Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.     

A strategy for the identification of site-specific glycosylation in glycoproteins using MALDI TOF MS

A strategy for investigation of site-specific glycosylation of glycoproteins has been developed, based on peptide mass fingerprinting using matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF MS). The glycoprotein is subjected to sequential digestion with a protease and glycan-specific endoglycosidases or with the glycan-specific endoglycosidases followed by the protease. Peptides with characteristic masses are detected for sequences containing glycosylated asparagine residues. By using a panel of three proteases, chymotrypsin, protease V8 and trypsin, and endoglycosidases F3 and H and peptide N-glycanase F, it was possible to monitor the state of glycosylation of all putative N-glycosylation sites on three glycoproteins. It was deduced that all potential N-glycosylation sites in human serum transferrin (two) and α1-antitrypsin (three) were occupied by non-fucosylated, biantennary, disialylated, complex glycans. In contrast, only four (asparagines 19, 59, 146 and 270) out of the five potential sites were glycosylated in recombinant human β-glucosylceramidase, with the site nearest the C-terminal (asparagine 462) being unoccupied. The glycans at each site consisted of a mixture of non-fucosylated and core α1–6 fucosylated oligomannose glycans (Man₃ GlcNAc₂), derived from the enzymic truncation of complex glycans.     

Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and drbitrap mass spectrometry

Protein arginine (Arg) methylation serves an important functional role in eucaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron-transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679–695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z=2–4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.     

Fast liquid chromatography coupled to electrospray tandem mass spectrometry peptide sequencing for cross-species protein identification

Using a parallel microcolumn switching liquid chromatography set-up coupled to a quadrupole time-of-flight mass spectrometer, a rapid liquid chromatography/mass spectrometric (LC/MS) protein identification method is presented. Without prior sample clean-up up to 300 protein digest samples a day can be processed. Using data-directed acquisition, up to 10 fragmentation analyses for each protein sample can be acquired in the same chromatographic run that can be used for database searching. Using internal peptide sequence information, protein databases and the various nucleic acid databases can both be queried for cross-species identification of the protein sample. The method was evaluated and put into force to generate data for a tobacco cell culture protein database.     

Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method is described for relative quantification of phosphoproteins to simultaneously compare the phosphorylation status of proteins under two different conditions. Quantification was achieved by beta-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d(5)-thiol selectively at the vinyl moiety of dehydroalanine and dehydroamino-2-butyric acid. The method was evaluated using the model phosphoprotein alpha(S1)-casein, for which three phosphopeptides were found after tryptic digestion. Reproducibility of the relative quantification of seven independent replicates was found to be 11% SD. The dynamic range covered two orders of magnitude, and quantification was linear for mixtures of 0 to 100% alpha(S1)-casein and dephospho-alpha(S1)-casein (R(2) = 0.986). Additionally, the method allowed protein identification and determination of the phosphorylation sites via MS/MS fragmentation.     

Chemical and metabolic profiling of Codonopsis Radix extract with an integrated strategy using ultra-high-performance liquid chromatography coupled with mass spectrometry

Codonopsis radix was commonly used as food materials or herbal medicines in many countries. However, the comprehensive analysis of chemical constituents, and in vivo xenobiotics of Codonopsis radix remain unclear. In the present study, an integrated strategy with feature-based molecular networking using ultra-high-performance liquid chromatography coupled with mass spectrometry was established to systematically screen the chemical constituents and the in vivo xenobiotics of Codonopsis radix. A step-by-step manner based on a composition database, visual structure classification, discriminant ions, and metabolite software prediction was proposed to overcome the complexities due to the similar structure of chemical constituents and metabolites of Codonopsis radix. As a result, 103 compounds were tentatively characterized, 20 of which were identified by reference standards. Besides, a total of 50 xenobiotics were detected in vivo, including 26 prototypes and 24 metabolites, while the metabolic features of the pyrrolidine alkaloids were elucidated for the first time. The metabolism reactions of pyrrolidine alkaloids and sesquiterpene lactones included oxidation, methylation, hydration, hydrogenation, demethylation, glucuronidation, and sulfation. This study provided a generally applicable approach to the comprehensive investigation of the chemical and metabolic profile of traditional Chinese medicine and offered reasonable guidelines for further screening of quality control indicators and pharmacodynamics mechanism of Codonopsis radix.     

