On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Structural analysis of naphthoquinone protein adducts with liquid chromatography/tandem mass spectrometry and the scoring algorithm for spectral analysis (SALSA)

The relative reactivities of various naphthoquinone isomers (1,4-, 1,2- and 2-methyl-1,4-naphthoquinone) to two test proteins, apomyoglobin and human hemoglobin, were evaluated via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The structural characterization of the resulting adducts was also obtained by LC/ESI-MS analysis of the intact proteins. The reactive sites of apomyoglobin and human hemoglobin with 1,4-naphthoquinone and 1,2-naphthoquinone were also identified through characterization of adducted tryptic peptides by use of high-pressure liquid chromatography/electrospray ionization with tandem mass spectrometry (HPLC/ESI-MS/MS), TurboSEQUEST, and the scoring algorithm for spectral analysis (SALSA). Four adducted peptides, which were formed by nucleophilic addition of a lysine amino acid residue to 1,4-naphthoquinone, were also identified, as was an adducted peptide from incubation of 1,2-naphthoquinone with apomyoglobin. In the case of incubation of human hemoglobin with the two naphthoquinones, two adducted peptides were identified from the N-terminal valine modification of the alpha and beta chains of human hemoglobin. The adducted protein formation may imply that naphthalene produces its in vivo toxicity through 1,2- and 1,4-naphthoquinone metabolites reacting with biomolecular proteins.     

LC/MS and LC/MS/MS determination of protein tryptic digests

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.     

Determination of theophylline in serum by high performance liquid chromatography/mass spectrometry with atmospheric pressure chemical-ionization (APCI HPLC/MS)

 A method for the determination of theophylline (TH), without derivatization, in serum by isotope dilution mass spectrometry using labelled [1, 3-¹⁵N₂-2-¹³C]theophylline (LTH) as internal standard is described. After deproteinization, the analyte is directly injected into a high performance liquid chromatography – mass spectrometer operating with atmospheric-pressure chemical-ionization (APCI HPLC/MS). The concentrations of TH in sera measured by APCI HPLC/MS are compared with results from gas chromatography – isotope dilution mass spectrometry (GC-ID/MS), high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the APCI HPLC/MS and GC-ID/MS methods are discussed. The coefficient of variation (CV) determined from duplicate samples was less than 2%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1. Received: 17 January 1996/Revised: 26 March 1996/Accepted: 5 April 1996     

Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

液相色谱-电喷雾电离质谱与电子轰击质谱联用筛选百合中的甾体皂甙

 利用高效液相色谱 电喷雾电离质谱联用仪 (HPLC/ESI MS)、电子轰击质谱 (EI MS)和半制备型高效液相色谱 ,从卷丹百合中筛选出了两种甾体皂甙 ,其中一种为含有 3个糖基与提果皂甙元的甾体皂甙 ,另一种为含有 3个糖基和薯蓣皂甙元的甾体皂甙。结果表明 :在线的HPLC/ESI MS能够准确快速地提供糖甙类化合物的分子质量和糖链部分的有益信息 ,但对甙元部分提供的信息极少 ;离线的EI MS只需极少量 (1mg～ 2mg)的纯品就能准确地提供甙元部分的有益信息 ,但很难获得糖甙的分子离子峰与糖链部分的信息 ,两者有机地结合起来能快速地从植物中筛选甾体皂甙。     

Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins.     

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3‐μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI). The captured material was processed using a single‐pot solid‐phase‐enhanced sample preparation (SP3) method and analyzed by LC‐MS/MS using ion mobility (IM) enhanced data independent acquisition (DIA) to identify and quantify proteins; more than 600 proteins were identified. Using a modified database that included isoform and the post‐translational modifications chain, loss of the initial methionine, and acetylation, 14 MALDI MSI peaks were identified. Comparison of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified proteins was achieved through an evolutionary relationships classification system.     

高效液相色谱-三重四级杆质谱分析海洋甲壳动物淋巴和肌肉中的20-羟基蜕皮酮

在三疣梭子蟹(Portunus trituberculatus)血淋巴中加入高油菜素内酯作为内标,用乙腈去除蛋白,采用高效液相色谱-三重四级杆质谱(HPLC-QqQ MS)对20-羟基蜕皮酮进行分析;对3种甲壳动物(-三疣梭子蟹、脊尾白虾(PaJaemon carincaua'a Holthuis)和口虾蛄(Orato...     

Evaluating the utility of ion mobility separation in combination with high-pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products

Many formulated products contain complex polymeric excipients such as polyethylene glycols (PEGs). Such excipients can be readily ionized by electrospray and may be present at very high concentrations, thus making it very difficult to identify trace level impurities such as degradants in samples, even if hyphenated techniques such as liquid chromatography/mass spectrometry (LC/MS) are used. Ion mobility (IM) spectrometry is a very rapid gas-phase separation technique and offers additional separation capability within the LC timeframe. This work investigates the use of an IM separator in combination with high-pressure liquid chromatography (HPLC) and MS, to improve the separation of drug-related materials from excipients, thus aiding the identification of trace-level impurities in an anti-HIV medication, Combivir.     

Analysis of aliphatic and aromatic carbonyl compounds in ambient air by LC/MS/MS.

