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opencc-by-4.0Jan 2022View details →

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches.

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3′ to P6′ was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.

Fig. 7 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 7. Levels of major glucosinolates in leaves of first year rosette plants of the Gtype (A) and P-type (B) of Barbarea vulgaris in plants subjected to various challenges or no challenge as control. The contrasting general profile of the types is evident from dominance of BAR in the G-type and EBAR in the P-type. Treatment codes are: Control, un-challenged plants harvested after 7 days; Pieris 3d and Pieris 7d, herbivory by Pieris brassicae larvae until harvest at either day 3 or day 7; Plutella, herbivory by Plutella xylostella for 4 days; CuCl2, spraying of leaves with 10 mM CuCl2 (aq.) followed by recovery for 4 days. Bars represent means, whiskers indicate standard deviation (N = 3 for each group).

Fig. 6 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 6. Lack of the aliphatic glucosinolate (GSL) Pren (107) in B. vulgaris and spiking of pure (intact) Pren for establishing the limit of detection. A. Total ion chromatogram for the three dominating peaks in G-type B. vulgaris (dGSL preparation). B. Extracted ion trace for desulfo Pren in the same extract as A, showing lack of detection. C, D, E. Results of serial spiking of the crude extract with serial 10-fold dilutions of Pren before the desulfation procedure, showing linearity also at low levels and ability to detect trace levels.

Fig. 9 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 9. Extracted ion HPLC-MS chromatograms of desulfoglucosinolates prepared from glucosinolates (GSLs) in seeds (A–F) or leaves (G) of Reseda luteola and seeds of Reseda odorata (H). The three major peaks (A, B, C) represent PE, IM and BAR, much like many Barbarea spp. Focus on minor peaks (D) allowed conclusive identification of EBAR, confirmed by tR and the characteristic MS2 spectrum. A range of putative derivatives were not detected (E, F), but an unidentified hydroxybutylGSL was present (G), as was a known glycoside in R. odorata.

Fig. 5 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 5. MS2 spectra of desulfated glucosinolates (GSLs) confirming the identity of two 2-3homoMet-derived GSLs in C. hirsuta. While the spectrum of desulfo Buen contains only the usual fragments for this type of dGSL (a, [anhydroGlc+Na]+; b, [thioGlc+Na]+), the spectrum of desulfo Peen contains an additional usual fragment (c, [M-anhydroGlc Na]+) and two unusual + fragment ions suggesting a structure-specific cyclization and exchange of O during fragmentation: 201, [gluconolactone Na]+ and 152, [C H NS Na]+. + 6 11 + The unusual fragments can be rationalized as fragment a plus O and fragment c minus O, respectively.

Fig. 3 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 3. HPLC-MS chromatograms of desulfoglucosinolates (dGSLs) prepared from glucosinolates (GSLs) in Planodes virginica (A) and Nasturtium officinale (B–C) seeds, showing qualitative similarities and quantitative contrasts. Major peaks (B) from N. officinale revealed many of the same GSLs as in A, but levels of EBAR (40R) were much lower while levels were much higher for the biosynthetic precursor PE (105). A focus on trace peaks from N. officinale (C) revealed sharp peaks representing a range of minor constituents. Due to the closely eluting peaks, the latter chromatograms (B–C) were made by combining extracted ion chromatograms corresponding to [M+Na]+ of the indicated dGSLs. In C, the m/z 366 signal of d105 was omitted to allow visualization of minor coeluting peaks. An asterisk after a peak number indicates tentative identification. HPLC-MS conditions as in Olsen et al. (2016). TIC, total ion chromatogram, EIC, extracted ion chromatogram.

