Analysis of sesquiterpene lactones in Eupatorium lindleyanum by HPLC‐PDA‐ESI‐MS/MS

Introduction – The aerial part Eupatorium lindleyanum is commonly used as an antipyretic and detoxicant clinically in traditional Chinese medicine. Our previous research showed that germacrane sesquiterpene lactones were its main active constituents, so the development of rapid and accurate methods for the identification of the sesquiterpene lactones is of great significance. Objective – To develop an HPLC‐PDA‐ESI‐MS/MS method capable for simple and rapid analysis of germacrane sesquiterpene lactones in the aerial part E. lindleyanum. Methodology – High‐performance liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used to analyze germacrane sesquiterpene lactones of Eupatorium lindleyanum. The fragmentation behavior of germacrane sesquiterpene lactones in a Micromass Q/TOF Mass Spectrometer was discussed, and 9 germacrane sesquiterpene lactones were identified by comparison of their characteristic data of HPLC and MS analyses with those obtained from reference compounds. Results – The investigated germacrane sesquiterpene lactones were identified as eupalinolides C (1), 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐14‐hydroxy‐costunolide (2), eupalinolides A (3), eupalinolides B (4), eupalinolides E (5), 3β‐acetoxy‐8β‐(4′‐oxo‐tigloyloxy)‐14‐hydroxy‐heliangolide (6), 3β‐acetoxy‐8β‐(4′‐oxo‐ tigloyloxy)‐14‐hydroxy‐costunolide (7), hiyodorilactone B (8), and 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐ costunolide (9). Compounds 6, 7 and 9 were reported for the first time. Conclusion – HPLC‐PDA‐ESI‐MS/MS provides a new powerful approach to identify germacrane sesquiterpene lactones in E. lindleyanum rapidly and accurately. Copyright © 2009 John Wiley & Sons, Ltd.     

Integrated and convenient procedure for protein extraction from formalin‐fixed,paraffin‐embedded tissues for LC‐MS/MS analysis

Because fresh‐frozen tissue samples associated with long‐term clinical data and of rare diseases are often unobtainable at the present time, formalin‐fixed paraffin‐embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross‐link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97–99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high‐yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic.     

Cyclodimerization of immunosuppressive fragment of HLA‐DR molecule. Design,synthesis and ESI‐MS/MS analysis

The nonapeptide fragment of the HLA‐DR molecule, located in the exposed loop of the alpha‐chain (164–172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three‐dimensional structure of the HLA‐DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C‐termini by spacer of (Gly₅)₂‐Lys‐NH₂ and the N‐termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG₅)₂K‐resin analog was synthesized using solid‐phase peptide synthesis protocols. The cyclization was achieved by cross‐linking the N‐terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega‐bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Enhanced detection of in‐gel released N‐glycans by MALDI‐TOF‐MS

Many biologically relevant glycoproteins need to be separated on 1D‐ or 2D‐gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in‐gel digestions with MALDI‐TOF‐MS. In this technical report, we investigated how the detection of in‐gel released N‐glycans could be improved by MALDI‐TOF‐MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D‐arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl‐β‐glucopyranoside, a nonionic detergent, was studied during in‐gel peptide‐N⁴‐(acetyl‐ß‐glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl‐β‐glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N‐glycans. A LOD of under 7 pmol glycoprotein could be achieved.     

On the use of ESI‐QqTOF‐MS/MS for the comparative sequencing of nucleic acids

The usability of a quadrupole—quadrupole—time‐of‐flight (QqTOF) instrument for the tandem mass spectrometric sequencing of oligodeoxynuleotides was investigated. The sample set consisted of 21 synthetic oligodeoxynucleotides ranging in length from 5 to 42 nucleotides. The sequences were randomly selected. For the majority of tested oligonucleotides, two or three different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 282 fragment ion mass spectra were acquired. Computer‐aided interpretation of fragment ion mass spectra was accomplished with a recently introduced comparative sequencing algorithm (COMPAS). The applied version of COMPAS was specifically optimized for the interpretation of information‐rich spectra obtained on the QqTOF. Sequences of oligodeoxynucleotides as large as 26‐mers were correctly verified in >94% of cases (182 of 192 spectra acquired). Fragment ion mass spectra of larger oligonucleotides were not specific enough for sequencing. Because of the occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences, tandem mass spectra obtained from oligonucleotides consisting of 30 and more nucleotides could not be used for sequence verification neither manually nor with COMPAS. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 401–409, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com