Revolving hollow fiber–liquid phase microextraction coupled to GC/MS using electron ionization for quantification of five aromatic hydrocarbon isomers

A dynamic liquid phase microextraction method using a revolving hollow fiber was demonstrated for coupling to GC/MS [using EI (electronic ionization) and selected ion monitor (SIM)] as a concentrating probe for rapid analysis and quantitative determination of five aromatic hydrocarbon isomers (cumene; propylbenzene; 2‐ethyltoluene; 1,2,3‐trimethylbenzene; and 1,2,4‐trimethylbenzene) from biological matrices (human urine and human plasma). This technique was named as revolving hollow fiber–liquid phase microextraction (RHF–LPME). The optimized parameters of RHF–LPME coupled to GC/MS experiments were extraction solvent, toluene; extraction time, 2 min; sample agitation rate, 700 rpm; salt concentration, 0%; rotating speed for motor driving rotator, 250 rpm; and the rotator was operated in a reversed direction with the stirrer. The linear range of calibration curve of RHF–LPME was from 0.002 to 0.4 μg/mL with R² > 0.9916 and the RSD values were from 4.5 to 5.2%. Additionally, comparing to single drop microextraction (SDME), this method offers better limits of detection (LODs) and EF (enrichment factor). This approach exhibits many advantages including simplicity, rapid detection with high reproducibility and high extraction efficiency, easy to operate and fast to reach equilibrium for analyzing biological samples. This approach has the potential to be widely used because it only requires simple devices to perform all extraction processes. We believe that this technique can be a powerful tool for GC/MS analysis of biological samples and clinical applications in the near future.     

Strategies for compound identification and quantification using fused silica capillary column GC/MS

A test mixture containing 28 compounds plus their stable isotopically labeled analogs was analyzed on a daily basis for one month during a series of routine fused silica capillary column GC/MS analyses in order to establish the precision with which retention times and responses were produced. These long-term precision data were evaluated to determine how to best predict retention times and how to best reproduce quantification. Results clearly indicate that relative retention times should be calculated using the reference compound eluted most closely to the target compound and that quantification should be based on relative response using a chemically “similar” compound.     

A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins,and then chelation of lanthanide metal ions as tags,we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry.The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level.The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads.We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75°C,among which N-terminal peptides of 24 proteins are partially acetylated.Moreover,metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified;these make up 55percent of the identified proteins.Finally,we developed a general approach for the relative quantification of proteins based on N-terminal peptides.We adopted lysozyme and ribonuclease B as model proteins;the correlation coefficients(R2)of the standard curves for the quantitative method were 0.9994 and 0.9997,respectively,with each concentration ratio ranging from0.1 to 10 and both relative standard derivations(RSD)measured at less than 5%.In T.tengcongensis at two incubation temperatures,80 proteins possess quantitative information.In addition,compared with the proteins of T.tengcongensis incubated at 55°C,in T.tengcongensis incubated at 75°C,7 proteins upregulate whereas 16 proteins downregulate,and most differential proteins are related to protein synthesis.     

An integrated coupled column liquid chromatography system for the determination of leukotriene metabolites in biological samples

Summary A fully integrated chromatographic system was developed for the determination of leukotrienes in biological samples using photodiode-array detection (PDAD), which eliminates time consuming manual sample handling steps. A special solid phase extraction, (SPE) methodology for leucotriene metabolite stability was developed which increased the recoveries and eliminates the contamination risk of biological samples. The inherent instability (autooxidation) of many of the leukotriene mediators, and the adsorption effects onto exposed surfaces in vials and in the chromatographic system were found to be very important parameters to control in order to circumvent high loss of sample analytes. By binding the cell supernatants to the functionalities of the SPE support stabilised these mediators. Cell culture samples were eluted through a disposable C₁₈ SPE column. The SPE columns were allowed to thaw and deposited in an automated sample handling unit (ASPEC XL). Desorption of the analytes was followed by a second on-line SPE step, to eliminate remaining interfering matrix compounds. Typical recoveries when stored at −70°C were in-between 55–97% except for (LTE₄) which was found to be around 40% after 72 days of storage. Seven reversed-phase packings were studied. Selectivity factors, as well as the separation efficiencies, were found to differ for the various C₁₈ bonded silica stationary phases. This integrated on-line column liquid chromatographic system was applied to the determination of leukotriene B₄, leukotriene C₄, leukotriene D₄, leukotriene and E₄ in human cell extracts using prostaglandin B₂ as the internal standard. More than 1500 biological samples were analysed. Some validation data are presented for unattended operations.     

Profiling and quantification of Drosophila melanogaster lipids using liquid chromatography/mass spectrometry