Fig. 4 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 4. HPLC-MS chromatogram of desulfoglucosinolates (dGSLs) prepared from glucosinolates (GSLs) from leaves (A) and petioles (B) of horseradish (Armoracia rusticana), focusing on trace level GSLs. The chromatograms were made by combining extracted ion chromatograms corresponding to [M+Na]+ of the indicated dGSLs, from analyses that were much overloaded with respect to the dominating dGSL d107 from Pren. In panel A, an insert shows magnification of the chromatogram from 5.2 to 5.8 min. Neither suggested BAR nor EBAR were detectable. In panel B, only extracted ion chromatograms of m/z 382 (BAR/EBAR), 352 (BZ), 366 (PE), 380 (3PP), 394 (4PB), 408 (5PP at high tR and 6mSOh at 5.4 min), 422 (7mSOh) and 436 (8mSOo) are included. Unlabeled trace peaks did not exhibit a combination of tR and m/z suitable for any of the mentioned candidates. HPLC conditions as in Olsen et al. (2016). Panel A depicts analysis of the Copenhagen garden accession; panel B from the naturalized population at Lake Fures¨o. An asterisk after a peak number indicates tentative identification.

Fig. 2 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 2. Detection of thioglucose-acylated glucosinolates (GSLs) by HPLC-MS of desulfated derivatives prepared from the indicated species. (A–C) Analysis of seeds of Arabidopsis thaliana Col-0 used as reference material for characteristic GSLs. Shown are the total ion chromatogram (A) and extracted ion chromatograms for sodium adducts of desulfo 6′Bz 4BzOb (d125) (B) and desulfo 6′Bz 4mSb (d127) (C). (D–F) Analysis of seeds of Barbarea grayi for dominating GSLs. Shown are total ion chromatograms (D), and extracted ion chromatograms for sodium adducts of desulfo 6'iF BAR (d131S) (E) and desulfo 6'iF PE (d129) (F).

Fig. 1 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 1. MS2 spectra of pairs of desulfoglucosinolates with and without a side chain double bond. Four short chain desulfoglucosinolates were investigated, including Na+ adducts of all (A–D) and in addition H+ adducts of the methylthio substituted (E–F), as indicated in each spectrum. The desulfo derivative of the putative 9mSn ([89]), poorly characterized in the literature, was also investigated (G).

Fig. 5. Proposed fragmentation scheme for 2-aminobenzoyl O in Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS/MS

Fig. 5. Proposed fragmentation scheme for 2-aminobenzoyl O-β-D-apiofuranosyl-(1 → 6)- β-D-glucopyranoside (m/z 432.15 when protonated) in positive ion mode ESI to m/z 300.11, 138.06, and 120.04 fragments.

Fig. 2 in Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS/MS

Fig. 2. Common esterification patterns between necic acids and necine bases observed in retronecine-type PAs.

opencc-zeroDec 2016View details →

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Data from: Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding, TLC and HPLC-MS

Many herbal products have a long history of use, but there are increasing concerns over product efficacy, safety and quality in the wake of recent cases exposing discrepancies between labeling and constituents. When it comes to St. John's wort (Hypericum perforatum L.) herbal products, there is limited oversight, frequent off-label use and insufficient monitoring of adverse drug reactions. In this study, we use amplicon metabarcoding (AMB) to authenticate 78 H. perforatum herbal products and evaluate its ability to detect substitution compared to standard methods using thin-layer chromatography (TLC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Hypericum perforatum was detected in 68% of the products using AMB. Furthermore, AMB detected incongruence between constituent species and those listed on the label in all products. Neither TLC nor HPLC-MS could be used to unambiguously identify H. perforatum. They are accurate methods for authenticating presence of the target compounds, but have limited efficiency in detecting infrageneric substitution and do not yield any information on other plant ingredients in the products. Random post-marketing AMB of herbal products by regulatory agencies could raise awareness among consumers of substitution and would provide an incentive to manufacturers to increase quality control from raw ingredients to commercialized products.

opencc-by-4.0Jun 2024View details →

HPLC-MS data collection results

HPLC-MS数据采集结果

Fig. 4 in Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS/MS

Fig. 4. Fragmentation patterns according to esterification pattern in retronecine-type PAs.

Fig. 1 in Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS/MS

Fig. 1. Varied necine base structures found in pyrrolizidine alkaloids.

Fig. 3 in Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS/MS

Fig. 3. Subtypes of lycopsamine-type PAs containing a retronecine core.