN Good NJ Wang T He J Bowie JH Pukala TL 《Journal of the American Society for Mass Spectrometry》2012,23(8):1364-1375
Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies. 相似文献
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Lisa J. Alcock Kyle D. Farrell Mawey T. Akol Gregory H. Jones Matthew M. Tierney Holger B. Kramer Tara L. Pukala Gonçalo J.L. Bernardes Michael V. Perkins Justin M. Chalker 《Tetrahedron》2018,74(12):1220-1228
Cysteine residues on proteins can react with cellular oxidants such as hydrogen peroxide. While this process is important for scavenging excess reactive oxygen species, the products of this oxidation may also mediate cell signalling. To understand the role of cysteine oxidation in biology, selective probes are required to detect and quantify its occurrence. Cysteine oxidation products such as sulfenic acids are sometimes unstable and therefore short-lived. If such cysteine derivatives are to be analysed, rapid reaction with the probe is required. Here we introduce norbornene derivatives as probes for cysteine oxidation, and demonstrate their ability to trap sulfenic acids. The synthesis of norbornene derivatives containing alkyne or biotin affinity tags are also reported to facilitate the use of these probes in chemical biology and proteomics. 相似文献
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Wang T Andreazza HJ Pukala TL Sherman PJ Calabrese AN Bowie JH 《Rapid communications in mass spectrometry : RCM》2011,25(9):1209-1221
Anuran peptides which contain His, including caerin 1.8 (GLFKVLGSVAKHLLPHVVPVIAEKL‐NH2), caerin 1.2 (GLLGVLGSVAKHVLPHVVPVIAEHL‐NH2), Ala15 maculatin 1.1 (GLFGVLAKVAAHVVAIEHF‐NH2), fallaxidin 4.1 (GLLSFLPKVIGHLIHPPS‐OH), riparin 5.1 (IVSYPDDAGEHAHKMG‐NH2) and signiferin 2.1 (IIGHLIKTALGMLGL‐NH2), all form MMet2+ and (M + Met2+‐2H+)2+ cluster ions (where Met is Cu, Mg and Zn) following electrospray ionisation (ESI) in a Waters QTOF 2 mass spectrometer. Peaks due to Cu(II) complexes are always the most abundant relative to other metal complexes. Information concerning metal2+ connectivity in a complex has been obtained (at least in part) using b and y fragmentation data from ESI collision‐induced dissociation tandem mass spectrometry (CID MS/MS). Theoretical calculations, using AMBER version 10, show that MCu2+ complexes with the membrane active caerin 1.8, Ala15 maculatin 1.1 and fallaxidin 4.1 are four‐coordinate and approximating square planar, with ligands including His and Lys, together with the carbonyl oxygens of particular backbone amide groups. When binding can occur through two His, or one His and one Lys, the His/Lys ligand structure is the more stable for the studied systems. The three‐dimensional (3D) structures of the complexes are always different from the previously determined structures of the uncomplexed model peptides (using 2D nuclear magnetic resonance (NMR) spectroscopy in membrane‐mimicking solvents like trifluoroethanol/water). Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
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Eric Yi‐Hsiu Huang Shou‐Chia Chu He Guei Chen Wayne Yen‐Hwa Chang Ying‐Ju Kuo Chin‐Chen Pan Allen W. Chiu Alex TL Lin Huihua Kenny Chiang 《Journal of Raman spectroscopy : JRS》2014,45(9):788-793
Renal cell carcinoma (RCC) accounts for 85% of all primary renal cancers. The definitive diagnosis of RCC relies exclusively on the subjective pathological interpretation of the surgical specimen. In this study, we aimed to analyze renal tissue using objective Raman spectroscopy (RS). We obtained 15 pairs of RCC (T) and corresponding normal renal parenchymal tissues (N) from our biobank. There are three subtypes of RCC: clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (cRCC). Five pairs of tissue of each subtype were enrolled. Fresh‐frozen sliced tissues were used for the RS detection. The Raman spectra between T and N were compared and analyzed using partial least squares (PLS) regression. Data for a total of 55 T and 58 N analyzable RS samples were obtained. The spectra were normalized by dividing the intensity of the characteristic peak at 1003 cm−1 using phenylalanine's Raman peak. After further analysis with PLS, the sensitivity and specificity for discriminating T from N were 95% and 93%, respectively. The RCC subtypes can be discriminated at an accuracy of 72% for ccRCC, 88% for cRCC, and 86% for pRCC. This study demonstrates the feasibility of analyzing renal tissue using RS. RS, with its advantages of easy and objective tissue assessment, may be applied to aid intraoperative decision making and pathological tissue assessment. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
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