We present here the findings of global profiling of Drosophila lipids using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on an LTQ-Orbitrap instrument. In addition, we present a multiple reaction monitoring (LC-MRM) method for the absolute quantification of the major phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids of Drosophila. Using both normal- and reversed-phase LC followed by accurate mass analysis and MS/MS on an LTQ-Orbitrap instrument, we evaluated the lipid composition of the fruit fly Drosophila melanogaster. A total of 74 lipid species were identified consisting of glycerphospholipids belonging to the PE, PC, phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes including several plasmanyl PE species, as well as triacylglycerides, cardiolipins, ceramides, and PE ceramides. Individual PE and PC phospholipids were then quantified using an LC-MRM approach. Reversed-phase chromatography followed by monitoring on a QTrap 4000 instrument of 21 MRM transitions combined with calibration curves constructed using internal standards enabled the absolute quantification of 28 PE and PC lipid species with limits of quantification of 3 and 5 pg/μL, respectively. Internal standards accounted for the differences in ionization efficiencies of PE and PC phospholipids, facilitating more accurate lipid abundance measurements. The method presented here builds on previous Drosophila work by making the quantification of absolute lipid abundance possible and will be of interest to scientists who study variation and changes in the degree of unsaturation, fatty acid carbon length, and head-group concentration among individuals of different genotypes in response to environmental, genetic, or physiological perturbation in small insects. It will also be particularly useful to biologists interested in adaptation and acclimation of cellular membranes in response to thermal heterogeneity.     

Determination of nalbuphine using high-performance liquid chromatography coupled to photodiode-array detection and gas chromatography coupled to mass spectrometry.

A procedure involving capillary column gas chromatography coupled to mass spectrometry and a method involving liquid chromatography coupled to a diode-array detector have been developed for the analysis of nalbuphine. The extraction step is the same for both techniques and involves extraction under alkaline conditions in chloroform-2-propanol-n-heptane (50:17:33, v/v/v) with levallorphan as the internal standard. After purification by acidic extraction and back alkaline extraction, drugs are derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane for gas chromatography-mass spectrometry and directly injected for high-performance liquid chromatography-diode-array detection. The limits of detection are 2.0 and 25.0 ng/mg, respectively.     

Simultaneous identification of three pseudoallergic components in Danshen injection by using high‐expression Mas‐related G protein coupled receptor X2 cell membrane chromatography coupled online to HPLC–ESI‐MS/MS

Adverse drug reactions of Danshen injection mainly manifested as pseudoallergic reactions. In the present study, salvianolic acid A and a pair of geometric isomers (isosalvianolic acid C and salvianolic acid C) were identified as pseudoallergic components in Danshen injection by a high‐expression Mas‐related G protein coupled receptor X2 cell membrane chromatography coupled online with high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry. Their pseudoallergic activities were evaluated by in vitro assay, which were consistent with the retention times on the cell membrane chromatography column. Salvianolic acid C, the most outstanding compound, was further found to induce pseudoallergic reaction through Mas‐related G protein coupled receptor X2. All the results above indicated that the system developed in this study is an effective method for simultaneously analyzing pseudoallergic components, even those with similar structures and the microcomponents in complex samples (salvianolic acid C in Danshen injection).     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.     

Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using high-performance liquid chromatography coupled to orbitrap mass spectrometry

A simple and accurate analytical method was developed for simultaneous and quantitative analysis of six triterpenoid saponins in Schefflera kwangsiensis via high-performance liquid chromatography (HPLC) with mass spectrometry (MS) in this study. Separation was performed on a Thermo hypersil GOLD C₁₈ column (150 mm × 2.1 mm, 5 μm). A mobile phase consisting of methanol/acetonitrile/8 mM ammonium acetate in water was used with a flow rate of 0.3 mL/min. The analytes were detected by MS with the electrospray ionisation (ESI) source combined with negative monitoring and full scan mode, and were analysed by extracted ion chromatography. This established HPLC-ESI-MS analysis demonstrated good linearity, sensitivity, stability, precision, accuracy and recovery. Therefore, this analytical method has great potential to be a novel tool to qualify S. kwangsiensis.     

Development of an absolute quantification method targeting growth hormone biomarkers using liquid chromatography coupled to isotope dilution mass spectrometry

A method to perform absolute quantification of two biomarkers (IGF-1 and IGFBP-3) of growth hormone abuse has been developed. Isotope dilution is used with synthetically labelled peptides as internal standards. Peptide selection and multiple reaction monitoring design are discussed. A simple sample preparation based on the reduction and alkylation of cysteine residues followed by tryptic digestion provides a sufficient digestion of proteins. Serum samples fortified with increasing amounts of target proteins are analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer. Specificity is ensured by the selection of sequences with no homology in BLAST, as well as retention time deviation check, and ion ratio monitoring. Linearity is studied in terms of calibration curves. These curves for IGFBP-3 and IGF-1 are generated with mean slopes of 0.055 and 0.065, intercepts of 0.107 and -0.011, and with coefficients of correlation of 0.95 and 0.98, respectively. These curves result from the addition of proteins to the serum. Risks of variations related to potential matrix effects are therefore reduced, as well as probable variations related to the digestion steps. The working concentration ranges are 4-10 ng/microl for IGFBP-3 and 2-8 ng/microl for IGF-1. Preliminary data regarding repeatability show that relative standard deviations (RSDs) range between 13 and 32% for IGFBP-3 and between 7 and 29% for IGF-1.