A sensitive liquid chromatograph/tandem mass spectrometric technique (LC/MS/MS) was applied to determine aliphatic and aromatic carbonyl compounds in ambient air. Traces of the carbonyl compounds were sampled by passing through a Sep-Pak DNPH-silica cartridge. Their derivatives were thus eluted with acetonitrile, separated by reversed-phase liquid chromatography and determined by quadrupole tandem mass spectrometry in an atmospheric pressure chemical ionization (APCI) mode with multiple reaction monitoring (MRM). The detection limits (DL) of the carbonyl compounds were 0.8 - 15 ng/m3. A number of the carbonyl compounds were detected at n.d.- 14 microg/m3 levels. The precursor ion scanning analysis was applied to identify the unknown compounds.     

Mass spectrometry of doping preparations of a new generation: Peroxisome proliferator-activated receptor agonists

The mass spectral properties of the peroxisome proliferator-activated receptor agonists (PPARδ) have been studied by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) and high-performance liquid chromatography/high resolution mass spectrometry (HPLC/HRMS). Dissociation pathways of the investigated compounds under the conditions of collision activation have been proposed on the basis of the consolidated analysis of the data provided by these methods.     

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Characterization and sequence identification of angiotensin II by a novel method involving ultra-fast liquid chromatography assay coupled with matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight five tandem mass spectrometry analysis

High-throughput proteomics aims to investigate dynamically changing proteins expressed by a full organism, specific tissue or cellular compartment under certain conditions. High-sensitivity mass spectrometry has gradually become a significant tool for characterizing peptides. Here, we analyzed angiotensin II using ultra-fast liquid chromatography (UFLC) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). First, we applied UFLC in isolating and collecting the angiotensin II, and then Axima-Resonance (MALDI-QIT-ToF MS(5)) was adopted, which enables collision-induced dissociation-MS(5) analysis for fine structural characterization of angiotensin II. Resultant MS, MS(2), MS(3) and MS(4) spectra of interested [M+H](+) ions selected as precursor ions yielded detailed information about the sites of fragmentation as well as the amino acid sequence for angiotensin II; meanwhile, the average deviation between theoretical mass and actually measured mass from MS to MS(5) spectra was only 0.32 Da. It indicated that Axima-Resonance was capable of analyzing the peptide sequence accurately and provide the corresponding fragmentation information thoroughly, thus suggesting a potential strategy involving UFLC assay coupled with MALDI-QIT-ToF MS(5) analysis on high-throughput proteomics study in future.     

Combination of different liquid chromatography/mass spectrometry technologies for the identification of transformation products of rhodamine B in groundwater

Rhodamine B and its five de‐ethylated transformation products could be identified in a groundwater sample. Using high‐performance thin‐layer chromatography (HPTLC) six fluorescent zones were detected in the sample. In order to identify the compounds in the zones by exact mass mass spectrometry (MS) measurements and tandem mass spectrometry (MS/MS), they were extracted from the HPTLC plate for subsequent analysis by nano‐chip high‐performance liquid chromatography quadrupole‐time‐of‐flight mass spectrometry (nano‐chip HPLC/QTOFMS). In addition, chemical derivatisation experiments on HPTLC plates were applied to detect the presence of a primary amino group in the transformation products. From the combined analytical results it was possible to allocate rhodamine B and its five de‐ethylated transformation products to the six different HPTLC zones. The quantification of rhodamine B in different groundwater samples was carried out by a high‐performance liquid chromatography/triple quadrupole mass spectrometry (HPLC/MS/MS). The maximum detected concentration of rhodamine B was 83 µg L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of liquid chromatography-tandem mass spectrometry to the analysis of benzodiazepines in blood

A sensitive and selective high-performance liquid chromatography/atmospheric pressure chemical ionisation tandem mass spectrometry (HPLC/APCI-MS/MS) method for the simultaneous detection of 18 benzodiazepines and metabolites in human blood is described. The procedure utilises butyl chloride extraction at alkaline pH followed by reversed-phase liquid chromatography. The technique is suitable for screening analyses and confirmation of identity of the benzodiazepines at their lowest reported therapeutic concentrations using 500 microL of blood. The method has been successfully applied in forensic cases involving low concentrations of benzodiazepines.     

Qualitative and quantitative analysis of the chemical constituents in Mahuang‐Fuzi‐Xixin decoction based on high performance liquid chromatography combined with time‐of‐flight mass spectrometry and triple quadrupole mass spectrometers

High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry (HPLC‐TOF/MS) and high‐performance liquid chromatography–triple quadrupole mass spectrometry (HPLC‐QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang‐Fuzi‐Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC‐QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC‐QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter‐ and intra‐day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang‐Fuzi‐Xixin decoction.     

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of bacteriochlorophylls from Chlorobiaceae: characteristic fragmentations

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry/mass spectrometry (APCI-LC/MS/MS) has been applied to the study of bacteriochlorophylls c, d, and e of phototrophic prokaryotes. Cultures of Chlorobiaceae containing bacteriochlorophyll c, d or e were examined using a high-resolution high-performance liquid chromatography (HPLC) method and APCI-LC/MS/MS employing post-column addition of formic acid. The results reveal complex distributions of bacteriochlorophyll homologues, with some closely eluting species giving isobaric protonated molecules. On-line LC/MS/MS studies reveal characteristic fragment ions for bacteriochlorophylls c, d, and e. Fragmentations involving loss of the extended alkyl substituents that are unique to bacteriochlorophylls c, d and e and their derivatives have been rationalised by studying the phaeophorbides and the results applied to the direct study of the bacteriochlorophylls.