Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

Conventional and microwave‐assisted SPPS approach: a comparative synthesis of PTHrP(1–34)NH2

Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we compared Fmoc/tBu MW‐assisted SPPS of 1–34 N‐terminal fragment of parathyroid hormone‐related peptide (PTHrP) with its conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the stepwise elongation of the resin‐bound peptide, monitoring was conducted by performing MW‐assisted mini‐cleavages and analyzing them by UPLC‐ESI‐MS. Identification of some deletion sequences was helpful to recognize critical couplings and as such helped to guide the introduction of MW irradiations to these stages. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

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.     

Identification and comparative analysis of the structural proteomes of ϕKZ and EL,two giant Pseudomonas aeruginosa bacteriophages

Giant bacteriophages ?KZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI‐MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless ?KZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The ?KZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201?2‐1. Phage EL is more distantly related, shown by its 26 non‐conserved structural proteins and the presence of genomic inversions.     

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.     

An acylated flavonol glycoside and hydrolysable tannins from Callistemon lanceolatus flowers and leaves

Introduction –  Callistemon lanceolatus DC. (Myrtaceae) is a plant rich in polyphenols, and is used as anticough, antibronchitis and insecticide in folk medicine. Because of the biological importance of plant polyphenols, particularly tannins, a phytochemical study was of interest to investigate the constitutive poyphenols in the extracts of flowers and leaves. Objective –  To avoid time‐consuming methodology for isolation of a complex mixture of known metabolites, HPLC‐ESI/MS was employed for fast picking up of the new compounds followed by identification of the structures with UV and one‐ and two‐dimensional NMR. Methodology –  Flowers and leaves were separately extracted with hot aqueous methanol under reflux (70°C). Pre‐isolation of the total extracts was achieved through column chromatographic fractionation on polyamide with water–methanol for gradient elution. The main fractions were purified using repeated column chromatography on cellulose and/or Sephadex LH‐20 with suitable eluents. HPLC‐ESI/MS analyses were carried out in the single ion monitoring (SIM) and negative ion modes. The pure compounds in methanol–water (1:1) were analysed by direct infusion ESI/MS. Final structure elucidation was obtained by one‐ and two‐dimensional NMR. Results –  Two new metabolites namely quercetin 3‐O‐β‐D‐glucuronopyranoside n‐butyl ester ( 1 ) and n‐butylgallate 4‐O‐(2′,6′‐di‐O‐galloyl)‐β‐d ‐glucopyranoside ( 4 ) along with nine known ones were identified from the aqueous methanol extracts of flowers and leaves. Conclusion –  The study has shown that Callistemon lanceolatus is rich in polyphenols. HPLC‐ESI/MS may be used, in negative ion mode, as an efficient and rapid analytical tool for investigating complex plant extracts. Copyright © 2008 John Wiley & Sons, Ltd.     

SDS‐PAGE‐free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS‐PAGE‐free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1‐butyl‐3‐methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D‐SCX‐RPLC‐ESI‐MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.     

A simple and efficient frit preparation method for one‐end tapered‐fused silica‐packed capillary columns in nano‐LC‐ESI MS

A novel frit preparation method for one‐end tapered‐fused silica‐packed capillary columns in nano‐LC‐ESI MS was developed. A hollow‐fused silica capillary column with a tapered tip as nano‐spray emitter was filled with 5 μm C₁₈ beads, and then a sintered frit about 0.25 mm in length was prepared at the tip by butane flame. A stainless steel protection tube with 0.5 mm id was used to control the length of the frit and to protect the packed C₁₈ beads behind the sintered frit during the sintering. C₁₈ sintered frits were evaluated by BSA tryptic digests with nano‐LC‐LTQ. The sintered frits did not produce post‐column band broadening due to very small volume (about 0.2 nL) and did not produce adsorption to sample. The sintered frit columns had good separation reproducibility and separation performance compared with self‐assembled particles frit columns and commercial columns.     

EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.     

New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.     

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.     

The 4‐pyridylmethyl ester as a protecting group for glutamic and aspartic acids: ‘flipping’ peptide charge states for characterization by positive ion mode ESI‐MS

Use of the 4‐pyridylmethyl ester group for side‐chain protection of glutamic acid residues in solid‐phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI‐MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4‐pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI‐MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Age‐related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old‐age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C‐terminus with significantly higher frequency compared to other residues, suggesting that trypsin‐like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA‐crystallin peptide (αA_57‐65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA_57‐65 formation escalated significantly. Using 2D gel electrophoresis/nanoLC‐ESI‐MS/MS, 12 protein complexes of 40–150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross‐linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes. Proteins 2015; 83:1878–1886. © 2015 Wiley Periodicals, Inc.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

Synthesis and mass spectrometry analysis of quaternary cryptando‐peptidic conjugates

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando‐peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando‐peptidic conjugates at the peptide N‐terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision‐induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α‐carbon in the quantitative LC‐MS analysis was analyzed. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC,HPLC, UPLC‐ESI‐QTOF‐MS and LC‐SPE‐NMR Fingerprints Analyses

Introduction

The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown.

Objective

The present study aims finding suitable conditions to identify the bioactive compounds from different fractions.

Methodology

Chromatographic fingerprint profiles of different fractions were developed using high‐performance liquid chromatography (HPLC) and then these conditions were transferred to thin‐layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra‐performance liquid chromatography‐quadrupole time of flight‐tandem mass spectrometry (UPLC‐QTOF‐MS) and liquid chromatography‐solid phase extraction‐nuclear magnetic resonance (LC‐SPE‐NMR) spectroscopy.

Results

The HPLC fingerprints, developed on two coupled Chromolith RP‐18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin‐3‐O‐rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di‐pentoside) were tentatively identified by QTOF‐MS, while NMR confirmed the structure of rutin and nicotiflorin.

Conclusion

The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd.     

A comparison of nLC-ESI-MS/MS and nLC-MALDI-MS/MS for GeLC-based protein identification and iTRAQ-based shotgun quantitative proteomics.

The use of nLC-ESI-MS/MS in shotgun proteomics experiments and GeLC-MS/MS analysis is well accepted and routinely available in most proteomics laboratories. However, the same cannot be said for nLC-MALDI MS/MS, which has yet to experience such widespread acceptance, despite the fact that the MALDI technology offers several critical advantages over ESI. As an illustration, in an analysis of moderately complex sample of E. coli proteins, the use MALDI in addition to ESI in GeLC-MS/MS resulted in a 16% average increase in protein identifications, while with more complex samples the number of additional protein identifications increased by an average of 45%. The size of the unique peptides identified by MALDI was, on average, 25% larger than the unique peptides identified by ESI, and they were found to be slightly more hydrophilic. The insensitivity of MALDI to the presence of ionization suppression agents was shown to be a significant advantage, suggesting it be used as a complement to ESI when ion suppression is a possibility. Furthermore, the higher resolution of the TOF/TOF instrument improved the sensitivity, accuracy, and precision of the data over that obtained using only ESI-based iTRAQ experiments using a linear ion trap. Nevertheless, accurate data can be generated with either instrument. These results demonstrate that coupling nanoLC with both ESI and MALDI ionization interfaces improves proteome coverage, reduces the deleterious effects of ionization suppression agents, and improves quantitation, particularly in complex samples.